SUBTILISIN VARIANTS HAVING IMPROVED STABILITY

Abstract

Disclosed herein is one or more subtilisin variant, nucleic acid encoding same, and compositions and methods related to the production and use thereof, including one or more subtilisin variant that has improved stability compared to one or more reference subtilisin.

Description

Disclosed herein is one or more subtilisin variant, and compositions and methods related to the production and use thereof, including one or more subtilisin variant that has improved stability and/or soil removal compared to one or more reference subtilisin.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the sequence listing electronically submitted with the application as an ASCII text file (Name: 20191120_NB41589PCT_ST25_SeqLst.txt; Size: 84 KB; Created: Nov. 20, 2019) forms part of the application and is hereby incorporated herein by reference in its entirety.

BACKGROUND

A protease (also known as a proteinase) is an enzyme that has the ability to break down other proteins. A protease has the ability to conduct proteolysis, by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein. This activity of a protease as a protein-digesting enzyme is termed a proteolytic activity. Many well-known procedures exist for measure ng proteolytic activity (Kalisz, “Microbial Proteinases,” In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology, (1988)). For example, proteolytic activity may be ascertained by comparative assays which analyze the respective protease's ability to hydrolyze a commercial substrate. Exemplary substrates useful in the analysis of protease or proteolytic activity, include, but are not limited to, di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, e.g., WO 99/34011 and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference).

Serine proteases are enzymes (EC No. 3.4.21) possessing an active site serine that initiates hydrolysis of peptide bonds of proteins. Serine proteases comprise a diverse class of enzymes having a wide range of specificities and biological functions that are further divided based on their structure into chymotrypsin-like (trypsin-like) and subtilisin-like. The prototypical subtilisin (EC No. 3.4.21.62) was initially obtained from Bacillus subtilis. Subtilisins (also sometimes referred to as subtilases) and their homologues are members of the S8 peptidase family of the MEROPS classification scheme. Members of family S8 have a catalytic triad in the order Asp, His and Ser in their amino acid sequence. Although a number of useful variant proteases have been developed for cleaning applications, there remains a need for improved subtilisin variants.

BRIEF SUMMARY

In one embodiment, the present disclosure provides one or more subtilisin variant having at least 70% amino acid sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, where the variant has at least one, two, three, four, or more features selected from the group consisting of: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), where the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

In another embodiment, the disclosure provides one or more subtilisin variant having at least 70% amino acid sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, where the variant has at least one, two or more features selected from the group consisting of X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X217L, X218S, X248D, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), where the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

In another embodiment, the disclosure provides one or more subtilisin variant having at least 70% amino acid sequence identity to SEQ ID NO: 8, 10, 13, 14, 16, 17, or 19, where the variant has at least one, two or more features selected from the group consisting of X003V, X009E, X040E, X069S, X076D, X078N, X166Q, X185Q, X218S, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), where the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence. In another embodiment, the disclosure provides one or more subtilisin variant having at least 70% amino acid sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, where the variant has at least one feature selected from the group consisting of X003V-X009E, X003V-X024Q, X003V-X040E, X003V-X069S, X003V-X076D, X003V-X078N, X003V-X079I, X003V-X087D, X003V-X118R, X003V-X124I, X003V-X128S, X003V-X129P, X003V-X130S, X003V-X145R, X003V-X166Q, X003V-X185Q, X003V-X210I, X003V-X217L, X003V-X218S, X003V-X248D, X003V-X259P, X009E-X024Q, X009E-X040E, X009E-X069S, X009E-X076D, X009E-X078N, X009E-X079I, X009E-X087D, X009E-X118R, X009E-X124I, X009E-X128S, X009E-X129P, X009E-X130S, X009E-X145R, X009E-X166Q, X009E-X185Q, X009E-X210I, X009E-X217L, X009E-X218S, X009E-X248D, X009E-X259P, X024Q-X040E, X024Q-X069S, X024Q-X076D, X024Q-X078N, X024Q-X079I, X024Q-X087D, X024Q-X118R, X024Q-X124I, X024Q-X128S, X024Q-X129P, X024Q-X130S, X024Q-X145R, X024Q-X166Q, X024Q-X185Q, X024Q-X210I, X024Q-X217L, X024Q-X218S, X024Q-X248D, X024Q-X259P, X040E-X069S, X040E-X076D, X040E-X078N, X040E-X079I, X040E-X087D, X040E-X118R, X040E-X124I, X040E-X128S, X040E-X129P, X040E-X130S, X040E-X145R, X040E-X166Q, X040E-X185Q, X040E-X210I, X040E-X217L, X040E-X218S, X040E-X248D, X040E-X259P, X069S-X076D, X069S-X078N, X069S-X079I, X069S-X087D, X069S-X118R, X069S-X124I, X069S-X128S, X069S-X129P, X069S-X130S, X069S-X145R, X069S-X166Q, X069S-X185Q, X069S-X210I, X069S-X217L, X069S-X218S, X069S-X248D, X069S-X259P, X076D-X078N, X076D-X079I, X076D-X087D, X076D-X118R, X076D-X124I, X076D-X128S, X076D-X129P, X076D-X130S, X076D-X145R, X076D-X166Q, X076D-X185Q, X076D-X210I, X076D-X217L, X076D-X218S, X076D-X248D, X076D-X259P, X078N-X079I, X078N-X087D, X078N-X118R, X078N-X124I, X078N-X128S, X078N-X129P, X078N-X130S, X078N-X145R, X078N-X166Q, X078N-X185Q, X078N-X210I, X078N-X217L, X078N-X218S, X078N-X248D, X078N-X259P, X079I-X087D, X079I-X118R, X079I-X124I, X079I-X128S, X079I-X129P, X079I-X130S, X079I-X145R, X079I-X166Q, X079I-X185Q, X079I-X210I, X079I-X217L, X079I-X218S, X079I-X248D, X079I-X259P, X087D-X118R, X087D-X124I, X087D-X128S, X087D-X129P, X087D-X130S, X087D-X145R, X087D-X166Q, X087D-X185Q, X087D-X210I, X087D-X217L, X087D-X218S, X087D-X248D, X087D-X259P, X118R-X124I, X118R-X128S, X118R-X129P, X118R-X130S, X118R-X145R, X118R-X166Q, X118R-X185Q, X118R-X210I, X118R-X217L, X118R-X218S, X118R-X248D, X118R-X259P, X124I-X128S, X124I-X129P, X124I-X130S, X124I-X145R, X124I-X166Q, X124I-X185Q, X124I-X210I, X124I-X217L, X124I-X218S, X124I-X248D, X124I-X259P, X128S-X129P, X128S-X130S, X128S-X145R, X128S-X166Q, X128S-X185Q, X128S-X210I, X128S-X217L, X128S-X218S, X128S-X248D, X128S-X259P, X129P-X130S, X129P-X145R, X129P-X166Q, X129P-X185Q, X129P-X210I, X129P-X217L, X129P-X218S, X129P-X248D, X129P-X259P, X130S-X145R, X130S-X166Q, X130S-X185Q, X130S-X210I, X130S-X217L, X130S-X218S, X130S-X248D, X130S-X259P, X145R-X166Q, X145R-X185Q, X145R-X210I, X145R-X217L, X145R-X218S, X145R-X248D, X145R-X259P, X166Q-X185Q, X166Q-X210I, X166Q-X217L, X166Q-X218S, X166Q-X248D, X166Q-X259P, X185Q-X210I, X185Q-X217L, X185Q-X218S, X185Q-X248D, X185Q-X259P, X210I-X217L, X210I-X218S, X210I-X248D, X210I-X259P, X217L-X218S, X217L-X248D, X217L-X259P, X218S-X248D, X218S-X259P, and X248D-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), where the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

Some further embodiments are directed to a composition comprising one or more subtilisin variant described herein. Further embodiments are directed to a method of cleaning comprising contacting a surface or an item in need of cleaning with an effective amount of one or more subtilisin variant described herein or one or more composition described herein.

Still other embodiments are directed to a method for producing a variant described herein, comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding one or more subtilisin variant described herein. Still further embodiments are directed to a polynucleotide comprising a nucleic acid sequence encoding one or more subtilisin variant described herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the locations on the structure of AprE (Subtilisin E from B. subtilis, strain 168) (PDB entry 1SCJ) for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability. The main chain fold of AprE (Subtilisin E) is schematically represented in light gray (only mature polypeptide region is shown, excluding the propeptide segment), the catalytic tried is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence, SEQ ID NO:1).

FIG. 2 depicts the locations on a structural homology model of Bacillus sp LG12 SprC subtilisin (described in WO2015038792) for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability. The main chain fold of LG12 is schematically represented in light gray, the catalytic triad is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence). The subtilisins Chemgen_164A, CP474, and ZP-00454 evaluated in this study are close homologs of LG12 (with amino acid sequence identity of 81.8%, 79.6% and 90.2%, respectively) and would be expected to adopt a similar fold.

FIG. 3 depicts the locations on a structural homology model of B. gibsonii DSM14391 subtilisin for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability. The homology model was built based on the structure of BSP-00801 (described in WO2016205755), which is a variant of the Bgi02446 subtilisin from B. gibsonii clade. The DSM14391 subtilisin, evaluated in this study is a close homolog of the Bgi02446 subtilisin and of the BSP-00801 variant subtilisin (with amino acid sequence identity of 90% and 89.6%, respectively). The main chain fold of DSM14391 is schematically represented in light gray, the catalytic triad is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence).

FIG. 4 depicts the locations on the structure of BPN′ subtilisin from B. amyloliquefaciens (PDB entry 2ST1) for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability at corresponding positions in other subtilisins. The main chain fold of BPN′ is schematically represented in light gray, the catalytic triad is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence).

FIG. 5 depicts the locations on the structure of AprL (subtilisin Carlsberg) from B. licheniformis (PDB entry 1CSE) for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability at corresponding positions in other subtilisins. The main chain fold of AprL is schematically represented in light gray, the catalytic triad is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence).

FIG. 6 depicts the locations on the structure of B. lentus GG36 subtilisin (PDB entry 1JEA) for a subset of sites where certain amino acid residues evaluated in this study show benefit in increasing stability at corresponding positions in other subtilisins, including Bpan01744 (with amino acid sequence identity of 89.6%). The main chain fold of GG36 is schematically represented in light gray, the catalytic triad is shown as gray spheres, and the sites evaluated for stability improvement are shown as black stick figures (numbered with respect to BPN′ subtilisin sequence).

DETAILED DESCRIPTION

The present disclosure provides subtilisin variants having amino acid sequences with one, two, three or more features (e.g. substitutions) at positions in the polypeptide sequence that provide for improved stability of the variant subtilisin when compared to a reference subtilisin lacking the one, two, three, or more features. As provided in more detail below, the features are found at positions selected from 3, 9, 24, 40, 69, 76, 78, 79, 87, 118, 124, 128, 129, 130, 145, 166, 182, 185, 210, 211, 217, 218, 248, and 259, where positions are numbered by correspondence to the amino acid positions of BPN′ (SEQ ID NO: 1) using the multiple protein sequence alignment shown on Table 28 of this application. The features can be combinations at the positions listed above, and include substitutions or, in some cases, combinations of wildtype amino acids and substitutions at the identified positions that provide improved stability in comparison to a parent or reference subtilisin polypeptide. Also provided are compositions (e.g. enzyme compositions or detergent compositions (e.g. dishwashing and laundry detergent compositions)) containing such subtilisin variants and methods using such variants and compositions.

Terms and abbreviations not defined should be accorded their ordinary meaning as used in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein are to be interpreted in the context of the specification as a whole. As used herein, the singular “a,” “an” and “the” includes the plural unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acid sequences are written left to right in 5′ to 3′ orientation; and amino acid sequences are written left to right in amino to carboxy orientation. Each numerical range used herein includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein in connection with a numerical value, the term “about” refers to a range of +/−0.5 of the numerical value, unless the term is otherwise specifically defined in context. For instance, the phrase a “pH value of about 6” refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.

The nomenclature of the amino acid substitutions of the one or more subtilisin variants described herein uses one or more of the following: position; position:amino acid or amino acid substitution(s); or starting amino acid(s):position:amino acid substitution(s). Reference to a “position” (i.e. 5, 8, 17, 22, etc) encompasses any starting amino acid that may be present at such position, and any substitution that may be present at such position. Reference to a position can be recited in several forms, for example, position 003 can also be referred to as position 3. Reference to a “position: amino acid substitution(s)” (i.e. 1S/T/G, 3G, 17T, etc) encompasses any starting amino acid that may be present at such position and the one or more amino acid(s) with which such starting amino acid may be substituted. Reference to a starting or substituted amino acid may be further expressed as several starting, or substituted amino acids separated by a foreslash (“/”). For example, D275S/K indicates position 275 is substituted with serine (S) or lysine (K) and P/S197K indicates that starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K). Reference to an X as the amino acid in a position, refers to any amino acid at the recited position.

Unless otherwise indicated, the position of an amino acid residue in a given amino acid sequence is numbered by correspondence with the amino acid sequence of SEQ ID NO:1. That is, the amino acid sequence of BPN′ shown in SEQ ID NO:1 serves as a reference sequence. In one embodiment, the amino acid sequence of one or more subtilisin variant described herein is aligned with the amino acid sequence of SEQ ID NO:1 in accordance with Table 28 using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO:1 is conveniently numbered by reference to the numerical position of that corresponding amino acid residue. Sequence alignment algorithms, such as, for example, those described herein will identify the location or locations where insertions or deletions occur in a subject sequence when compared to a query sequence (also sometimes referred to as “reference sequence”).

The terms “protease” and “proteinase” refer to an enzyme that has the ability to break down proteins and peptides. A protease has the ability to conduct “proteolysis,” by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein. This activity of a protease as a protein-digesting enzyme is referred to as “proteolytic activity.” Many well-known procedures exist for measuring proteolytic activity. For example, proteolytic activity may be ascertained by comparative assays that analyze the respective protease's ability to hydrolyze a suitable substrate. Exemplary substrates useful in the analysis of protease or proteolytic activity, include, but are not limited to, di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (See e.g., WO99/34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (See e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) also finds use in determining the active enzyme concentration. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 405 or 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. The activity on substrate divided by protein concentration gives the enzyme specific activity.

The phrase “composition(s) substantially-free of boron” or “detergent(s) substantially-free of boron” refers to composition(s) or detergent(s), respectively, that contain trace amounts of boron, for example, less than about 1000 ppm (1 mg/kg or liter equals 1 ppm), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm, perhaps from other compositions or detergent constituents.

As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus”, or B. polymyxa, which is now “Paenibacillus polymyxa”. The production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

The term “vector” refers to a nucleic acid construct used to introduce or transfer nucleic acid(s) into a target cell or tissue. A vector is typically used to introduce foreign DNA into a cell or tissue. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vector), cosmids, expression vectors, shuttle vectors, and the like. A vector typically includes an origin of replication, a multicloning site, and a selectable marker. The process of inserting a vector into a target cell is typically referred to as transformation. The present invention includes, in some embodiments, a vector that comprises a DNA sequence encoding a serine protease polypeptide (e.g., precursor or mature serine protease polypeptide) that is operably linked to a suitable prosequence (e.g., secretory, signal peptide sequence, etc.) capable of effecting the expression of the DNA sequence in a suitable host, and the folding and translocation of the recombinant polypeptide chain.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell which results from the uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

The term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may also refer to translation of mRNA into a polypeptide. Thus, the term “expression” includes any step involved in the “production of the polypeptide” including, but not limited to, transcription, post-transcriptional modifications, translation, post-translational modifications, secretion and the like.

The phrases “expression cassette” or “expression vector” refer to a nucleic acid construct or vector generated recombinantly or synthetically for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or transgene) in a target cell. The nucleic acid of interest typically expresses a protein of interest. An expression vector or expression cassette typically comprises a promoter nucleotide sequence that drives or promotes expression of the foreign nucleic acid. The expression vector or cassette also typically includes other specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell or genome of the host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors for expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of those of skill in the art.

As used herein, a nucleic acid is “operably linked” with another nucleic acid sequence when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is positioned so as to facilitate translation of the coding sequence. Typically, “operably linked” DNA sequences are contiguous. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.

The term “gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions. In some instances a gene includes intervening sequences (introns) between individual coding segments (exons).

The term “recombinant”, when used with reference to a cell typically indicates that the cell has been modified by the introduction of a foreign nucleic acid sequence or that the cell is derived from a cell so modified. For example, a recombinant cell may comprise a gene not found in identical form within the native (non-recombinant) form of the cell, or a recombinant cell may comprise a native gene (found in the native form of the cell) that has been modified and re-introduced into the cell. A recombinant cell may comprise a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for the production of recombinant DNA in vitro and transfer of the recombinant DNA into cells where it may be expressed or propagated, thereby producing a recombinant polypeptide. “Recombination” and “recombining” of polynucleotides or nucleic acids refer generally to the assembly or combining of two or more nucleic acid or polynucleotide strands or fragments to generate a new polynucleotide or nucleic acid.

A nucleic acid or polynucleotide is said to “encode” a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a nucleic acid is also said to encode the sequence.

The terms “host strain” and “host cell” refer to a suitable host for an expression vector comprising a DNA sequence of interest.

A “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues. The terms “protein” and “polypeptide” are used interchangeably herein. The single and three-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

The terms “prosequence” or “propeptide sequence” refer to an amino acid sequence between the signal peptide sequence and mature protease sequence that is involved in the proper folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in a mature active protease. Bacterial serine proteases are often expressed as pro-enzymes. Examples of modified propeptides are provided, for example, in WO 2016/205710.

The terms “signal sequence” and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursor form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase after the protein is transported.

The term “mature” form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide sequence and propeptide sequence.

The term “precursor” form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a “signal” sequence operably linked to the amino terminus of the prosequence. The precursor may also have additional polypeptides that are involved in post-translational activity (e.g., polypeptides cleaved therefrom to leave the mature form of a protein or peptide).

The term “wildtype”, with respect to a polypeptide, refers to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions. Similarly, the term “wildtype”, with respect to a polynucleotide, refers to a naturally-occurring polynucleotide that does not include a man-made substitution, insertion, or deletion at one or more nucleotides. A polynucleotide encoding a wildtype polypeptide is, however, not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wildtype or parental polypeptide.

The term “parent”, with respect to a polypeptide, includes reference to a naturally-occurring, or wildtype, polypeptide or to a naturally-occurring polypeptide in which a man-made substitution, insertion, or deletion at one or more amino acid positions has been made. The term “parent” with respect to a polypeptide also includes any polypeptide that has protease activity that serves as the starting polypeptide for alteration, such as substitutions, additions, and/or deletions, to result in a variant having one or more alterations in comparison to the starting polypeptide. That is, a parental, or reference polypeptide is not limited to a naturally-occurring wildtype polypeptide, and encompasses any wildtype, parental, or reference polypeptide. Similarly, the term “parent,” with respect to a polynucleotide, can refer to a naturally-occurring polynucleotide or to a polynucleotide that does include a man-made substitution, insertion, or deletion at one or more nucleotides. The term “parent” with respect to a polynucleotide also includes any polynucleotide that encodes a polypeptide having protease activity that serves as the starting polynucleotide for alteration to result in a variant protease having a modification, such as substitutions, additions, and/or deletions, in comparison to the starting polynucleotide. That is, a polynucleotide encoding a wildtype, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wildtype, parental, or reference polypeptide.

The term “naturally-occurring” refers to, for example, a sequence and residues contained therein (e.g., polypeptide sequence and amino acids contained therein or nucleic acid sequence and nucleotides contained therein) that are found in nature. Conversely, the term “non-naturally occurring” refers to, for example, a sequence and residues contained therein (e.g., polypeptide sequences and amino acids contained therein or nucleic acid sequence and nucleotides contained therein) that are not found in nature.

As used herein with regard to amino acid residue positions, “corresponding to” or “corresponds to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, “corresponding region” generally refers to an analogous position in a related protein or a reference protein.

The terms “derived from” and “obtained from” refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question. To exemplify, “proteases derived from Bacillus” refers to those enzymes having proteolytic activity that are naturally produced by Bacillus, as well as to serine proteases like those produced by Bacillus sources but which through the use of genetic engineering techniques are produced by other host cells transformed with a nucleic acid encoding the serine proteases.

The term “identical” in the context of two polynucleotide or polypeptide sequences refers to nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art.

The phrases “% identity” or “percent identity” or “PID” refer to protein sequence identity. Percent identity may be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using a program such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). A percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence including any gaps created by the program for optimal/maximum alignment. BLAST algorithms refer to the “reference” sequence as the “query” sequence.

As used herein, “homologous proteins” or “homologous proteases” refers to proteins that have distinct similarity in primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, MUSCLE, or CLUSTAL. Homologous search of protein sequences can be done using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value cut-off) at 0.001. (Altschul et al., “Gapped BLAST and PSI BLAST a new generation of protein database search programs”, Nucleic Acids Res, Set 1; 25(17):3389-402(1997)). The BLAST program uses several search parameters, most of which are set to the default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for a nucleic acid sequence searches include: Neighboring words threshold=11; E-value cutoff=10; Scoring Matrix=NUC.3.1 (match=1, mismatch=−3); Gap Opening=5; and Gap Extension=2. Exemplary default BLAST parameters for amino acid sequence searches include: Word size=3; E-value cutoff=10; Scoring Matrix=BLOSUM62; Gap Opening=11; and Gap extension=1. Using this information, protein sequences can be grouped and/or a phylogenetic tree built therefrom. Amino acid sequences can be entered in a program such as the Vector NTI Advance suite and a Guide Tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). The tree construction can be calculated using Kimura's correction for sequence distance and ignoring positions with gaps. A program such as AlignX can display the calculated distance values in parentheses following the molecule name displayed on the phylogenetic tree.

In embodiments where three-dimensional structures of proteins have been determined or homology models created, structurally homologous amino acid positions between two or more molecules can be determined. For molecules with significant structural similarities, it might be expected that introducing substitutions that confer improvement in one molecule at structurally homologous sites in another molecule could confer similar improvements in performance and/or stability to these molecules. Structurally homologous amino acid positions can be identified by performing a structural alignment, which attempts to determine homology between two or more protein structures based on their shape and three-dimensional conformation. Structural alignment can produce a superposition of the atomic coordinate sets and a minimal root mean square deviation between the structures. Examples of methods for creating structural alignments are the distance alignment matrix method (DALI) (Holm L, Sander C (1996) “Mapping the protein universe”, Science, 273 (5275): 595-603), combinatorial extension (CE) (Shindyalov, I. N.; Bourne P. E. (1998) “Protein structure alignment by incremental combinatorial extension (CE) of the optimal path”, Protein Engineering, 11 (9): 739-747), and Sequential Structure Alignment Program (S SAP) (Taylor W R, Flores T P, Orengo C A (1994) “Multiple protein structure alignment”, Protein Sci., 3 (10): 1858-70). By combining multiple sequence alignments with structural alignments, structurally homologous amino acid positions can be identified in molecules for which the three-dimensional structure has not been determined. Examples of methods for creating multiple sequence alignment-based structural alignments are 3DCoffee (Poirot O et al (2004) “3DCoffee@igs: a web server for combining sequences and structures into a multiple sequence alignment” Nucleic Acids Res., 2004 Jul. 1; 32:W37-40), PROMALS3D (Pei J et al. (2008) “PROMALS3D: a tool for multiple protein sequence and structure alignments.” Nucleic Acids Res., 36(7):2295-300), and 3DM (Kuipers, R K et al (2010) “3DM: Systematic analysis of heterogeneous superfamily data to discover protein functionalities” Proteins, 78(9):2101-13). Understanding the homology between molecules can reveal the evolutionary history of the molecules, as well as information about their function; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the new protein's biochemical function. Two molecules are said to be homologous if they have been derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes, paralogs and orthologs. Paralogs are homologs that are present within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that are present within different species and have very similar or identical functions. A protein superfamily is the largest grouping (clade) of proteins for which common ancestry can be inferred. Usually this common ancestry is based on sequence alignment and mechanistic similarity. Superfamilies typically contain several protein families which show sequence similarity within the family. The term “protein clan” is commonly used for protease superfamilies based on the MEROPS protease classification system. As used herein, the term “subtilisin” includes any member of the S8 serine protease family as described in MEROPS—The Peptidase Data base (Rawlings, N. D. et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350).

The CLUSTAL W algorithm is another example of a sequence alignment algorithm (See, Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994). Default parameters for the CLUSTAL W algorithm include: Gap opening penalty=10.0; Gap extension penalty=0.05; Protein weight matrix=BLOSUM series; DNA weight matrix=IUB; Delay divergent sequences %=40; Gap separation distance=8; DNA transitions weight=0.50; List hydrophilic residues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gap separation penalty=OFF. In CLUSTAL algorithms, deletions occurring at either terminus are included. For example, a variant with a five amino acid deletion at either terminus (or within the polypeptide) of a polypeptide of 500 amino acids would have a percent sequence identity of 99% (495/500 identical residues x 100) relative to the “reference” polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to the polypeptide.

A nucleic acid or polynucleotide is “isolated” when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein or peptide is “isolated” when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is more abundant than are other species in a composition. For example, an isolated species may comprise at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a nucleic acid or a protein sample, respectively, followed by visualization upon staining. If desired, a high-resolution technique, such as high performance liquid chromatography (HPLC) or a similar means can be utilized for purification of the material.

The term “purified” as applied to nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.” A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term “enriched” refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than in a starting composition.

The term “cleaning activity” refers to a cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions prevailing during the proteolytic, hydrolyzing, cleaning, or other process of the disclosure. In some embodiments, cleaning performance of a serine protease or reference subtilisin may be determined by using various assays for cleaning one or more enzyme sensitive stain on an item or surface (e.g., a stain resulting from food, grass, blood, ink, milk, oil, and/or egg protein). Cleaning performance of one or more subtilisin variant described herein or reference subtilisin can be determined by subjecting the stain on the item or surface to standard wash condition(s) and assessing the degree to which the stain is removed by using various chromatographic, spectrophotometric, or other quantitative methodologies. Exemplary cleaning assays and methods are known in the art and include, but are not limited to those described in WO99/34011 and U.S. Pat. No. 6,605,458, as well as those cleaning assays and methods included in the Examples provided below.

The terms “stable” and “stability” with regard to a protease variant refer to a protease that retains a greater amount of residual activity when compared to the parent or reference protease after exposure to altered temperatures over a given period of time under conditions (or “stress conditions”) prevailing during proteolytic, hydrolysing, cleaning or other process. Residual activity is the amount of activity remaining after the test compared to the initial activity of the sample and can be reported as a percentage e.g. % remaining activity. “Altered temperatures” encompass increased or decreased temperatures. In some embodiments, the proteases retain at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity (residual activity) in comparison to the respective parent or reference protease after exposure to altered temperatures over a given time period, for example, at least about 20 minutes, at least about 40 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes.

Alternatively, the terms “stable” and “stability” with regard to a protease variant also refer to a protease that, after exposure to altered temperatures over a given period of time under conditions (or “stress conditions”) prevailing during proteolytic, hydrolysing, cleaning or other process, retains a higher residual activity than a parent, or reference, protease. “Altered temperatures” encompass increased or decreased temperatures. A stability Performance Index (PI) for a variant protease can be obtained by dividing the residual activity of the variant protease by the residual activity of the parent, or reference protease when tested under the same conditions, stressed and non-stressed. In some embodiments, the protease variants have a PI of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, about 3, about 4, or higher than 4, after exposure to altered temperatures over a given time period, for example, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 40 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes. Altered temperatures for evaluation of protein stability can be between 28-85° C., e.g. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80° C. In some embodiments, stability (e.g. residual activity or performance index) may be measured in the presence of one or more protease stabilizers.

Alternatively, the terms “stable” and “stability” with regard to a protease variant also refer to a protease that, after exposure to altered temperatures over a given period of time under conditions (or “stress conditions”) prevailing during proteolytic, hydrolysing, cleaning or other process, exhibits longer half-lives for inactivation (T1/2) than a parent, or reference, protease. “Altered temperatures” encompass increased or decreased temperatures. “Half-lives for inactivation” with regard to a protease variant refers to the time period after which the protease retains one half of the initial enzymatic activity.

The term “stability” includes storage stability and stability during use, e.g. during a wash process and reflects the stability of the subtilisin variant according to the invention as a function of time, e.g. how much activity is retained when the subtilisin variants is kept in solution in particular in a detergent solution. The stability is influenced by many factors e.g. pH, temperature, detergent composition, e.g. amount of builder, surfactant, water content, protease inhibitors/stabilizers etc. The stability of the subtilisin variant may be measured using the assays described in Example 2. The term “improved stability” or “increased stability” is defined herein as a variant subtilisin displaying an increased stability in solutions, relative to the stability of the parent subtilisin. The terms “improved stability” and “increased stability” includes “improved chemical stability” or “improved detergent stability”.

The term “improved detergent stability” is defined herein as a variant subtilisin displaying retention of enzymatic activity after a period of incubation in the presence of a detergent or chemical component of a detergent, which reduces the enzymatic activity of the parent enzyme. Improved detergent stability may also result in variants being more able to catalyze a reaction in the presence of such detergent or chemical components. The term “detergent stability” or “improved detergent stability” is in particular an improved stability of the protease activity when a subtilisin variant of the present invention is mixed into a liquid detergent formulation and incubated at temperatures between 30-70° C., e.g. 35, 40, 45, 50, 55, 60, or 65° C. Detergent stability can be evaluated in a diluted liquid detergent composition, such as 10% detergent, where the commercial liquid detergent is diluted 10 fold in water or a liquid buffer solution prepared at a relevant pH.

The term “enhanced stability” or “improved stability” in the context of an oxidation, chelator, denaturant, surfactant, thermal and/or pH stable protease refers to a higher retained proteolytic activity over time as compared to a reference protease, for example, a wildtype protease or parent protease. Autolysis has been identified as one mode of subtilisin activity loss in liquid detergents. (Stoner et al., 2004 Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors, Enzyme and Microbial Technology 34:114-125.)

The term “effective amount” of one or more subtilisin variant described herein or reference subtilisin refers to the amount of protease that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, bar) composition is required, etc.

The term “adjunct material” refers to any liquid, solid, or gaseous material included in a cleaning composition, other than one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof. In some embodiments, the cleaning compositions of the present disclosure include one or more cleaning adjunct materials. Each cleaning adjunct material is typically selected depending on the particular type and form of cleaning composition (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or other composition). Preferably, each cleaning adjunct material is compatible with the protease enzyme used in the composition.

Cleaning compositions and cleaning formulations include any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object, item, and/or surface. Such compositions and formulations include, but are not limited to, for example, liquid and/or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, bar, granule, and/or solid laundry cleaning or detergent compositions and fine fabric detergent compositions); hard surface cleaning compositions and formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile, laundry booster cleaning or detergent compositions, laundry additive cleaning compositions, and laundry pre-spotter cleaning compositions; dishwashing compositions, including hand or manual dishwashing compositions (e.g., “hand” or “manual” dishwashing detergents) and automatic dishwashing compositions (e.g., “automatic dishwashing detergents”). Single dosage unit forms also find use with the present invention, including but not limited to pills, tablets, gelcaps, or other single dosage units such as pre-measured powders or liquids.

Cleaning composition or cleaning formulations, as used herein, include, unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, granular, gel, solid, tablet, paste, or unit dosage form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) detergent or heavy-duty dry (HDD) detergent types; liquid fine-fabric detergents; hand or manual dishwashing agents, including those of the high-foaming type; hand or manual dishwashing, automatic dishwashing (ADW), or dishware or tableware washing agents, including the various tablet, powder, solid, granular, liquid, gel, and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car shampoos, carpet shampoos, bathroom cleaners; hair shampoos and/or hair-rinses for humans and other animals; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries, such as bleach additives and “stain-stick” or pre-treat types. In some embodiments, granular compositions are in “compact” form; in some embodiments, liquid compositions are in a “concentrated” form.

The term “detergent composition” or “detergent formulation” is used in reference to a composition intended for use in a wash medium for the cleaning of soiled or dirty objects, including particular fabric and/or non-fabric objects or items. In some embodiments, the detergents of the disclosure comprise one or more subtilisin variant described herein and, in addition, one or more surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders (e.g., a builder salt), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and/or solubilizers. In some instances, a builder salt is a mixture of a silicate salt and a phosphate salt, preferably with more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some embodiments are directed to cleaning compositions or detergent compositions that do not contain any phosphate (e.g., phosphate salt or phosphate builder).

The term “bleaching” refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and/or under appropriate pH and/or temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, for example, ClO₂, H₂O₂, peracids, NO₂, etc. Bleaching agents also include enzymatic bleaching agents such as perhydrolase and arylesterases. Another embodiment is directed to a composition comprising one or more subtilisin variant described herein, and one or more perhydrolase, such as, for example, is described in WO2005/056782, WO2007/106293, WO 2008/063400, WO2008/106214, and WO2008/106215.

The term “wash performance” of a protease (e.g., one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof) refers to the contribution of one or more subtilisin variant described herein to washing that provides additional cleaning performance to the detergent as compared to the detergent without the addition of the one or more subtilisin variant described herein to the composition. Wash performance is compared under relevant washing conditions. In some test systems, other relevant factors, such as detergent composition, sud concentration, water hardness, washing mechanics, time, pH, and/or temperature, can be controlled in such a way that condition(s) typical for household application in a certain market segment (e.g., hand or manual dishwashing, automatic dishwashing, dishware cleaning, tableware cleaning, fabric cleaning, etc.) are imitated.

The phrase “relevant washing conditions” is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a hand dishwashing, automatic dishwashing, or laundry detergent market segment.

The term “disinfecting” refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items.

The term “compact” form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding about 15% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by weight of the composition. In some embodiments, the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

Disclosed herein is one or more subtilisin variants useful, for example, in cleaning compositions and applications and in methods of cleaning, as well as in a variety of industrial applications. Disclosed herein is one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants. In some embodiments, one or more subtilisin variants described herein is useful in cleaning applications and can be incorporated into cleaning compositions that are useful in methods of cleaning an item or a surface in need thereof.

In one embodiment, the disclosure provides one or more subtilisin variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, where the variant has at least one, two, three, four, or more features selected from the group consisting of: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′). In one embodiment, the variant does not have 100% sequence identity to a wildtype amino acid sequence (SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22).

In another embodiment, the disclosure provides one or more subtilisin variants having at least one, two, three or more features selected from the group consisting of X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128S, X129P, X130S, X166Q, X182E, X185Q, X217L, X218S, X248D, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In another embodiment, the subtilisin variant has at least one, two, three or more features selected from the group consisting of X003V, X009E, X040E, X069S, X076D, X078N, X166Q, X185Q, X218S, and X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), where the variant does not have 100% sequence identity to a wildtype amino acid sequence.

In still another embodiment, the subtilisin variant has at least two or more features, where the combination of two more features are selected from the group consisting of X003V-X009E, X003V-X024Q, X003V-X040E, X003V-X069S, X003V-X076D, X003V-X078N, X003V-X079I, X003V-X087D, X003V-X118R, X003V-X124I, X003V-X128S, X003V-X129P, X003V-X130S, X003V-X145R, X003V-X166Q, X003V-X185Q, X003V-X210I, X003V-X217L, X003V-X218S, X003V-X248D, X003V-X259P, X009E-X024Q, X009E-X040E, X009E-X069S, X009E-X076D, X009E-X078N, X009E-X079I, X009E-X087D, X009E-X118R, X009E-X124I, X009E-X128S, X009E-X129P, X009E-X130S, X009E-X145R, X009E-X166Q, X009E-X185Q, X009E-X210I, X009E-X217L, X009E-X218S, X009E-X248D, X009E-X259P, X024Q-X040E, X024Q-X069S, X024Q-X076D, X024Q-X078N, X024Q-X079I, X024Q-X087D, X024Q-X118R, X024Q-X124I, X024Q-X128S, X024Q-X129P, X024Q-X130S, X024Q-X145R, X024Q-X166Q, X024Q-X185Q, X024Q-X210I, X024Q-X217L, X024Q-X218S, X024Q-X248D, X024Q-X259P, X040E-X069S, X040E-X076D, X040E-X078N, X040E-X079I, X040E-X087D, X040E-X118R, X040E-X124I, X040E-X128S, X040E-X129P, X040E-X130S, X040E-X145R, X040E-X166Q, X040E-X185Q, X040E-X210I, X040E-X217L, X040E-X218S, X040E-X248D, X040E-X259P, X069S-X076D, X069S-X078N, X069S-X079I, X069S-X087D, X069S-X118R, X069S-X124I, X069S-X128S, X069S-X129P, X069S-X130S, X069S-X145R, X069S-X166Q, X069S-X185Q, X069S-X210I, X069S-X217L, X069S-X218S, X069S-X248D, X069S-X259P, X076D-X078N, X076D-X079I, X076D-X087D, X076D-X118R, X076D-X124I, X076D-X128S, X076D-X129P, X076D-X130S, X076D-X145R, X076D-X166Q, X076D-X185Q, X076D-X210I, X076D-X217L, X076D-X218S, X076D-X248D, X076D-X259P, X078N-X079I, X078N-X087D, X078N-X118R, X078N-X124I, X078N-X128S, X078N-X129P, X078N-X130S, X078N-X145R, X078N-X166Q, X078N-X185Q, X078N-X210I, X078N-X217L, X078N-X218S, X078N-X248D, X078N-X259P, X079I-X087D, X079I-X118R, X079I-X124I, X079I-X128S, X079I-X129P, X079I-X130S, X079I-X145R, X079I-X166Q, X079I-X185Q, X079I-X210I, X079I-X217L, X079I-X218S, X079I-X248D, X079I-X259P, X087D-X118R, X087D-X124I, X087D-X128S, X087D-X129P, X087D-X130S, X087D-X145R, X087D-X166Q, X087D-X185Q, X087D-X210I, X087D-X217L, X087D-X218S, X087D-X248D, X087D-X259P, X118R-X124I, X118R-X128S, X118R-X129P, X118R-X130S, X118R-X145R, X118R-X166Q, X118R-X185Q, X118R-X210I, X118R-X217L, X118R-X218S, X118R-X248D, X118R-X259P, X124I-X128S, X124I-X129P, X124I-X130S, X124I-X145R, X124I-X166Q, X124I-X185Q, X124I-X210I, X124I-X217L, X124I-X218S, X124I-X248D, X124I-X259P, X128S-X129P, X128S-X130S, X128S-X145R, X128S-X166Q, X128S-X185Q, X128S-X210I, X128S-X217L, X128S-X218S, X128S-X248D, X128S-X259P, X129P-X130S, X129P-X145R, X129P-X166Q, X129P-X185Q, X129P-X210I, X129P-X217L, X129P-X218S, X129P-X248D, X129P-X259P, X130S-X145R, X130S-X166Q, X130S-X185Q, X130S-X210I, X130S-X217L, X130S-X218S, X130S-X248D, X130S-X259P, X145R-X166Q, X145R-X185Q, X145R-X210I, X145R-X217L, X145R-X218S, X145R-X248D, X145R-X259P, X166Q-X185Q, X166Q-X210I, X166Q-X217L, X166Q-X218S, X166Q-X248D, X166Q-X259P, X185Q-X210I, X185Q-X217L, X185Q-X218S, X185Q-X248D, X185Q-X259P, X210I-X217L, X210I-X218S, X210I-X248D, X210I-X259P, X217L-X218S, X217L-X248D, X217L-X259P, X218S-X248D, X218S-X259P, X248D-X259P where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In other embodiments, the subtilisin variants disclosed herein contain a combination of two or more features with respect to SEQ ID NO: 1, where the combination of two or more features are selected from the group consisting of X003V-X009E, X003V-X040E, X003V-X069S, X003V-X076D, X003V-X078N, X003V-X079I, X003V-X124I, X003V-X128S, X003V-X129P, X003V-X166Q, X003V-X185Q, X003V-X218S, X003V-X259P, X003V-X262L, X009E-X040E, X009E-X069S, X009E-X076D, X009E-X078N, X009E-X166Q, X009E-X185Q, X009E-X218S, X009E-X259P, X040E-X069S, X040E-X076D, X040E-X078N, X040E-X079I, X040E-X124I, X040E-X128S, X040E-X129P, X040E-X166Q, X040E-X185Q, X040E-X218S, X040E-X259P, X069S-X076D, X069S-X078N, X069S-X079I, X069S-X124I, X069S-X128S, X069S-X129P, X069S-X166Q, X069S-X185Q, X069S-X218S, X069S-X259P, X076D-X078N, X076D-X079I, X076D-X124I, X076D-X128S, X076D-X129P, X076D-X166Q, X076D-X185Q, X076D-X218S, X076D-X259P, X078N-X079I, X078N-X124I, X078N-X128S, X078N-X129P, X078N-X166Q, X078N-X185Q, X078N-X218S, X078N-X259P, X078T-X124I, X079I-X124I, X079I-X128S, X079I-X129P, X079I-X166Q, X079I-X185Q, X079I-X218S, X079I-X259P, X124I-X128S, X124I-X129P, X124I-X166Q, X124I-X185Q, X124I-X218S, X124I-X259K, X124I-X259P, X128S-X129P, X128S-X166Q, X128S-X185Q, X128S-X218S, X128S-X259P, X129P-X166Q, X129P-X185Q, X129P-X218S, X129P-X259P, X166Q-X185Q, X166Q-X218S, X166Q-X259P, X185Q-X218S, X185Q-X259P, and X218S-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In other embodiments, the subtilisin variants disclosed herein contain a combination of three or more features with respect to SEQ ID NO: 1, where the combination of three or more features are selected from the group consisting of X003V-X009E-X024Q, X003V-X009E-X040E, X003V-X009E-X069S, X003V-X009E-X076D, X003V-X009E-X078N, X003V-X009E-X079I, X003V-X009E-X087D, X003V-X009E-X118R, X003V-X009E-X124I, X003V-X009E-X128S, X003V-X009E-X129P, X003V-X009E-X130S, X003V-X009E-X145R, X003V-X009E-X166Q, X003V-X009E-X185Q, X003V-X009E-X210I, X003V-X009E-X217L, X003V-X009E-X218S, X003V-X009E-X248D, X003V-X009E-X259P, X003V-X024Q-X040E, X003V-X024Q-X069S, X003V-X024Q-X076D, X003V-X024Q-X078N, X003V-X024Q-X079I, X003V-X024Q-X087D, X003V-X024Q-X118R, X003V-X024Q-X124I, X003V-X024Q-X128S, X003V-X024Q-X129P, X003V-X024Q-X130S, X003V-X024Q-X145R, X003V-X024Q-X166Q, X003V-X024Q-X185Q, X003V-X024Q-X210I, X003V-X024Q-X217L, X003V-X024Q-X218S, X003V-X024Q-X248D, X003V-X024Q-X259P, X003V-X040E-X069S, X003V-X040E-X076D, X003V-X040E-X078N, X003V-X040E-X079I, X003V-X040E-X087D, X003V-X040E-X118R, X003V-X040E-X124I, X003V-X040E-X128S, X003V-X040E-X129P, X003V-X040E-X130S, X003V-X040E-X145R, X003V-X040E-X166Q, X003V-X040E-X185Q, X003V-X040E-X210I, X003V-X040E-X217L, X003V-X040E-X218S, X003V-X040E-X248D, X003V-X040E-X259P, X003V-X069S-X076D, X003V-X069S-X078N, X003V-X069S-X079I, X003V-X069S-X087D, X003V-X069S-X118R, X003V-X069S-X124I, X003V-X069S-X128S, X003V-X069S-X129P, X003V-X069S-X130S, X003V-X069S-X145R, X003V-X069S-X166Q, X003V-X069S-X185Q, X003V-X069S-X210I, X003V-X069S-X217L, X003V-X069S-X218S, X003V-X069S-X248D, X003V-X069S-X259P, X003V-X076D-X078N, X003V-X076D-X079I, X003V-X076D-X087D, X003V-X076D-X118R, X003V-X076D-X124I, X003V-X076D-X128S, X003V-X076D-X129P, X003V-X076D-X130S, X003V-X076D-X145R, X003V-X076D-X166Q, X003V-X076D-X185Q, X003V-X076D-X210I, X003V-X076D-X217L, X003V-X076D-X218S, X003V-X076D-X248D, X003V-X076D-X259P, X003V-X078N-X079I, X003V-X078N-X087D, X003V-X078N-X118R, X003V-X078N-X124I, X003V-X078N-X128S, X003V-X078N-X129P, X003V-X078N-X130S, X003V-X078N-X145R, X003V-X078N-X166Q, X003V-X078N-X185Q, X003V-X078N-X210I, X003V-X078N-X217L, X003V-X078N-X218S, X003V-X078N-X248D, X003V-X078N-X259P, X003V-X079I-X087D, X003V-X079I-X118R, X003V-X079I-X124I, X003V-X079I-X128S, X003V-X079I-X129P, X003V-X079I-X130S, X003V-X079I-X145R, X003V-X079I-X166Q, X003V-X079I-X185Q, X003V-X079I-X210I, X003V-X079I-X217L, X003V-X079I-X218S, X003V-X079I-X248D, X003V-X079I-X259P, X003V-X087D-X118R, X003V-X087D-X124I, X003V-X087D-X128S, X003V-X087D-X129P, X003V-X087D-X130S, X003V-X087D-X145R, X003V-X087D-X166Q, X003V-X087D-X185Q, X003V-X087D-X210I, X003V-X087D-X217L, X003V-X087D-X218S, X003V-X087D-X248D, X003V-X087D-X259P, X003V-X118R-X124I, X003V-X118R-X128S, X003V-X118R-X129P, X003V-X118R-X130S, X003V-X118R-X145R, X003V-X118R-X166Q, X003V-X118R-X185Q, X003V-X118R-X210I, X003V-X118R-X217L, X003V-X118R-X218S, X003V-X118R-X248D, X003V-X118R-X259P, X003V-X124I-X128S, X003V-X124I-X129P, X003V-X124I-X130S, X003V-X124I-X145R, X003V-X124I-X166Q, X003V-X124I-X185Q, X003V-X124I-X210I, X003V-X124I-X217L, X003V-X124I-X218S, X003V-X124I-X248D, X003V-X124I-X259P, X003V-X128S-X129P, X003V-X128S-X130S, X003V-X128S-X145R, X003V-X128S-X166Q, X003V-X128S-X185Q, X003V-X128S-X210I, X003V-X128S-X217L, X003V-X128S-X218S, X003V-X128S-X248D, X003V-X128S-X259P, X003V-X129P-X130S, X003V-X129P-X145R, X003V-X129P-X166Q, X003V-X129P-X185Q, X003V-X129P-X210I, X003V-X129P-X217L, X003V-X129P-X218S, X003V-X129P-X248D, X003V-X129P-X259P, X003V-X130S-X145R, X003V-X130S-X166Q, X003V-X130S-X185Q, X003V-X130S-X210I, X003V-X130S-X217L, X003V-X130S-X218S, X003V-X130S-X248D, X003V-X130S-X259P, X003V-X145R-X166Q, X003V-X145R-X185Q, X003V-X145R-X210I, X003V-X145R-X217L, X003V-X145R-X218S, X003V-X145R-X248D, X003V-X145R-X259P, X003V-X166Q-X185Q, X003V-X166Q-X210I, X003V-X166Q-X217L, X003V-X166Q-X218S, X003V-X166Q-X248D, X003V-X166Q-X259P, X003V-X185Q-X210I, X003V-X185Q-X217L, X003V-X185Q-X218S, X003V-X185Q-X248D, X003V-X185Q-X259P, X003V-X210I-X217L, X003V-X210I-X218S, X003V-X210I-X248D, X003V-X210I-X259P, X003V-X217L-X218S, X003V-X217L-X248D, X003V-X217L-X259P, X003V-X218S-X248D, X003V-X218S-X259P, X003V-X248D-X259P, X009E-X024Q-X040E, X009E-X024Q-X069S, X009E-X024Q-X076D, X009E-X024Q-X078N, X009E-X024Q-X079I, X009E-X024Q-X087D, X009E-X024Q-X118R, X009E-X024Q-X124I, X009E-X024Q-X128S, X009E-X024Q-X129P, X009E-X024Q-X130S, X009E-X024Q-X145R, X009E-X024Q-X166Q, X009E-X024Q-X185Q, X009E-X024Q-X210I, X009E-X024Q-X217L, X009E-X024Q-X218S, X009E-X024Q-X248D, X009E-X024Q-X259P, X009E-X040E-X069S, X009E-X040E-X076D, X009E-X040E-X078N, X009E-X040E-X079I, X009E-X040E-X087D, X009E-X040E-X118R, X009E-X040E-X124I, X009E-X040E-X128S, X009E-X040E-X129P, X009E-X040E-X130S, X009E-X040E-X145R, X009E-X040E-X166Q, X009E-X040E-X185Q, X009E-X040E-X210I, X009E-X040E-X217L, X009E-X040E-X218S, X009E-X040E-X248D, X009E-X040E-X259P, X009E-X069S-X076D, X009E-X069S-X078N, X009E-X069S-X079I, X009E-X069S-X087D, X009E-X069S-X118R, X009E-X069S-X124I, X009E-X069S-X128S, X009E-X069S-X129P, X009E-X069S-X130S, X009E-X069S-X145R, X009E-X069S-X166Q, X009E-X069S-X185Q, X009E-X069S-X210I, X009E-X069S-X217L, X009E-X069S-X218S, X009E-X069S-X248D, X009E-X069S-X259P, X009E-X076D-X078N, X009E-X076D-X079I, X009E-X076D-X087D, X009E-X076D-X118R, X009E-X076D-X124I, X009E-X076D-X128S, X009E-X076D-X129P, X009E-X076D-X130S, X009E-X076D-X145R, X009E-X076D-X166Q, X009E-X076D-X185Q, X009E-X076D-X210I, X009E-X076D-X217L, X009E-X076D-X218S, X009E-X076D-X248D, X009E-X076D-X259P, X009E-X078N-X079I, X009E-X078N-X087D, X009E-X078N-X118R, X009E-X078N-X124I, X009E-X078N-X128S, X009E-X078N-X129P, X009E-X078N-X130S, X009E-X078N-X145R, X009E-X078N-X166Q, X009E-X078N-X185Q, X009E-X078N-X210I, X009E-X078N-X217L, X009E-X078N-X218S, X009E-X078N-X248D, X009E-X078N-X259P, X009E-X079I-X087D, X009E-X079I-X118R, X009E-X079I-X124I, X009E-X079I-X128S, X009E-X079I-X129P, X009E-X079I-X130S, X009E-X079I-X145R, X009E-X079I-X166Q, X009E-X079I-X185Q, X009E-X079I-X210I, X009E-X079I-X217L, X009E-X079I-X218S, X009E-X079I-X248D, X009E-X079I-X259P, X009E-X087D-X118R, X009E-X087D-X124I, X009E-X087D-X128S, X009E-X087D-X129P, X009E-X087D-X130S, X009E-X087D-X145R, X009E-X087D-X166Q, X009E-X087D-X185Q, X009E-X087D-X210I, X009E-X087D-X217L, X009E-X087D-X218S, X009E-X087D-X248D, X009E-X087D-X259P, X009E-X118R-X124I, X009E-X118R-X128S, X009E-X118R-X129P, X009E-X118R-X130S, X009E-X118R-X145R, X009E-X118R-X166Q, X009E-X118R-X185Q, X009E-X118R-X210I, X009E-X118R-X217L, X009E-X118R-X218S, X009E-X118R-X248D, X009E-X118R-X259P, X009E-X124I-X128S, X009E-X124I-X129P, X009E-X124I-X130S, X009E-X124I-X145R, X009E-X124I-X166Q, X009E-X124I-X185Q, X009E-X124I-X210I, X009E-X124I-X217L, X009E-X124I-X218S, X009E-X124I-X248D, X009E-X124I-X259P, X009E-X128S-X129P, X009E-X128S-X130S, X009E-X128S-X145R, X009E-X128S-X166Q, X009E-X128S-X185Q, X009E-X128S-X210I, X009E-X128S-X217L, X009E-X128S-X218S, X009E-X128S-X248D, X009E-X128S-X259P, X009E-X129P-X130S, X009E-X129P-X145R, X009E-X129P-X166Q, X009E-X129P-X185Q, X009E-X129P-X210I, X009E-X129P-X217L, X009E-X129P-X218S, X009E-X129P-X248D, X009E-X129P-X259P, X009E-X130S-X145R, X009E-X130S-X166Q, X009E-X130S-X185Q, X009E-X130S-X210I, X009E-X130S-X217L, X009E-X130S-X218S, X009E-X130S-X248D, X009E-X130S-X259P, X009E-X145R-X166Q, X009E-X145R-X185Q, X009E-X145R-X210I, X009E-X145R-X217L, X009E-X145R-X218S, X009E-X145R-X248D, X009E-X145R-X259P, X009E-X166Q-X185Q, X009E-X166Q-X210I, X009E-X166Q-X217L, X009E-X166Q-X218S, X009E-X166Q-X248D, X009E-X166Q-X259P, X009E-X185Q-X210I, X009E-X185Q-X217L, X009E-X185Q-X218S, X009E-X185Q-X248D, X009E-X185Q-X259P, X009E-X210I-X217L, X009E-X210I-X218S, X009E-X210I-X248D, X009E-X210I-X259P, X009E-X217L-X218S, X009E-X217L-X248D, X009E-X217L-X259P, X009E-X218S-X248D, X009E-X218S-X259P, X009E-X248D-X259P, X024Q-X040E-X069S, X024Q-X040E-X076D, X024Q-X040E-X078N, X024Q-X040E-X079I, 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X076D-X217L-X259P, X076D-X218S-X248D, X076D-X218S-X259P, X076D-X248D-X259P, X078N-X079I-X087D, X078N-X079I-X118R, X078N-X079I-X124I, X078N-X079I-X128S, X078N-X079I-X129P, X078N-X079I-X130S, X078N-X079I-X145R, X078N-X079I-X166Q, X078N-X079I-X185Q, X078N-X079I-X210I, X078N-X079I-X217L, X078N-X079I-X218S, X078N-X079I-X248D, X078N-X079I-X259P, X078N-X087D-X118R, X078N-X087D-X124I, X078N-X087D-X128S, X078N-X087D-X129P, X078N-X087D-X130S, X078N-X087D-X145R, X078N-X087D-X166Q, X078N-X087D-X185Q, X078N-X087D-X210I, X078N-X087D-X217L, X078N-X087D-X218S, X078N-X087D-X248D, X078N-X087D-X259P, X078N-X118R-X124I, X078N-X118R-X128S, X078N-X118R-X129P, X078N-X118R-X130S, X078N-X118R-X145R, X078N-X118R-X166Q, X078N-X118R-X185Q, X078N-X118R-X210I, X078N-X118R-X217L, X078N-X118R-X218S, X078N-X118R-X248D, X078N-X118R-X259P, X078N-X124I-X128S, X078N-X124I-X129P, X078N-X124I-X130S, X078N-X124I-X145R, X078N-X124I-X166Q, X078N-X124I-X185Q, X078N-X124I-X210I, X078N-X124I-X217L, X078N-X124I-X218S, X078N-X124I-X248D, X078N-X124I-X259P, X078N-X128S-X129P, X078N-X128S-X130S, X078N-X128S-X145R, X078N-X128S-X166Q, X078N-X128S-X185Q, X078N-X128S-X210I, X078N-X128S-X217L, X078N-X128S-X218S, X078N-X128S-X248D, X078N-X128S-X259P, X078N-X129P-X130S, X078N-X129P-X145R, X078N-X129P-X166Q, X078N-X129P-X185Q, X078N-X129P-X210I, X078N-X129P-X217L, X078N-X129P-X218S, X078N-X129P-X248D, X078N-X129P-X259P, X078N-X130S-X145R, X078N-X130S-X166Q, X078N-X130S-X185Q, X078N-X130S-X210I, X078N-X130S-X217L, X078N-X130S-X218S, X078N-X130S-X248D, X078N-X130S-X259P, X078N-X145R-X166Q, X078N-X145R-X185Q, X078N-X145R-X210I, X078N-X145R-X217L, X078N-X145R-X218S, X078N-X145R-X248D, X078N-X145R-X259P, X078N-X166Q-X185Q, X078N-X166Q-X210I, X078N-X166Q-X217L, X078N-X166Q-X218S, X078N-X166Q-X248D, X078N-X166Q-X259P, X078N-X185Q-X210I, X078N-X185Q-X217L, X078N-X185Q-X218S, X078N-X185Q-X248D, X078N-X185Q-X259P, X078N-X210I-X217L, X078N-X210I-X218S, X078N-X210I-X248D, X078N-X210I-X259P, X078N-X217L-X218S, X078N-X217L-X248D, X078N-X217L-X259P, X078N-X218S-X248D, X078N-X218S-X259P, X078N-X248D-X259P, X079I-X087D-X118R, X079I-X087D-X124I, X079I-X087D-X128S, X079I-X087D-X129P, X079I-X087D-X130S, X079I-X087D-X145R, X079I-X087D-X166Q, X079I-X087D-X185Q, X079I-X087D-X210I, X079I-X087D-X217L, X079I-X087D-X218S, X079I-X087D-X248D, X079I-X087D-X259P, X079I-X118R-X124I, X079I-X118R-X128S, X079I-X118R-X129P, X079I-X118R-X130S, X079I-X118R-X145R, X079I-X118R-X166Q, X079I-X118R-X185Q, X079I-X118R-X210I, X079I-X118R-X217L, X079I-X118R-X218S, X079I-X118R-X248D, X079I-X118R-X259P, X079I-X124I-X128S, X079I-X124I-X129P, X079I-X124I-X130S, X079I-X124I-X145R, X079I-X124I-X166Q, X079I-X124I-X185Q, X079I-X124I-X210I, X079I-X124I-X217L, X079I-X124I-X218S, X079I-X124I-X248D, X079I-X124I-X259P, X079I-X128S-X129P, X079I-X128S-X130S, X079I-X128S-X145R, X079I-X128S-X166Q, X079I-X128S-X185Q, X079I-X128S-X210I, X079I-X128S-X217L, X079I-X128S-X218S, X079I-X128S-X248D, X079I-X128S-X259P, X079I-X129P-X130S, X079I-X129P-X145R, X079I-X129P-X166Q, X079I-X129P-X185Q, X079I-X129P-X210I, X079I-X129P-X217L, X079I-X129P-X218S, X079I-X129P-X248D, X079I-X129P-X259P, X079I-X130S-X145R, X079I-X130S-X166Q, X079I-X130S-X185Q, X079I-X130S-X210I, X079I-X130S-X217L, X079I-X130S-X218S, X079I-X130S-X248D, X079I-X130S-X259P, X079I-X145R-X166Q, X079I-X145R-X185Q, X079I-X145R-X210I, X079I-X145R-X217L, X079I-X145R-X218S, X079I-X145R-X248D, X079I-X145R-X259P, X079I-X166Q-X185Q, X079I-X166Q-X210I, X079I-X166Q-X217L, X079I-X166Q-X218S, X079I-X166Q-X248D, X079I-X166Q-X259P, X079I-X185Q-X210I, X079I-X185Q-X217L, X079I-X185Q-X218S, X079I-X185Q-X248D, X079I-X185Q-X259P, X079I-X210I-X217L, X079I-X210I-X218S, X079I-X210I-X248D, X079I-X210I-X259P, X079I-X217L-X218S, X079I-X217L-X248D, X079I-X217L-X259P, X079I-X218S-X248D, X079I-X218S-X259P, X079I-X248D-X259P, X087D-X118R-X124I, X087D-X118R-X128S, X087D-X118R-X129P, X087D-X118R-X130S, X087D-X118R-X145R, X087D-X118R-X166Q, X087D-X118R-X185Q, X087D-X118R-X210I, X087D-X118R-X217L, X087D-X118R-X218S, X087D-X118R-X248D, X087D-X118R-X259P, X087D-X124I-X128S, X087D-X124I-X129P, X087D-X124I-X130S, X087D-X124I-X145R, X087D-X124I-X166Q, X087D-X124I-X185Q, X087D-X124I-X210I, X087D-X124I-X217L, X087D-X124I-X218S, X087D-X124I-X248D, X087D-X124I-X259P, X087D-X128S-X129P, X087D-X128S-X130S, X087D-X128S-X145R, X087D-X128S-X166Q, X087D-X128S-X185Q, X087D-X128S-X210I, X087D-X128S-X217L, X087D-X128S-X218S, X087D-X128S-X248D, X087D-X128S-X259P, X087D-X129P-X130S, X087D-X129P-X145R, X087D-X129P-X166Q, X087D-X129P-X185Q, X087D-X129P-X210I, X087D-X129P-X217L, X087D-X129P-X218S, X087D-X129P-X248D, X087D-X129P-X259P, X087D-X130S-X145R, X087D-X130S-X166Q, X087D-X130S-X185Q, X087D-X130S-X210I, X087D-X130S-X217L, X087D-X130S-X218S, X087D-X130S-X248D, X087D-X130S-X259P, X087D-X145R-X166Q, X087D-X145R-X185Q, X087D-X145R-X210I, X087D-X145R-X217L, X087D-X145R-X218S, X087D-X145R-X248D, X087D-X145R-X259P, X087D-X166Q-X185Q, X087D-X166Q-X210I, X087D-X166Q-X217L, X087D-X166Q-X218S, X087D-X166Q-X248D, X087D-X166Q-X259P, X087D-X185Q-X210I, X087D-X185Q-X217L, X087D-X185Q-X218S, X087D-X185Q-X248D, X087D-X185Q-X259P, X087D-X210I-X217L, X087D-X210I-X218S, X087D-X210I-X248D, X087D-X210I-X259P, X087D-X217L-X218S, X087D-X217L-X248D, X087D-X217L-X259P, X087D-X218S-X248D, X087D-X218S-X259P, X087D-X248D-X259P, X118R-X124I-X128S, X118R-X124I-X129P, X118R-X124I-X130S, X118R-X124I-X145R, X118R-X124I-X166Q, X118R-X124I-X185Q, X118R-X124I-X210I, X118R-X124I-X217L, X118R-X124I-X218S, X118R-X124I-X248D, X118R-X124I-X259P, X118R-X128S-X129P, X118R-X128S-X130S, X118R-X128S-X145R, X118R-X128S-X166Q, X118R-X128S-X185Q, X118R-X128S-X210I, X118R-X128S-X217L, X118R-X128S-X218S, X118R-X128S-X248D, X118R-X128S-X259P, X118R-X129P-X130S, X118R-X129P-X145R, X118R-X129P-X166Q, X118R-X129P-X185Q, X118R-X129P-X210I, X118R-X129P-X217L, X118R-X129P-X218S, X118R-X129P-X248D, X118R-X129P-X259P, X118R-X130S-X145R, X118R-X130S-X166Q, X118R-X130S-X185Q, X118R-X130S-X210I, X118R-X130S-X217L, X118R-X130S-X218S, X118R-X130S-X248D, X118R-X130S-X259P, X118R-X145R-X166Q, X118R-X145R-X185Q, X118R-X145R-X210I, X118R-X145R-X217L, X118R-X145R-X218S, X118R-X145R-X248D, X118R-X145R-X259P, X118R-X166Q-X185Q, X118R-X166Q-X210I, X118R-X166Q-X217L, X118R-X166Q-X218S, X118R-X166Q-X248D, X118R-X166Q-X259P, X118R-X185Q-X210I, X118R-X185Q-X217L, X118R-X185Q-X218S, X118R-X185Q-X248D, X118R-X185Q-X259P, X118R-X210I-X217L, X118R-X210I-X218S, X118R-X210I-X248D, X118R-X210I-X259P, X118R-X217L-X218S, X118R-X217L-X248D, X118R-X217L-X259P, X118R-X218S-X248D, X118R-X218S-X259P, X118R-X248D-X259P, X124I-X128S-X129P, X124I-X128S-X130S, X124I-X128S-X145R, X124I-X128S-X166Q, X124I-X128S-X185Q, X124I-X128S-X210I, X124I-X128S-X217L, X124I-X128S-X218S, X124I-X128S-X248D, X124I-X128S-X259P, X124I-X129P-X130S, X124I-X129P-X145R, X124I-X129P-X166Q, X124I-X129P-X185Q, X124I-X129P-X210I, X124I-X129P-X217L, X124I-X129P-X218S, X124I-X129P-X248D, X124I-X129P-X259P, X124I-X130S-X145R, X124I-X130S-X166Q, X124I-X130S-X185Q, X124I-X130S-X210I, X124I-X130S-X217L, X124I-X130S-X218S, X124I-X130S-X248D, X124I-X130S-X259P, X124I-X145R-X166Q, X124I-X145R-X185Q, X124I-X145R-X210I, X124I-X145R-X217L, X124I-X145R-X218S, X124I-X145R-X248D, X124I-X145R-X259P, X124I-X166Q-X185Q, X124I-X166Q-X210I, X124I-X166Q-X217L, X124I-X166Q-X218S, X124I-X166Q-X248D, X124I-X166Q-X259P, X124I-X185Q-X210I, X124I-X185Q-X217L, X124I-X185Q-X218S, X124I-X185Q-X248D, X124I-X185Q-X259P, X124I-X210I-X217L, X124I-X210I-X218S, X124I-X210I-X248D, X124I-X210I-X259P, X124I-X217L-X218S, X124I-X217L-X248D, X124I-X217L-X259P, X124I-X218S-X248D, X124I-X218S-X259P, X124I-X248D-X259P, X128S-X129P-X130S, X128S-X129P-X145R, X128S-X129P-X166Q, X128S-X129P-X185Q, X128S-X129P-X210I, X128S-X129P-X217L, X128S-X129P-X218S, X128S-X129P-X248D, X128S-X129P-X259P, X128S-X130S-X145R, X128S-X130S-X166Q, X128S-X130S-X185Q, X128S-X130S-X210I, X128S-X130S-X217L, X128S-X130S-X218S, X128S-X130S-X248D, X128S-X130S-X259P, X128S-X145R-X166Q, X128S-X145R-X185Q, X128S-X145R-X210I, X128S-X145R-X217L, X128S-X145R-X218S, X128S-X145R-X248D, X128S-X145R-X259P, X128S-X166Q-X185Q, X128S-X166Q-X210I, X128S-X166Q-X217L, X128S-X166Q-X218S, X128S-X166Q-X248D, X128S-X166Q-X259P, X128S-X185Q-X210I, X128S-X185Q-X217L, X128S-X185Q-X218S, X128S-X185Q-X248D, X128S-X185Q-X259P, X128S-X210I-X217L, X128S-X210I-X218S, X128S-X210I-X248D, X128S-X210I-X259P, X128S-X217L-X218S, X128S-X217L-X248D, X128S-X217L-X259P, X128S-X218S-X248D, X128S-X218S-X259P, X128S-X248D-X259P, X129P-X130S-X145R, X129P-X130S-X166Q, X129P-X130S-X185Q, X129P-X130S-X210I, X129P-X130S-X217L, X129P-X130S-X218S, X129P-X130S-X248D, X129P-X130S-X259P, X129P-X145R-X166Q, X129P-X145R-X185Q, X129P-X145R-X210I, X129P-X145R-X217L, X129P-X145R-X218S, X129P-X145R-X248D, X129P-X145R-X259P, X129P-X166Q-X185Q, X129P-X166Q-X210I, X129P-X166Q-X217L, X129P-X166Q-X218S, X129P-X166Q-X248D, X129P-X166Q-X259P, X129P-X185Q-X210I, X129P-X185Q-X217L, X129P-X185Q-X218S, X129P-X185Q-X248D, X129P-X185Q-X259P, X129P-X210I-X217L, X129P-X210I-X218S, X129P-X210I-X248D, X129P-X210I-X259P, X129P-X217L-X218S, X129P-X217L-X248D, X129P-X217L-X259P, X129P-X218S-X248D, X129P-X218S-X259P, X129P-X248D-X259P, X130S-X145R-X166Q, X130S-X145R-X185Q, X130S-X145R-X210I, X130S-X145R-X217L, X130S-X145R-X218S, X130S-X145R-X248D, X130S-X145R-X259P, X130S-X166Q-X185Q, X130S-X166Q-X210I, X130S-X166Q-X217L, X130S-X166Q-X218S, X130S-X166Q-X248D, X130S-X166Q-X259P, X130S-X185Q-X210I, X130S-X185Q-X217L, X130S-X185Q-X218S, X130S-X185Q-X248D, X130S-X185Q-X259P, X130S-X210I-X217L, X130S-X210I-X218S, X130S-X210I-X248D, X130S-X210I-X259P, X130S-X217L-X218S, X130S-X217L-X248D, X130S-X217L-X259P, X130S-X218S-X248D, X130S-X218S-X259P, X130S-X248D-X259P, X145R-X166Q-X185Q, X145R-X166Q-X210I, X145R-X166Q-X217L, X145R-X166Q-X218S, X145R-X166Q-X248D, X145R-X166Q-X259P, X145R-X185Q-X210I, X145R-X185Q-X217L, X145R-X185Q-X218S, X145R-X185Q-X248D, X145R-X185Q-X259P, X145R-X210I-X217L, X145R-X210I-X218S, X145R-X210I-X248D, X145R-X210I-X259P, X145R-X217L-X218S, X145R-X217L-X248D, X145R-X217L-X259P, X145R-X218S-X248D, X145R-X218S-X259P, X145R-X248D-X259P, X166Q-X185Q-X210I, X166Q-X185Q-X217L, X166Q-X185Q-X218S, X166Q-X185Q-X248D, X166Q-X185Q-X259P, X166Q-X210I-X217L, X166Q-X210I-X218S, X166Q-X210I-X248D, X166Q-X210I-X259P, X166Q-X217L-X218S, X166Q-X217L-X248D, X166Q-X217L-X259P, X166Q-X218S-X248D, X166Q-X218S-X259P, X166Q-X248D-X259P, X185Q-X210I-X217L, X185Q-X210I-X218S, X185Q-X210I-X248D, X185Q-X210I-X259P, X185Q-X217L-X218S, X185Q-X217L-X248D, X185Q-X217L-X259P, X185Q-X218S-X248D, X185Q-X218S-X259P, X185Q-X248D-X259P, X210I-X217L-X218S, X210I-X217L-X248D, X210I-X217L-X259P, X210I-X218S-X248D, X210I-X218S-X259P, X210I-X248D-X259P, X217L-X218S-X248D, X217L-X218S-X259P, X217L-X248D-X259P, X218S-X248D-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In other embodiments, the subtilisin variants disclosed herein contain a combination of three or more features with respect to SEQ ID NO: 1, where the combination of three or more features are selected from the group consisting of X003V-X009E-X040E, X003V-X009E-X069S, X003V-X009E-X076D, X003V-X009E-X078N, X003V-X009E-X166Q, X003V-X009E-X185Q, X003V-X009E-X218S, X003V-X009E-X259P, X003V-X040E-X069S, X003V-X040E-X076D, X003V-X040E-X078N, X003V-X040E-X166Q, X003V-X040E-X185Q, X003V-X040E-X218S, X003V-X040E-X259P, X003V-X069S-X076D, X003V-X069S-X078N, X003V-X069S-X128S, X003V-X069S-X129P, X003V-X069S-X166Q, X003V-X069S-X185Q, X003V-X069S-X218S, X003V-X069S-X259P, X003V-X076D-X078N, X003V-X076D-X129P, X003V-X076D-X166Q, X003V-X076D-X185Q, X003V-X076D-X218S, X003V-X076D-X259P, X003V-X078N-X128S, X003V-X078N-X166Q, X003V-X078N-X185Q, X003V-X078N-X218S, X003V-X078N-X259P, X003V-X124I-X128S, X003V-X124I-X259P, X003V-X128S-X166Q, X003V-X128S-X259P, X003V-X129P-X166Q, X003V-X129P-X185Q, X003V-X129P-X259P, X003V-X166Q-X185Q, X003V-X166Q-X218S, X003V-X166Q-X259P, X003V-X185Q-X218S, X003V-X185Q-X259P, X003V-X218S-X259P, X009E-X040E-X069S, X009E-X040E-X076D, X009E-X040E-X078N, X009E-X040E-X166Q, X009E-X040E-X185Q, X009E-X040E-X218S, X009E-X040E-X259P, X009E-X069S-X076D, X009E-X069S-X078N, X009E-X069S-X166Q, X009E-X069S-X185Q, X009E-X069S-X218S, X009E-X069S-X259P, X009E-X076D-X078N, X009E-X076D-X166Q, X009E-X076D-X185Q, X009E-X076D-X218S, X009E-X076D-X259P, X009E-X078N-X166Q, X009E-X078N-X185Q, X009E-X078N-X218S, X009E-X078N-X259P, X009E-X166Q-X185Q, X009E-X166Q-X218S, X009E-X166Q-X259P, X009E-X185Q-X218S, X009E-X185Q-X259P, X009E-X218S-X259P, X040E-X069S-X076D, X040E-X069S-X078N, X040E-X069S-X166Q, X040E-X069S-X185Q, X040E-X069S-X218S, X040E-X069S-X259P, X040E-X076D-X078N, X040E-X076D-X166Q, X040E-X076D-X185Q, X040E-X076D-X218S, X040E-X076D-X259P, X040E-X078N-X129P, X040E-X078N-X166Q, X040E-X078N-X185Q, X040E-X078N-X218S, X040E-X078N-X259P, X040E-X166Q-X185Q, X040E-X166Q-X218S, X040E-X166Q-X259P, X040E-X185Q-X218S, X040E-X185Q-X259P, X040E-X218S-X259P, X069S-X076D-X078N, X069S-X076D-X128S, X069S-X076D-X166Q, X069S-X076D-X185Q, X069S-X076D-X218S, X069S-X076D-X259P, X069S-X078N-X124I, X069S-X078N-X166Q, X069S-X078N-X185Q, X069S-X078N-X218S, X069S-X078N-X259P, X069S-X128S-X185Q, X069S-X129P-X166Q, X069S-X129P-X185Q, X069S-X129P-X218S, X069S-X129P-X259P, X069S-X166Q-X185Q, X069S-X166Q-X218S, X069S-X166Q-X259P, X069S-X185Q-X218S, X069S-X185Q-X259P, X069S-X218S-X259P, X076D-X078N-X166Q, X076D-X078N-X185Q, X076D-X078N-X218S, X076D-X078N-X259P, X076D-X128S-X166Q, X076D-X129P-X218S, X076D-X166Q-X185Q, X076D-X166Q-X218S, X076D-X166Q-X259P, X076D-X185Q-X218S, X076D-X185Q-X259P, X076D-X218S-X259P, X078N-X124I-X166Q, X078N-X128S-X166Q, X078N-X129P-X259P, X078N-X166Q-X185Q, X078N-X166Q-X218S, X078N-X166Q-X259P, X078N-X185Q-X218S, X078N-X185Q-X259P, X078N-X218S-X259P, X124I-X128S-X166Q, X124I-X128S-X185Q, X124I-X129P-X185Q, X124I-X129P-X259P, X124I-X166Q-X259P, X124I-X185Q-X259P, X124I-X218S-X259P, X128S-X129P-X218S, X128S-X166Q-X185Q, X128S-X166Q-X259P, X128S-X185Q-X218S, X128S-X185Q-X259P, X166Q-X185Q-X218S, X166Q-X185Q-X259P, X166Q-X218S-X259P, and X185Q-X218S-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In other embodiments, the subtilisin variants disclosed herein contain a combination of four or more features with respect to SEQ ID NO: 1, where the combination of four or more features are selected from the group consisting of X003V-X009E-X024Q-X040E, X003V-X009E-X024Q-X069S, X003V-X009E-X024Q-X076D, X003V-X009E-X024Q-X078N, X003V-X009E-X024Q-X079I, X003V-X009E-X024Q-X087D, X003V-X009E-X024Q-X118R, X003V-X009E-X024Q-X124I, X003V-X009E-X024Q-X128S, X003V-X009E-X024Q-X129P, X003V-X009E-X024Q-X130S, X003V-X009E-X024Q-X145R, X003V-X009E-X024Q-X166Q, X003V-X009E-X024Q-X185Q, X003V-X009E-X024Q-X210I, X003V-X009E-X024Q-X217L, X003V-X009E-X024Q-X218S, X003V-X009E-X024Q-X248D, X003V-X009E-X024Q-X259P, X003V-X009E-X040E-X069S, X003V-X009E-X040E-X076D, X003V-X009E-X040E-X078N, X003V-X009E-X040E-X079I, X003V-X009E-X040E-X087D, X003V-X009E-X040E-X118R, X003V-X009E-X040E-X124I, X003V-X009E-X040E-X128S, X003V-X009E-X040E-X129P, X003V-X009E-X040E-X130S, X003V-X009E-X040E-X145R, X003V-X009E-X040E-X166Q, X003V-X009E-X040E-X185Q, X003V-X009E-X040E-X210I, X003V-X009E-X040E-X217L, X003V-X009E-X040E-X218S, X003V-X009E-X040E-X248D, X003V-X009E-X040E-X259P, X003V-X009E-X069S-X076D, X003V-X009E-X069S-X078N, X003V-X009E-X069S-X079I, X003V-X009E-X069S-X087D, X003V-X009E-X069S-X118R, X003V-X009E-X069S-X124I, X003V-X009E-X069S-X128S, X003V-X009E-X069S-X129P, X003V-X009E-X069S-X130S, X003V-X009E-X069S-X145R, X003V-X009E-X069S-X166Q, X003V-X009E-X069S-X185Q, X003V-X009E-X069S-X210I, X003V-X009E-X069S-X217L, X003V-X009E-X069S-X218S, X003V-X009E-X069S-X248D, X003V-X009E-X069S-X259P, X003V-X009E-X076D-X078N, X003V-X009E-X076D-X079I, X003V-X009E-X076D-X087D, X003V-X009E-X076D-X118R, X003V-X009E-X076D-X124I, X003V-X009E-X076D-X128S, X003V-X009E-X076D-X129P, X003V-X009E-X076D-X130S, X003V-X009E-X076D-X145R, X003V-X009E-X076D-X166Q, X003V-X009E-X076D-X185Q, X003V-X009E-X076D-X210I, X003V-X009E-X076D-X217L, X003V-X009E-X076D-X218S, X003V-X009E-X076D-X248D, X003V-X009E-X076D-X259P, X003V-X009E-X078N-X079I, X003V-X009E-X078N-X087D, X003V-X009E-X078N-X118R, X003V-X009E-X078N-X124I, X003V-X009E-X078N-X128S, X003V-X009E-X078N-X129P, X003V-X009E-X078N-X130S, X003V-X009E-X078N-X145R, X003V-X009E-X078N-X166Q, X003V-X009E-X078N-X185Q, X003V-X009E-X078N-X210I, X003V-X009E-X078N-X217L, X003V-X009E-X078N-X218S, X003V-X009E-X078N-X248D, X003V-X009E-X078N-X259P, X003V-X009E-X079I-X087D, X003V-X009E-X079I-X118R, X003V-X009E-X079I-X124I, X003V-X009E-X079I-X128S, X003V-X009E-X079I-X129P, X003V-X009E-X079I-X130S, X003V-X009E-X079I-X145R, X003V-X009E-X079I-X166Q, X003V-X009E-X079I-X185Q, X003V-X009E-X079I-X210I, X003V-X009E-X079I-X217L, X003V-X009E-X079I-X218S, X003V-X009E-X079I-X248D, X003V-X009E-X079I-X259P, X003V-X009E-X087D-X118R, X003V-X009E-X087D-X124I, X003V-X009E-X087D-X128S, X003V-X009E-X087D-X129P, X003V-X009E-X087D-X130S, X003V-X009E-X087D-X145R, X003V-X009E-X087D-X166Q, X003V-X009E-X087D-X185Q, X003V-X009E-X087D-X210I, X003V-X009E-X087D-X217L, X003V-X009E-X087D-X218S, X003V-X009E-X087D-X248D, X003V-X009E-X087D-X259P, X003V-X009E-X118R-X124I, X003V-X009E-X118R-X128S, X003V-X009E-X118R-X129P, X003V-X009E-X118R-X130S, X003V-X009E-X118R-X145R, X003V-X009E-X118R-X166Q, X003V-X009E-X118R-X185Q, X003V-X009E-X118R-X210I, X003V-X009E-X118R-X217L, X003V-X009E-X118R-X218S, X003V-X009E-X118R-X248D, X003V-X009E-X118R-X259P, X003V-X009E-X124I-X128S, X003V-X009E-X124I-X129P, X003V-X009E-X124I-X130S, X003V-X009E-X124I-X145R, X003V-X009E-X124I-X166Q, X003V-X009E-X124I-X185Q, X003V-X009E-X124I-X210I, X003V-X009E-X124I-X217L, X003V-X009E-X124I-X218S, X003V-X009E-X124I-X248D, X003V-X009E-X124I-X259P, X003V-X009E-X128 S-X129P, X003V-X009E-X128 S-X130S, X003V-X009E-X128 S-X145R, X003V-X009E-X128 S-X166Q, X003V-X009E-X128 S-X185Q, X003V-X009E-X128 S-X210I, X003V-X009E-X128S-X217L, X003V-X009E-X128S-X218S, X003V-X009E-X128S-X248D, X003V-X009E-X128S-X259P, X003V-X009E-X129P-X130S, X003V-X009E-X129P-X145R, X003V-X009E-X129P-X166Q, X003V-X009E-X129P-X185Q, X003V-X009E-X129P-X210I, X003V-X009E-X129P-X217L, X003V-X009E-X129P-X218S, X003V-X009E-X129P-X248D, X003V-X009E-X129P-X259P, X003V-X009E-X130S-X145R, X003V-X009E-X130S-X166Q, X003V-X009E-X130S-X185Q, X003V-X009E-X130S-X210I, X003V-X009E-X130S-X217L, X003V-X009E-X130S-X218S, X003V-X009E-X130S-X248D, X003V-X009E-X130S-X259P, X003V-X009E-X145R-X166Q, X003V-X009E-X145R-X185Q, X003V-X009E-X145R-X210I, X003V-X009E-X145R-X217L, X003V-X009E-X145R-X218S, X003V-X009E-X145R-X248D, X003V-X009E-X145R-X259P, X003V-X009E-X166Q-X185Q, X003V-X009E-X166Q-X210I, X003V-X009E-X166Q-X217L, X003V-X009E-X166Q-X218S, X003V-X009E-X166Q-X248D, X003V-X009E-X166Q-X259P, X003V-X009E-X185Q-X210I, X003V-X009E-X185Q-X217L, X003V-X009E-X185Q-X218S, X003V-X009E-X185Q-X248D, X003V-X009E-X185Q-X259P, X003V-X009E-X210I-X217L, X003V-X009E-X210I-X218S, X003V-X009E-X210I-X248D, X003V-X009E-X210I-X259P, X003V-X009E-X217L-X218S, X003V-X009E-X217L-X248D, X003V-X009E-X217L-X259P, X003V-X009E-X218S-X248D, X003V-X009E-X218S-X259P, X003V-X009E-X248D-X259P, X003V-X024Q-X040E-X069S, X003V-X024Q-X040E-X076D, X003V-X024Q-X040E-X078N, X003V-X024Q-X040E-X079I, X003V-X024Q-X040E-X087D, X003V-X024Q-X040E-X118R, X003V-X024Q-X040E-X124I, X003V-X024Q-X040E-X128S, X003V-X024Q-X040E-X129P, X003V-X024Q-X040E-X130S, X003V-X024Q-X040E-X145R, X003V-X024Q-X040E-X166Q, X003V-X024Q-X040E-X185Q, X003V-X024Q-X040E-X210I, X003V-X024Q-X040E-X217L, X003V-X024Q-X040E-X218S, X003V-X024Q-X040E-X248D, X003V-X024Q-X040E-X259P, X003V-X024Q-X069S-X076D, X003V-X024Q-X069S-X078N, X003V-X024Q-X069S-X079I, X003V-X024Q-X069S-X087D, X003V-X024Q-X069S-X118R, X003V-X024Q-X069S-X124I, X003V-X024Q-X069S-X128S, X003V-X024Q-X069S-X129P, X003V-X024Q-X069S-X130S, X003V-X024Q-X069S-X145R, X003V-X024Q-X069S-X166Q, X003V-X024Q-X069S-X185Q, X003V-X024Q-X069S-X210I, X003V-X024Q-X069S-X217L, X003V-X024Q-X069S-X218S, X003V-X024Q-X069S-X248D, X003V-X024Q-X069S-X259P, X003V-X024Q-X076D-X078N, X003V-X024Q-X076D-X079I, X003V-X024Q-X076D-X087D, X003V-X024Q-X076D-X118R, 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X129P-X130S-X145R-X210I, X129P-X130S-X145R-X217L, X129P-X130S-X145R-X218S, X129P-X130S-X145R-X248D, X129P-X130S-X145R-X259P, X129P-X130S-X166Q-X185Q, X129P-X130S-X166Q-X210I, X129P-X130S-X166Q-X217L, X129P-X130S-X166Q-X218S, X129P-X130S-X166Q-X248D, X129P-X130S-X166Q-X259P, X129P-X130S-X185Q-X210I, X129P-X130S-X185Q-X217L, X129P-X130S-X185Q-X218S, X129P-X130S-X185Q-X248D, X129P-X130S-X185Q-X259P, X129P-X130S-X210I-X217L, X129P-X130S-X210I-X218S, X129P-X130S-X210I-X248D, X129P-X130S-X210I-X259P, X129P-X130S-X217L-X218S, X129P-X130S-X217L-X248D, X129P-X130S-X217L-X259P, X129P-X130S-X218S-X248D, X129P-X130S-X218S-X259P, X129P-X130S-X248D-X259P, X129P-X145R-X166Q-X185Q, X129P-X145R-X166Q-X210I, X129P-X145R-X166Q-X217L, X129P-X145R-X166Q-X218S, X129P-X145R-X166Q-X248D, X129P-X145R-X166Q-X259P, X129P-X145R-X185Q-X210I, X129P-X145R-X185Q-X217L, X129P-X145R-X185Q-X218S, X129P-X145R-X185Q-X248D, X129P-X145R-X185Q-X259P, X129P-X145R-X210I-X217L, X129P-X145R-X210I-X218S, X129P-X145R-X210I-X248D, X129P-X145R-X210I-X259P, X129P-X145R-X217L-X218S, X129P-X145R-X217L-X248D, X129P-X145R-X217L-X259P, X129P-X145R-X218S-X248D, X129P-X145R-X218S-X259P, X129P-X145R-X248D-X259P, X129P-X166Q-X185Q-X210I, X129P-X166Q-X185Q-X217L, X129P-X166Q-X185Q-X218S, X129P-X166Q-X185Q-X248D, X129P-X166Q-X185Q-X259P, X129P-X166Q-X210I-X217L, X129P-X166Q-X210I-X218S, X129P-X166Q-X210I-X248D, X129P-X166Q-X210I-X259P, X129P-X166Q-X217L-X218S, X129P-X166Q-X217L-X248D, X129P-X166Q-X217L-X259P, X129P-X166Q-X218S-X248D, X129P-X166Q-X218S-X259P, X129P-X166Q-X248D-X259P, X129P-X185Q-X210I-X217L, X129P-X185Q-X210I-X218S, X129P-X185Q-X210I-X248D, X129P-X185Q-X210I-X259P, X129P-X185Q-X217L-X218S, X129P-X185Q-X217L-X248D, X129P-X185Q-X217L-X259P, X129P-X185Q-X218S-X248D, X129P-X185Q-X218S-X259P, X129P-X185Q-X248D-X259P, X129P-X210I-X217L-X218S, X129P-X210I-X217L-X248D, X129P-X210I-X217L-X259P, X129P-X210I-X218S-X248D, X129P-X210I-X218S-X259P, X129P-X210I-X248D-X259P, X129P-X217L-X218S-X248D, X129P-X217L-X218S-X259P, X129P-X217L-X248D-X259P, X129P-X218S-X248D-X259P, X130S-X145R-X166Q-X185Q, X130S-X145R-X166Q-X210I, X130S-X145R-X166Q-X217L, X130S-X145R-X166Q-X218S, X130S-X145R-X166Q-X248D, X130S-X145R-X166Q-X259P, X130S-X145R-X185Q-X210I, X130S-X145R-X185Q-X217L, X130S-X145R-X185Q-X218S, X130S-X145R-X185Q-X248D, X130S-X145R-X185Q-X259P, X130S-X145R-X210I-X217L, X130S-X145R-X210I-X218S, X130S-X145R-X210I-X248D, X130S-X145R-X210I-X259P, X130S-X145R-X217L-X218S, X130S-X145R-X217L-X248D, X130S-X145R-X217L-X259P, X130S-X145R-X218S-X248D, X130S-X145R-X218S-X259P, X130S-X145R-X248D-X259P, X130S-X166Q-X185Q-X210I, X130S-X166Q-X185Q-X217L, X130S-X166Q-X185Q-X218S, X130S-X166Q-X185Q-X248D, X130S-X166Q-X185Q-X259P, X130S-X166Q-X210I-X217L, X130S-X166Q-X210I-X218S, X130S-X166Q-X210I-X248D, X130S-X166Q-X210I-X259P, X130S-X166Q-X217L-X218S, X130S-X166Q-X217L-X248D, X130S-X166Q-X217L-X259P, X130S-X166Q-X218S-X248D, X130S-X166Q-X218S-X259P, X130S-X166Q-X248D-X259P, X130S-X185Q-X210I-X217L, X130S-X185Q-X210I-X218S, X130S-X185Q-X210I-X248D, X130S-X185Q-X210I-X259P, X130S-X185Q-X217L-X218S, X130S-X185Q-X217L-X248D, X130S-X185Q-X217L-X259P, X130S-X185Q-X218S-X248D, X130S-X185Q-X218S-X259P, X130S-X185Q-X248D-X259P, X130S-X210I-X217L-X218S, X130S-X210I-X217L-X248D, X130S-X210I-X217L-X259P, X130S-X210I-X218S-X248D, X130S-X210I-X218S-X259P, X130S-X210I-X248D-X259P, X130S-X217L-X218S-X248D, X130S-X217L-X218S-X259P, X130S-X217L-X248D-X259P, X130S-X218S-X248D-X259P, X145R-X166Q-X185Q-X210I, X145R-X166Q-X185Q-X217L, X145R-X166Q-X185Q-X218S, X145R-X166Q-X185Q-X248D, X145R-X166Q-X185Q-X259P, X145R-X166Q-X210I-X217L, X145R-X166Q-X210I-X218S, X145R-X166Q-X210I-X248D, X145R-X166Q-X210I-X259P, X145R-X166Q-X217L-X218S, X145R-X166Q-X217L-X248D, X145R-X166Q-X217L-X259P, X145R-X166Q-X218S-X248D, X145R-X166Q-X218S-X259P, X145R-X166Q-X248D-X259P, X145R-X185Q-X210I-X217L, X145R-X185Q-X210I-X218S, X145R-X185Q-X210I-X248D, X145R-X185Q-X210I-X259P, X145R-X185Q-X217L-X218S, X145R-X185Q-X217L-X248D, X145R-X185Q-X217L-X259P, X145R-X185Q-X218S-X248D, X145R-X185Q-X218S-X259P, X145R-X185Q-X248D-X259P, X145R-X210I-X217L-X218S, X145R-X210I-X217L-X248D, X145R-X210I-X217L-X259P, X145R-X210I-X218S-X248D, X145R-X210I-X218S-X259P, X145R-X210I-X248D-X259P, X145R-X217L-X218S-X248D, X145R-X217L-X218S-X259P, X145R-X217L-X248D-X259P, X145R-X218S-X248D-X259P, X166Q-X185Q-X210I-X217L, X166Q-X185Q-X210I-X218S, X166Q-X185Q-X210I-X248D, X166Q-X185Q-X210I-X259P, X166Q-X185Q-X217L-X218S, X166Q-X185Q-X217L-X248D, X166Q-X185Q-X217L-X259P, X166Q-X185Q-X218S-X248D, X166Q-X185Q-X218S-X259P, X166Q-X185Q-X248D-X259P, X166Q-X210I-X217L-X218S, X166Q-X210I-X217L-X248D, X166Q-X210I-X217L-X259P, X166Q-X210I-X218S-X248D, X166Q-X210I-X218S-X259P, X166Q-X210I-X248D-X259P, X166Q-X217L-X218S-X248D, X166Q-X217L-X218S-X259P, X166Q-X217L-X248D-X259P, X166Q-X218S-X248D-X259P, X185Q-X210I-X217L-X218S, X185Q-X210I-X217L-X248D, X185Q-X210I-X217L-X259P, X185Q-X210I-X218S-X248D, X185Q-X210I-X218S-X259P, X185Q-X210I-X248D-X259P, X185Q-X217L-X218S-X248D, X185Q-X217L-X218S-X259P, X185Q-X217L-X248D-X259P, X185Q-X218S-X248D-X259P, X210I-X217L-X218S-X248D, X210I-X217L-X218S-X259P, X210I-X217L-X248D-X259P, X210I-X218S-X248D-X259P, X217L-X218S-X248D-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In other embodiments, the subtilisin variants disclosed herein contain a combination of four or more features with respect to SEQ ID NO: 1, where the combination of four or more features are selected from the group consisting of X003V-X009E-X040E-X076D, X003V-X009E-X040E-X166Q, X003V-X009E-X040E-X185Q, X003V-X009E-X069S-X078N, X003V-X009E-X069S-X166Q, X003V-X009E-X069S-X185Q, X003V-X009E-X076D-X166Q, X003V-X009E-X076D-X218S, X003V-X009E-X166Q-X185Q, X003V-X009E-X166Q-X259P, X003V-X009E-X218S-X259P, X003V-X040E-X069S-X076D, X003V-X040E-X069S-X166Q, X003V-X040E-X076D-X078N, X003V-X040E-X076D-X129P, X003V-X040E-X076D-X185Q, X003V-X040E-X076D-X218S, X003V-X040E-X078N-X124I, X003V-X040E-X124I-X218S, X003V-X040E-X166Q-X185Q, X003V-X040E-X166Q-X259P, X003V-X069S-X076D-X078N, X003V-X069S-X076D-X128S, X003V-X069S-X076D-X185Q, X003V-X069S-X076D-X218S, X003V-X069S-X076D-X259P, X003V-X069S-X078N-X128S, X003V-X069S-X078N-X129P, X003V-X069S-X078N-X166Q, X003V-X069S-X078N-X185Q, X003V-X069S-X078N-X218S, X003V-X069S-X078N-X259P, X003V-X069S-X124I-X218S, X003V-X069S-X128S-X166Q, X003V-X069S-X128S-X185Q, X003V-X069S-X128S-X259P, X003V-X069S-X129P-X185Q, X003V-X069S-X129P-X218S, X003V-X069S-X129P-X259P, X003V-X069S-X166Q-X185Q, X003V-X069S-X166Q-X218S, X003V-X069S-X185Q-X218S, X003V-X069S-X185Q-X259P, X003V-X069S-X218S-X259P, X003V-X076D-X078N-X128S, X003V-X076D-X078N-X166Q, X003V-X076D-X078N-X259P, X003V-X076D-X124I-X166Q, X003V-X076D-X128S-X259P, X003V-X076D-X129P-X166Q, X003V-X076D-X129P-X185Q, X003V-X076D-X129P-X259P, X003V-X076D-X166Q-X185Q, X003V-X076D-X166Q-X259P, X003V-X076D-X185Q-X259P, X003V-X076D-X218S-X259P, X003V-X078N-X124I-X166Q, X003V-X078N-X128S-X185Q, X003V-X078N-X128S-X218S, X003V-X078N-X129P-X185Q, X003V-X078N-X129P-X259P, X003V-X078N-X166Q-X259P, X003V-X078N-X185Q-X218S, X003V-X078N-X185Q-X259P, X003V-X078N-X218S-X259P, X003V-X124I-X128S-X166Q, X003V-X124I-X128S-X218S, X003V-X124I-X128S-X259P, X003V-X124I-X166Q-X259P, X003V-X124I-X185Q-X259P, X003V-X128S-X129P-X185Q, X003V-X128S-X166Q-X218S, X003V-X128S-X185Q-X218S, X003V-X128S-X185Q-X259P, X003V-X128S-X218S-X259P, X003V-X129P-X185Q-X218S, X003V-X166Q-X185Q-X218S, X003V-X166Q-X185Q-X259P, X003V-X166Q-X218S-X259P, X003V-X185Q-X218S-X259P, X009E-X040E-X069S-X076D, X009E-X040E-X069S-X078N, X009E-X040E-X069S-X185Q, X009E-X040E-X069S-X218S, X009E-X040E-X069S-X259P, X009E-X040E-X076D-X078N, X009E-X040E-X076D-X185Q, X009E-X040E-X078N-X185Q, X009E-X040E-X078N-X259P, X009E-X040E-X166Q-X218S, X009E-X040E-X166Q-X259P, X009E-X040E-X185Q-X218S, X009E-X040E-X185Q-X259P, X009E-X040E-X218S-X259P, X009E-X069S-X076D-X078N, X009E-X069S-X076D-X166Q, X009E-X069S-X076D-X185Q, X009E-X069S-X076D-X218S, X009E-X069S-X076D-X259P, X009E-X069S-X078N-X166Q, X009E-X069S-X078N-X218S, X009E-X069S-X166Q-X185Q, X009E-X069S-X166Q-X218S, X009E-X069S-X166Q-X259P, X009E-X069S-X185Q-X218S, X009E-X069S-X218S-X259P, X009E-X076D-X078N-X166Q, X009E-X076D-X078N-X185Q, X009E-X076D-X078N-X259P, X009E-X076D-X166Q-X185Q, X009E-X076D-X166Q-X218S, X009E-X076D-X166Q-X259P, X009E-X076D-X185Q-X218S, X009E-X076D-X185Q-X259P, X009E-X076D-X218S-X259P, X009E-X078N-X166Q-X185Q, X009E-X078N-X166Q-X218S, X009E-X078N-X185Q-X218S, X009E-X078N-X185Q-X259P, X009E-X078N-X218S-X259P, X009E-X166Q-X185Q-X218S, X009E-X166Q-X185Q-X259P, X009E-X166Q-X218S-X259P, X009E-X185Q-X218S-X259P, X040E-X069S-X076D-X078N, X040E-X069S-X076D-X185Q, X040E-X069S-X078N-X166Q, X040E-X069S-X078N-X218S, X040E-X069S-X078N-X259P, X040E-X069S-X129P-X218S, X040E-X069S-X166Q-X218S, X040E-X069S-X166Q-X259P, X040E-X069S-X185Q-X218S, X040E-X069S-X185Q-X259P, X040E-X069S-X218S-X259P, X040E-X076D-X078N-X166Q, X040E-X076D-X078N-X185Q, X040E-X076D-X078N-X218S, X040E-X076D-X124I-X218S, X040E-X076D-X166Q-X185Q, X040E-X076D-X166Q-X218S, X040E-X076D-X166Q-X259P, X040E-X076D-X185Q-X218S, X040E-X076D-X185Q-X259P, X040E-X076D-X218S-X259P, X040E-X078N-X166Q-X218S, X040E-X078N-X185Q-X259P, X040E-X078N-X218S-X259P, X040E-X124I-X218S-X259P, X040E-X166Q-X185Q-X218S, X040E-X185Q-X218S-X259P, X069S-X076D-X078N-X128S, X069S-X076D-X078N-X185Q, X069S-X076D-X078N-X218S, X069S-X076D-X078N-X259P, X069S-X076D-X124I-X128S, X069S-X076D-X129P-X166Q, X069S-X076D-X129P-X259P, X069S-X076D-X166Q-X185Q, X069S-X076D-X166Q-X218S, X069S-X076D-X166Q-X259P, X069S-X076D-X185Q-X218S, X069S-X076D-X185Q-X259P, X069S-X076D-X218S-X259P, X069S-X078N-X128S-X218S, X069S-X078N-X129P-X185Q, X069S-X078N-X166Q-X185Q, X069S-X078N-X166Q-X218S, X069S-X078N-X185Q-X218S, X069S-X078N-X185Q-X259P, X069S-X078N-X218S-X259P, X069S-X124I-X128S-X185Q, X069S-X124I-X128S-X218S, X069S-X124I-X129P-X259P, X069S-X124I-X185Q-X218S, X069S-X128S-X166Q-X218S, X069S-X128S-X218S-X259P, X069S-X129P-X166Q-X185Q, X069S-X129P-X166Q-X218S, X069S-X129P-X166Q-X259P, X069S-X129P-X185Q-X218S, X069S-X166Q-X185Q-X218S, X069S-X166Q-X185Q-X259P, X069S-X166Q-X218S-X259P, X069S-X185Q-X218S-X259P, X076D-X078N-X124I-X218S, X076D-X078N-X128S-X259P, X076D-X078N-X129P-X185Q, X076D-X078N-X166Q-X185Q, X076D-X078N-X166Q-X218S, X076D-X078N-X166Q-X259P, X076D-X078N-X185Q-X218S, X076D-X078N-X185Q-X259P, X076D-X078N-X218S-X259P, X076D-X124I-X128S-X166Q, X076D-X124I-X128S-X259P, X076D-X128S-X166Q-X218S, X076D-X129P-X166Q-X259P, X076D-X129P-X185Q-X218S, X076D-X166Q-X185Q-X218S, X076D-X166Q-X185Q-X259P, X076D-X166Q-X218S-X259P, X076D-X185Q-X218S-X259P, X078N-X124I-X128S-X218S, X078N-X124I-X128S-X259P, X078N-X124I-X166Q-X259P, X078N-X128S-X166Q-X218S, X078N-X128S-X218S-X259P, X078N-X129P-X166Q-X259P, X078N-X129P-X185Q-X218S, X078N-X129P-X185Q-X259P, X078N-X166Q-X185Q-X218S, X078N-X166Q-X185Q-X259P, X078N-X166Q-X218S-X259P, X078N-X185Q-X218S-X259P, X124I-X128S-X166Q-X259P, X124I-X129P-X166Q-X259P, X124I-X166Q-X218S-X259P, X124I-X185Q-X218S-X259P, X128S-X129P-X166Q-X259P, X128S-X166Q-X185Q-X218S, X128S-X166Q-X185Q-X259P, X129P-X166Q-X185Q-X259P, X129P-X166Q-X218S-X259P, X129P-X185Q-X218S-X259P, and X166Q-X185Q-X218S-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides a subtilisin variant having at least two or more features selected from the group consisting of X003V-X009E, X003V-X040E, X003V-X069S, X003V-X076D, X003V-X078N, X003V-X166Q, X003V-X185Q, X003V-X218S, X003V-X259P, X009E-X040E, X009E-X069S, X009E-X076D, X009E-X078N, X009E-X166Q, X009E-X185Q, X009E-X218S, X009E-X259P, X040E-X069S, X040E-X076D, X040E-X078N, X040E-X166Q, X040E-X185Q, X040E-X218S, X040E-X259P, X069S-X076D, X069S-X078N, X069S-X166Q, X069S-X185Q, X069S-X218S, X069S-X259P, X076D-X078N, X076D-X166Q, X076D-X185Q, X076D-X218S, X076D-X259P, X078N-X166Q, X078N-X185Q, X078N-X218S, X078N-X259P, X166Q-X185Q, X166Q-X218S, X166Q-X259P, X185Q-X218S, X185Q-X259P, and X218S-X259P.

In yet another embodiment, the disclosure provides a subtilisin variant having at least three or more features selected from the group consisting of X003V-X009E-X040E, X003V-X009E-X069S, X003V-X009E-X076D, X003V-X009E-X078N, X003V-X009E-X166Q, X003V-X009E-X185Q, X003V-X009E-X218S, X003V-X009E-X259P, X003V-X040E-X069S, X003V-X040E-X076D, X003V-X040E-X078N, X003V-X040E-X166Q, X003V-X040E-X185Q, X003V-X040E-X218S, X003V-X040E-X259P, X003V-X069S-X076D, X003V-X069S-X078N, X003V-X069S-X166Q, X003V-X069S-X185Q, X003V-X069S-X218S, X003V-X069S-X259P, X003V-X076D-X078N, X003V-X076D-X166Q, X003V-X076D-X185Q, X003V-X076D-X218S, X003V-X076D-X259P, X003V-X078N-X166Q, X003V-X078N-X185Q, X003V-X078N-X218S, X003V-X078N-X259P, X003V-X166Q-X185Q, X003V-X166Q-X218S, X003V-X166Q-X259P, X003V-X185Q-X218S, X003V-X185Q-X259P, X003V-X218S-X259P, X009E-X040E-X069S, X009E-X040E-X076D, X009E-X040E-X078N, X009E-X040E-X166Q, X009E-X040E-X185Q, X009E-X040E-X218S, X009E-X040E-X259P, X009E-X069S-X076D, X009E-X069S-X078N, X009E-X069S-X166Q, X009E-X069S-X185Q, X009E-X069S-X218S, X009E-X069S-X259P, X009E-X076D-X078N, X009E-X076D-X166Q, X009E-X076D-X185Q, X009E-X076D-X218S, X009E-X076D-X259P, X009E-X078N-X166Q, X009E-X078N-X185Q, X009E-X078N-X218S, X009E-X078N-X259P, X009E-X166Q-X185Q, X009E-X166Q-X218S, X009E-X166Q-X259P, X009E-X185Q-X218S, X009E-X185Q-X259P, X009E-X218S-X259P, X040E-X069S-X076D, X040E-X069S-X078N, X040E-X069S-X166Q, X040E-X069S-X185Q, X040E-X069S-X218S, X040E-X069S-X259P, X040E-X076D-X078N, X040E-X076D-X166Q, X040E-X076D-X185Q, X040E-X076D-X218S, X040E-X076D-X259P, X040E-X078N-X166Q, X040E-X078N-X185Q, X040E-X078N-X218S, X040E-X078N-X259P, X040E-X166Q-X185Q, X040E-X166Q-X218S, X040E-X166Q-X259P, X040E-X185Q-X218S, X040E-X185Q-X259P, X040E-X218S-X259P, X069S-X076D-X078N, X069S-X076D-X166Q, X069S-X076D-X185Q, X069S-X076D-X218S, X069S-X076D-X259P, X069S-X078N-X166Q, X069S-X078N-X185Q, X069S-X078N-X218S, X069S-X078N-X259P, X069S-X166Q-X185Q, X069S-X166Q-X218S, X069S-X166Q-X259P, X069S-X185Q-X218S, X069S-X185Q-X259P, X069S-X218S-X259P, X076D-X078N-X166Q, X076D-X078N-X185Q, X076D-X078N-X218S, X076D-X078N-X259P, X076D-X166Q-X185Q, X076D-X166Q-X218S, X076D-X166Q-X259P, X076D-X185Q-X218S, X076D-X185Q-X259P, X076D-X218S-X259P, X078N-X166Q-X185Q, X078N-X166Q-X218S, X078N-X166Q-X259P, X078N-X185Q-X218S, X078N-X185Q-X259P, X078N-X218S-X259P, X166Q-X185Q-X218S, X166Q-X185Q-X259P, X166Q-X218S-X259P, and X185Q-X218S-X259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides subtilisin variants with one or more mutations at E003, Q003, S003, T003, P009, S009, T009, A024, N024, S024, A040, P040, S040, A069, N076, D078, S078, T078, E079, L079, T079, V079, E087, N087, Q087, S087, G118, M118, N118, L124, M124, G128, I128, T128, A129, D129, S129, A130, M130, Q130, T130, V130, E145, Q145, S145, G166, S166, Q182, S182, N185, R185, S185, V185, L210, P210, F217, M217, Y217, N218, P218, T218, A248, N248, Q248, S248, D259, G259, and N259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′). In yet another embodiment, the disclosure provides variants of subtilisin AprE with one or more mutations at S003, S009, S024, P040, A069, N076, S078, S087, N118, M124, G128, T130, S145, G166, S182, P210, Y217, N218, and N259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin AprE from the group consisting of 5003V, N076D, S078N, G166Q, Y217L, N218S, N259P, S009E, P040E, S003V-N259P, S003V-P040E, S003V-M124I, S003V-S078N, S003V-N076D, S003V-G166Q, S003V-G128S, A069S-N076D, A069S-N218S, A069S-G166Q, A069S-N259P, A069S-S078N, A069S-G128S, A069S-M124I, N076D-G128S, N076D-S078N, N076D-N218S, N076D-G166Q, N076D-M124I, S078T-M124I, S078N-G128S, S078N-N259P, S078N-N218S, S078N-G166Q, M124I-G166Q, M124I-N259P, M124I-G128S, M124I-N218S, G128S-N259P, G128S-N218S, G128S-G166Q, G166Q-N259P, G166Q-N218S, N218S-N259P, S003V-S009E, S003V-A069S, S003V-N218S, S009E-P040E, S009E-A069S, S009E-N076D, S009E-S078N, S009E-G166Q, S009E-N218S, S009E-N259P, P040E-A069S, P040E-N076D, P040E-S078N, P040E-G166Q, P040E-N218S, P040E-N259P, N076D-N259P, S003V-N076D-S078N, S003V-S078N-N218S, S003V-M124I-N259P, S003V-G128S-G166Q, S003V-G166Q-N218S, S003V-N218S-N259P, P040E-N076D-S078N, G128S-G166Q-N259P, S003V-S009E-P040E, S003V-S009E-A069S, S003V-S009E-N076D, S003V-S009E-S078N, S003V-S009E-G166Q, S003V-S009E-N218S, S003V-S009E-N259P, S003V-P040E-A069S, S003V-P040E-N076D, S003V-P040E-N218S, S003V-P040E-N259P, S003V-A069S-N076D, S003V-A069S-S078N, S003V-A069S-G166Q, S003V-A069S-N218S, S003V-A069S-N259P, S003V-N076D-G166Q, S003V-N076D-N218S, S003V-N076D-N259P, S003V-S078N-N259P, S003V-G166Q-N259P, S009E-P040E-A069S, S009E-P040E-N076D, S009E-P040E-S078N, S009E-P040E-G166Q, S009E-P040E-N218S, S009E-P040E-N259P, S009E-A069S-N076D, S009E-A069S-S078N, S009E-A069S-G166Q, S009E-A069S-N218S, S009E-A069S-N259P, S009E-N076D-S078N, S009E-N076D-G166Q, S009E-N076D-N218S, S009E-N076D-N259P, S009E-S078N-G166Q, S009E-S078N-N218S, S009E-S078N-N259P, S009E-G166Q-N218S, S009E-G166Q-N259P, S009E-N218S-N259P, P040E-A069S-S078N, P040E-A069S-G166Q, P040E-A069S-N218S, P040E-A069S-N259P, P040E-N076D-G166Q, P040E-N076D-N218S, P040E-N076D-N259P, P040E-S078N-G166Q, P040E-S078N-N218S, P040E-S078N-N259P, P040E-G166Q-N218S, P040E-G166Q-N259P, P040E-N218S-N259P, A069S-N076D-S078N, A069S-N076D-G166Q, A069S-N076D-N218S, A069S-N076D-N259P, A069S-S078N-G166Q, A069S-S078N-N218S, A069S-S078N-N259P, A069S-G166Q-N218S, A069S-G166Q-N259P, A069S-N218S-N259P, N076D-S078N-G166Q, N076D-S078N-N218S, N076D-S078N-N259P, N076D-G166Q-N218S, N076D-G166Q-N259P, N076D-N218S-N259P, S078N-G166Q-N218S, S078N-G166Q-N259P, S078N-N218S-N259P, G166Q-N218S-N259P, S003V-P040E-N076D-S078N, S003V-P040E-S078N-M124I, S003V-P040E-M124I-N218S, S003V-N076D-S078N-G128S, S003V-N076D-M124I-G166Q, S003V-N076D-G128S-N259P, S003V-N076D-G166Q-N259P, S003V-N076D-N218S-N259P, S003V-M124I-G128S-N218S, S003V-G128S-G166Q-N218S, P040E-N076D-S078N-G166Q, P040E-N076D-M124I-N218S, P040E-N076D-G166Q-N218S, P040E-N076D-G166Q-N259P, N076D-S078N-M124I-N218S, N076D-S078N-G128S-N259P, N076D-S078N-G166Q-N259P, N076D-M124I-G128S-G166Q, N076D-M124I-G128S-N259P, N076D-G128S-G166Q-N218S, S078N-G128S-G166Q-N218S, S078N-G128S-N218S-N259P, M124I-G166Q-N218S-N259P, S003V-P040E-A069S-G166Q, S003V-P040E-N076D-N218S, S003V-A069S-G166Q-N218S, S009E-P040E-A069S-N076D, S009E-P040E-A069S-N259P, S009E-P040E-S078N-N259P, S009E-P040E-G166Q-N218S, S009E-P040E-G166Q-N259P, S009E-P040E-N218S-N259P, S009E-A069S-S078N-G166Q, S009E-S078N-N218S-N259P, S009E-G166Q-N218S-N259P, P040E-A069S-N076D-S078N, P040E-A069S-S078N-G166Q, P040E-A069S-S078N-N259P, P040E-A069S-G166Q-N218S, N076D-S078N-G166Q-N218S, and N076D-G166Q-N218S-N259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′). In yet another embodiment, the disclosure provides variants of subtilisin Bad02409 with one or more mutations at T003, P009, S024, A069, N076, S078, V079, N087, G118, M124, G128, S129, M130, S145, G166, S182, N185, P210, Y217, N218, N248, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Bad02409 from the group consisting of N076D, S078N, G128S, S182E, T003V-N218S, T003V-A069S, T003V-D259P, T003V-M124I, T003V-N076D, T003V-N185Q, T003V-G166Q, T003V-S078N, T003V-S129P, A069S-S129P, A069S-G166Q, A069S-N076D, A069S-S078N, N076D-S129P, S078N-N185Q, M124I-S129P, S129P-D259P, S129P-G166Q, G166Q-N218S, T003V-A069S-G166Q, T003V-N076D-D259P, T003V-S078N-N185Q, T003V-S129P-N185Q, T003V-G166Q-D259P, T003V-N185Q-D259P, A069S-N076D-G166Q, N076D-G166Q-N185Q, T003V-A069S-S078N-S129P, T003V-N076D-N185Q-D259P, T003V-S078N-S129P-N185Q, T003V-S078N-G166Q-D259P, T003V-G166Q-N185Q-D259P, A069S-N076D-S129P-D259P, A069S-S078N-S129P-N185Q, A069S-S129P-G166Q-N185Q, N076D-S078N-S129P-N185Q, N076D-S129P-G166Q-D259P, and N076D-G166Q-N185Q-D259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Bba02069 with one or more mutations at Q003, T009, N024, P040, A069, N076, Q087, G118, M124, G128, S129, G166, S182, V185, P210, Y217, N218, Q248, and S259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Bba02069 from the group consisting of S129P, G118R, Q087D, N076D, M124I, Q248D, A069S, G128S, N024Q, Q003V, P040E, T009E, N218S, G166Q, S259P, Q003V-A069S, Q003V-V185Q, Q003V-S129P, Q003V-M124I, Q003V-G166Q, Q003V-G128S, Q003V-S259P, Q003V-N076D, P040E-V185Q, P040E-G166Q, A069S-G128S, A069S-S259P, A069S-M124I, A069S-G166Q, A069S-N076D, N076D-G128S, N076D-G166Q, N076D-M124I, N076D-S259P, N076D-S129P, M124I-G128S, M124I-V185Q, M124I-S129P, M124I-S259P, M124I-G166Q, M124I-N218S, G128S-S129P, G128S-G166Q, G128S-S259P, G128S-V185Q, S129P-G166Q, S129P-V185Q, S129P-S259P, G166Q-S259P, G166Q-V185Q, V185Q-S259P, V185Q-N218S, N218S-S259P, Q003V-T009E, Q003V-P040E, Q003V-N218S, T009E-P040E, T009E-A069S, T009E-N076D, T009E-G166Q, T009E-V185Q, T009E-N218S, T009E-S259P, P040E-A069S, P040E-N076D, P040E-N218S, P040E-S259P, A069S-V185Q, A069S-N218S, N076D-V185Q, N076D-N218S, G166Q-N218S, Q003V-P040E-G166Q, Q003V-G166Q-S259P, A069S-S129P-G166Q, A069S-S129P-V185Q, G128S-S129P-N218S, G128S-V185Q-N218S, Q003V-T009E-P040E, Q003V-T009E-A069S, Q003V-T009E-N076D, Q003V-T009E-G166Q, Q003V-T009E-V185Q, Q003V-T009E-N218S, Q003V-T009E-S259P, Q003V-P040E-A069S, Q003V-P040E-N076D, Q003V-P040E-V185Q, Q003V-P040E-N218S, Q003V-P040E-S259P, Q003V-A069S-N076D, Q003V-A069S-G166Q, Q003V-A069S-N218S, Q003V-A069S-S259P, Q003V-N076D-G166Q, Q003V-N076D-V185Q, Q003V-N076D-N218S, Q003V-N076D-S259P, Q003V-G166Q-V185Q, Q003V-G166Q-N218S, Q003V-V185Q-S259P, Q003V-N218S-S259P, T009E-P040E-A069S, T009E-P040E-N076D, T009E-P040E-G166Q, T009E-P040E-V185Q, T009E-P040E-N218S, T009E-P040E-S259P, T009E-A069S-N076D, T009E-A069S-G166Q, T009E-A069S-V185Q, T009E-A069S-N218S, T009E-A069S-S259P, T009E-N076D-G166Q, T009E-N076D-V185Q, T009E-N076D-N218S, T009E-N076D-S259P, T009E-G166Q-V185Q, T009E-G166Q-N218S, T009E-G166Q-S259P, T009E-V185Q-N218S, T009E-V185Q-S259P, T009E-N218S-S259P, P040E-A069S-N076D, P040E-A069S-G166Q, P040E-A069S-V185Q, P040E-A069S-N218S, P040E-A069S-S259P, P040E-N076D-G166Q, P040E-N076D-V185Q, P040E-N076D-N218S, P040E-N076D-S259P, P040E-G166Q-V185Q, P040E-G166Q-N218S, P040E-G166Q-S259P, P040E-V185Q-N218S, P040E-V185Q-S259P, P040E-N218S-S259P, A069S-N076D-G166Q, A069S-N076D-V185Q, A069S-N076D-N218S, A069S-N076D-S259P, A069S-G166Q-V185Q, A069S-G166Q-N218S, A069S-G166Q-S259P, A069S-V185Q-N218S, A069S-V185Q-S259P, A069S-N218S-S259P, N076D-G166Q-V185Q, N076D-G166Q-N218S, N076D-G166Q-S259P, N076D-V185Q-N218S, N076D-V185Q-S259P, N076D-N218S-S259P, G166Q-V185Q-N218S, G166Q-V185Q-S259P, G166Q-N218S-S259P, V185Q-N218S-S259P, Q003V-A069S-S129P-S259P, Q003V-M124I-G128S-G166Q, Q003V-G128S-S129P-V185Q, P040E-A069S-S129P-N218S, P040E-M124I-N218S-S259P, A069S-N076D-S129P-G166Q, A069S-M124I-S129P-S259P, A069S-M124I-V185Q-N218S, G128S-G166Q-V185Q-N218S, S129P-G166Q-V185Q-S259P, Q003V-T009E-P040E-G166Q, Q003V-T009E-P040E-V185Q, Q003V-T009E-N076D-N218S, Q003V-T009E-G166Q-V185Q, Q003V-P040E-N076D-V185Q, T009E-P040E-A069S-V185Q, T009E-P040E-A069S-N218S, T009E-P040E-N076D-V185Q, T009E-A069S-N076D-N218S, T009E-A069S-G166Q-V185Q, T009E-A069S-G166Q-N218S, T009E-A069S-V185Q-N218S, T009E-N076D-V185Q-S259P, T009E-V185Q-N218S-S259P, P040E-A069S-N076D-V185Q, P040E-A069S-G166Q-N218S, P040E-A069S-V185Q-N218S, P040E-A069S-V185Q-S259P, P040E-A069S-N218S-S259P, P040E-N076D-G166Q-V185Q, P040E-N076D-V185Q-N218S, P040E-N076D-V185Q-S259P, P040E-G166Q-V185Q-N218S, P040E-V185Q-N218S-S259P, A069S-N076D-G166Q-V185Q, A069S-N076D-G166Q-N218S, A069S-N076D-G166Q-S259P, A069S-N076D-V185Q-N218S, A069S-N076D-N218S-S259P, A069S-G166Q-V185Q-N218S, N076D-G166Q-V185Q-S259P, N076D-G166Q-N218S-S259P, and G166Q-V185Q-N218S-S259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Bpan01744 with one or more mutations at 5003, S009, S024, A069, N076, S078, N087, G118, M124, T128, S129, A130, G166, Q182, N185, P210, N218, N248, and N259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Bpan01744 from the group consisting of 5003V, S078N, S009E, N076D, N185Q, N218S, N259P, S003V-N185Q, S003V-N076D, S003V-A069S, S003V-S078N, S003V-N218S, S003V-N259P, A069S-N076D, A069S-G166Q, A069S-N185Q, A069S-S078N, A069S-N218S, N076D-S078N, N076D-S129P, S078N-G166Q, S078N-M124I, S078N-S129P, M124I-N218S, M124I-S129P, M124I-N185Q, S129P-N259P, S129P-N185Q, S129P-N218S, S129P-G166Q, G166Q-N259P, G166Q-N218S, G166Q-N185Q, N185Q-N218S, N185Q-N259P, N218S-N259P, S003V-S009E, S003V-G166Q, S009E-N076D, S009E-S078N, S009E-G166Q, S009E-N185Q, S009E-N218S, S009E-N259P, N076D-G166Q, N076D-N185Q, N076D-N218S, N076D-N259P, S078N-N185Q, S078N-N218S, S078N-N259P, S003V-A069S-N076D, S003V-A069S-S129P, S003V-A069S-N185Q, S003V-N076D-S129P, S003V-N076D-N185Q, S003V-S078N-G166Q, S003V-S129P-N259P, S003V-G166Q-N218S, A069S-N076D-N185Q, A069S-G166Q-N185Q, N076D-S078N-G166Q, N076D-S129P-N218S, S078N-S129P-N259P, S078N-N185Q-N259P, S003V-S009E-A069S, S003V-S009E-N076D, S003V-S009E-S078N, S003V-S009E-N185Q, S003V-S009E-N218S, S003V-S009E-N259P, S003V-A069S-S078N, S003V-A069S-N218S, S003V-N076D-S078N, S003V-N076D-G166Q, S003V-N076D-N218S, S003V-S078N-N185Q, S003V-S078N-N218S, S003V-S078N-N259P, S003V-N185Q-N218S, S003V-N185Q-N259P, S003V-N218S-N259P, S009E-A069S-N076D, S009E-A069S-S078N, S009E-A069S-G166Q, S009E-A069S-N259P, S009E-N076D-S078N, S009E-N076D-G166Q, S009E-N076D-N185Q, S009E-N076D-N218S, S009E-N076D-N259P, S009E-S078N-N185Q, S009E-S078N-N218S, S009E-S078N-N259P, S009E-G166Q-N185Q, S009E-G166Q-N218S, S009E-N185Q-N218S, S009E-N185Q-N259P, S009E-N218S-N259P, A069S-N076D-S078N, A069S-N076D-G166Q, A069S-N076D-N218S, A069S-N076D-N259P, A069S-S078N-N185Q, A069S-S078N-N218S, A069S-S078N-N259P, A069S-G166Q-N218S, A069S-N185Q-N218S, A069S-N218S-N259P, N076D-S078N-N185Q, N076D-S078N-N218S, N076D-S078N-N259P, N076D-G166Q-N218S, N076D-N185Q-N218S, N076D-N185Q-N259P, S078N-G166Q-N218S, S078N-N185Q-N218S, S078N-N218S-N259P, G166Q-N185Q-N218S, N185Q-N218S-N259P, S003V-A069S-N076D-S078N, S003V-A069S-N076D-N185Q, S003V-A069S-N076D-N259P, S003V-A069S-S078N-S129P, S003V-A069S-S078N-N185Q, S003V-A069S-S129P-N218S, S003V-A069S-N185Q-N259P, S003V-N076D-S129P-G166Q, S003V-N076D-S129P-N185Q, S003V-N076D-S129P-N259P, S003V-S078N-N185Q-N218S, S003V-S078N-N185Q-N259P, S003V-G166Q-N185Q-N259P, S003V-N185Q-N218S-N259P, A069S-N076D-S078N-N218S, A069S-N076D-N185Q-N259P, A069S-S078N-N185Q-N218S, A069S-S129P-G166Q-N218S, A069S-S129P-N185Q-N218S, A069S-G166Q-N218S-N259P, A069S-N185Q-N218S-N259P, N076D-S078N-S129P-N185Q, N076D-S078N-N185Q-N218S, N076D-S129P-N185Q-N218S, N076D-G166Q-N185Q-N259P, S078N-S129P-G166Q-N259P, S078N-S129P-N185Q-N259P, S078N-G166Q-N218S-N259P, S129P-G166Q-N218S-N259P, S129P-N185Q-N218S-N259P, S003V-S009E-A069S-N185Q, S003V-S009E-N218S-N259P, S003V-A069S-N076D-N218S, S003V-A069S-N185Q-N218S, S003V-A069S-N218S-N259P, S003V-N076D-N218S-N259P, S003V-G166Q-N185Q-N218S, S009E-A069S-N076D-S078N, S009E-A069S-S078N-G166Q, S009E-A069S-S078N-N218S, S009E-A069S-G166Q-N218S, S009E-A069S-N218S-N259P, S009E-N076D-S078N-G166Q, S009E-N076D-S078N-N185Q, S009E-N076D-N218S-N259P, S009E-S078N-N185Q-N218S, S009E-S078N-N218S-N259P, S009E-G166Q-N185Q-N218S, A069S-N076D-S078N-N259P, A069S-S078N-G166Q-N218S, A069S-S078N-N185Q-N259P, A069S-S078N-N218S-N259P, N076D-S078N-G166Q-N185Q, N076D-S078N-G166Q-N218S, N076D-S078N-N218S-N259P, N076D-G166Q-N185Q-N218S, N076D-G166Q-N218S-N259P, N076D-N185Q-N218S-N259P, S078N-G166Q-N185Q-N218S, and S078N-G166Q-N185Q-N259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspAI02518 with one or more mutations at T003, S009, A069, N076, S078, S087, G118, M124, G128, T130, S166, S182, N185, P210, N218, Q248, and N259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspAI02518 from the group consisting of S003V, S009E, N076D, S078N, M124I, S182E, N185Q, N218S, N259P, S166Q, S003V-N259P, S003V-N218S, S003V-A069S, S003V-N185Q, S003V-G128S, S003V-M124I, S003V-N076D, S003V-S166Q, A069S-N218S, A069S-N076D, A069S-S078N, A069S-N185Q, A069S-S166Q, N076D-N218S, N076D-N259P, N076D-G128S, N076D-M124I, N076D-N185Q, N076D-S078N, S078N-M124I, S078N-N218S, S078N-N259P, S078N-G128S, S078N-N185Q, M124I-G128S, M124I-S166Q, M124I-N185Q, S166Q-N218S, S166Q-N259P, N185Q-N259P, N218S-N259P, S003V-S009E, S003V-S078N, S009E-A069S, S009E-N076D, S009E-S078N, S009E-S166Q, S009E-N185Q, S009E-N218S, S009E-N259P, A069S-N259P, N076D-S166Q, S078N-S166Q, S166Q-N185Q, N185Q-N218S, S003V-A069S-N185Q, S003V-S078N-G128S, S003V-N185Q-N259P, A069S-N076D-G128S, A069S-N076D-N218S, A069S-S078N-M124I, N076D-S078N-N185Q, N076D-S078N-N218S, N076D-G128S-S166Q, M124I-G128S-N185Q, G128S-S166Q-N185Q, S003V-S009E-A069S, S003V-S009E-N076D, S003V-S009E-S078N, S003V-S009E-S166Q, S003V-S009E-N185Q, S003V-S009E-N218S, S003V-S009E-N259P, S003V-A069S-N076D, S003V-A069S-S078N, S003V-A069S-N218S, S003V-N076D-S078N, S003V-N076D-S166Q, S003V-N076D-N185Q, S003V-N076D-N218S, S003V-N076D-N259P, S003V-S078N-S166Q, S003V-S078N-N185Q, S003V-S078N-N218S, S003V-S078N-N259P, S003V-S166Q-N185Q, S003V-S166Q-N259P, S003V-N185Q-N218S, S003V-N218S-N259P, S009E-A069S-N076D, S009E-A069S-N185Q, S009E-A069S-N218S, S009E-N076D-S078N, S009E-N076D-S166Q, S009E-N076D-N185Q, S009E-N076D-N218S, S009E-N076D-N259P, S009E-S078N-S166Q, S009E-S078N-N185Q, S009E-S078N-N218S, S009E-S078N-N259P, S009E-S166Q-N185Q, S009E-S166Q-N218S, S009E-S166Q-N259P, S009E-N185Q-N218S, S009E-N185Q-N259P, S009E-N218S-N259P, A069S-N076D-S078N, A069S-N076D-S166Q, A069S-N076D-N185Q, A069S-N076D-N259P, A069S-S078N-S166Q, A069S-S078N-N218S, A069S-S166Q-N185Q, A069S-S166Q-N218S, A069S-S166Q-N259P, A069S-N185Q-N218S, A069S-N218S-N259P, N076D-S078N-5166Q, N076D-S078N-N259P, N076D-5166Q-N185Q, N076D-5166Q-N218S, N076D-5166Q-N259P, N076D-N185Q-N218S, N076D-N185Q-N259P, N076D-N218S-N259P, S078N-S166Q-N185Q, S078N-S166Q-N218S, S078N-S166Q-N259P, S078N-N185Q-N218S, S078N-N185Q-N259P, S078N-N218S-N259P, S166Q-N185Q-N218S, S166Q-N185Q-N259P, S166Q-N218S-N259P, S003V-A069S-N076D-G128S, S003V-A069S-S078N-G128S, S003V-A069S-S078N-S166Q, S003V-A069S-M124I-N218S, S003V-A069S-G128S-S166Q, S003V-A069S-G128S-N259P, S003V-N076D-S166Q-N185Q, S003V-S078N-M124I-S166Q, S003V-S078N-G128S-N185Q, S003V-S078N-G128S-N218S, S003V-M124I-S166Q-N259P, S003V-M124I-N185Q-N259P, S003V-G128S-N185Q-N218S, S003V-G128S-N218S-N259P, S003V-S166Q-N218S-N259P, A069S-N076D-S078N-G128S, A069S-N076D-M124I-G128S, A069S-N076D-S166Q-N218S, A069S-N076D-N185Q-N259P, A069S-N076D-N218S-N259P, A069S-S078N-G128S-N218S, A069S-S078N-N185Q-N259P, A069S-M124I-G128S-N185Q, A069S-M124I-G128S-N218S, A069S-G128S-S166Q-N218S, A069S-G128S-N218S-N259P, N076D-S166Q-N185Q-N259P, S078N-M124I-G128S-N218S, S078N-N185Q-N218S-N259P, M124I-G128S-S166Q-N259P, M124I-S166Q-N218S-N259P, G128S-S166Q-N185Q-N259P, S003V-S009E-N076D-S166Q, S003V-A069S-S078N-N259P, S003V-A069S-S166Q-N218S, S003V-N076D-S078N-S166Q, S003V-N076D-S078N-N259P, S003V-N076D-S166Q-N259P, S003V-S166Q-N185Q-N259P, S009E-A069S-N076D-S166Q, S009E-A069S-N076D-N218S, S009E-A069S-N076D-N259P, S009E-A069S-S078N-S166Q, S009E-A069S-S078N-N218S, S009E-A069S-S166Q-N259P, S009E-N076D-S078N-N259P, S009E-N076D-S166Q-N185Q, S009E-N076D-S166Q-N259P, S009E-N076D-N185Q-N218S, S009E-N076D-N185Q-N259P, S009E-S078N-S166Q-N218S, S009E-S078N-N185Q-N259P, S009E-S078N-N218S-N259P, A069S-N076D-S078N-N218S, A069S-N076D-S166Q-N185Q, A069S-N076D-N185Q-N218S, A069S-S078N-S166Q-N218S, A069S-S078N-N185Q-N218S, A069S-S078N-N218S-N259P, A069S-S166Q-N185Q-N218S, A069S-S166Q-N185Q-N259P, N076D-S078N-S166Q-N218S, N076D-S078N-N185Q-N218S, N076D-S078N-N185Q-N259P, N076D-S078N-N218S-N259P, N076D-S166Q-N185Q-N218S, N076D-S166Q-N218S-N259P, N076D-N185Q-N218S-N259P, S078N-S166Q-N185Q-N218S, S078N-S166Q-N185Q-N259P, and S166Q-N185Q-N218S-N259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspAK01305 with one or more mutations at S003, S024, S040, A069, D078, E079, E087, N118, L124, S145, G166, Q182, S185, P210, S248, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspAK01305 from the group consisting of S003V, S040E, G166Q, S185Q, P210I, S003V-S185Q, S003V-G166Q, S003V-R262L, S003V-S040E, S040E-G166Q, S040E-S185Q, and G166Q-S185Q, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspZ00056 with one or more mutations at T003, P009, A024, A069, D078, L079, Q087, G118, M124, G128, S129, Q130, E145, G166, Q182, N185, P210, F217, N218, N248, and G259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspZ00056 from the group consisting of Q182E, G128S, E145R, M124I, N218S, S129P, T003V, P009E, A069S, G166Q, N185Q, G259P, G128S-S129P, G128S-N218S, A069S-N185Q, A069S-D078N, D078N-G166Q, G166Q-N185Q, T003V-M124I, G128S-N185Q, A069S-G128S, M124I-N185Q, G166Q-G259P, A069S-G259P, S129P-G166Q, T003V-G259P, T003V-N185Q, D078N-G259P, M124I-N218S, G128S-G166Q, A069S-M124I, S129P-N185Q, G128S-G259P, T003V-G166Q, T003V-G128S, T003V-A069S, M124I-G166Q, M124I-S129P, N218S-G259P, N185Q-G259P, D078N-S129P, M124I-G128S, T003V-N218S, A069S-G166Q, P009E-G166Q, P009E-G259P, A069S-N218S, G166Q-N218S, N185Q-N218S, T003V-S129P-G166Q, T003V-S129P-N185Q, T003V-S129P-G259P, T003V-N185Q-N218S, A069S-G128S-N185Q, A069S-S129P-G259P, A069S-G166Q-N185Q, A069S-G166Q-N218S, M124I-S129P-N185Q, M124I-S129P-G259P, M124I-N185Q-G259P, M124I-N218S-G259P, G128S-G166Q-G259P, G128S-N185Q-G259P, G166Q-N218S-G259P, T003V-P009E-G166Q, T003V-A069S-G166Q, T003V-A069S-G259P, T003V-G166Q-N185Q, T003V-G166Q-N218S, T003V-G166Q-G259P, T003V-N185Q-G259P, T003V-N218S-G259P, P009E-A069S-G166Q, P009E-A069S-G259P, P009E-G166Q-N185Q, P009E-G166Q-N218S, P009E-G166Q-G259P, P009E-N218S-G259P, A069S-G166Q-G259P, A069S-N185Q-G259P, A069S-N218S-G259P, G166Q-N185Q-G259P, N185Q-N218S-G259P, T003V-A069S-G128S-N185Q, T003V-A069S-N185Q-G259P, T003V-A069S-N218S-G259P, T003V-M124I-G166Q-G259P, T003V-N185Q-N218S-G259P, A069S-S129P-G166Q-G259P, M124I-S129P-G166Q-G259P, M124I-G166Q-N218S-G259P, M124I-N185Q-N218S-G259P, G128S-S129P-G166Q-G259P, S129P-G166Q-N185Q-G259P, T003V-P009E-A069S-G166Q, T003V-P009E-G166Q-G259P, T003V-A069S-G166Q-N185Q, T003V-G166Q-N218S-G259P, P009E-A069S-G166Q-N218S, P009E-G166Q-N185Q-N218S, P009E-G166Q-N185Q-G259P, P009E-G166Q-N218S-G259P, P009E-N185Q-N218S-G259P, A069S-G166Q-N185Q-N218S, A069S-G166Q-N185Q-G259P, A069S-G166Q-N218S-G259P, and G166Q-N185Q-N218S-G259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspZ00258 with one or more mutations at E003, N024, A069, D078, L079, N118, M124, G128, A129, V130, Q145, G166, S182, N185, L210, N218, A248, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin BspZ00258 from the group consisting of E003V, G166Q, D259P, A069S-N185Q, A069S-N218S, A129P-D259P, G166Q-N185Q, G166Q-D259P, E003V-A069S-G128S, E003V-G128S-D259P, E003V-A129P-N185Q, A069S-A129P-G166Q, E003V-A069S-G128S-N185Q, E003V-A069S-A129P-N185Q, E003V-G128S-N185Q-D259P, and E003V-A129P-N185Q-N218S, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Chemgen_164A with one or more mutations at T003, T009, S024, P040, A069, N076, T078, N087, N118, M124, G128, S129, 5145, G166, S182, N185, P210, Y217, N218, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin Chemgen_164A from the group consisting of T003V, A069S, N087D, N118R, S129P, G166Q, S182E, N218S, P040E, N076D, T078N, N185Q, T003V-N076D, T003V-N185Q, T003V-G128S, T003V-S129P, T003V-G166Q, T003V-T078N, T003V-A069S, T003V-M124I, T003V-N218S, P040E-M124I, A069S-G128S, A069S-G166Q, A069S-T078N, A069S-D259P, A069S-S129P, A069S-N076D, A069S-N218S, A069S-N185Q, N076D-G166Q, N076D-N218S, N076D-M124I, N076D-T078N, N076D-D259P, N076D-S129P, N076D-G128S, T078N-G128S, T078N-N185Q, T078N-N218S, T078N-D259P, T078N-S129P, T078N-M124I, T078N-G166Q, G128S-N218S, G128S-D259P, G128S-N185Q, S129P-N185Q, S129P-G166Q, S129P-N218S, G166Q-D259P, G166Q-N185Q, G166Q-N218S, N185Q-N218S, N185Q-D259P, N218S-D259P, T003V-T009E, T003V-P040E, T003V-D259P, T009E-A069S, T009E-N076D, T009E-T078N, T009E-G166Q, T009E-N185Q, T009E-D259P, P040E-T078N, P040E-G166Q, P040E-N185Q, P040E-N218S, N076D-N185Q, T003V-T009E-P040E, T003V-T009E-A069S, T003V-T009E-N185Q, T003V-P040E-N076D, T003V-P040E-G166Q, T003V-P040E-N185Q, T003V-P040E-D259P, T003V-A069S-N076D, T003V-A069S-T078N, T003V-A069S-G166Q, T003V-A069S-N185Q, T003V-A069S-N218S, T003V-A069S-D259P, T003V-N076D-T078N, T003V-N076D-G166Q, T003V-N076D-N185Q, T003V-N076D-N218S, T003V-N076D-D259P, T003V-T078N-G166Q, T003V-T078N-N185Q, T003V-T078N-D259P, T003V-G166Q-N185Q, T003V-G166Q-D259P, T003V-N185Q-N218S, T003V-N185Q-D259P, T003V-N218S-D259P, T009E-P040E-A069S, T009E-P040E-N076D, T009E-P040E-T078N, T009E-P040E-G166Q, T009E-P040E-N218S, T009E-P040E-D259P, T009E-A069S-N076D, T009E-A069S-T078N, T009E-A069S-G166Q, T009E-A069S-N218S, T009E-A069S-D259P, T009E-N076D-T078N, T009E-N076D-G166Q, T009E-N076D-N185Q, T009E-N076D-N218S, T009E-N076D-D259P, T009E-T078N-G166Q, T009E-T078N-N185Q, T009E-T078N-N218S, T009E-T078N-D259P, T009E-G166Q-N218S, T009E-G166Q-D259P, T009E-N185Q-N218S, T009E-N185Q-D259P, P040E-A069S-N076D, P040E-A069S-T078N, P040E-A069S-G166Q, P040E-A069S-D259P, P040E-N076D-T078N, P040E-N076D-G166Q, P040E-N076D-N185Q, P040E-N076D-N218S, P040E-T078N-N185Q, P040E-T078N-D259P, P040E-G166Q-N185Q, P040E-G166Q-N218S, P040E-G166Q-D259P, P040E-N185Q-N218S, P040E-N185Q-D259P, P040E-N218S-D259P, A069S-N076D-T078N, A069S-N076D-G166Q, A069S-N076D-N185Q, A069S-N076D-N218S, A069S-N076D-D259P, A069S-T078N-G166Q, A069S-T078N-N185Q, A069S-T078N-N218S, A069S-T078N-D259P, A069S-G166Q-N218S, A069S-G166Q-D259P, A069S-N185Q-N218S, A069S-N185Q-D259P, A069S-N218S-D259P, N076D-T078N-G166Q, N076D-T078N-N218S, N076D-T078N-D259P, N076D-G166Q-N218S, N076D-G166Q-D259P, N076D-N185Q-N218S, N076D-N218S-D259P, T078N-G166Q-N185Q, T078N-G166Q-N218S, T078N-N185Q-D259P, T078N-N218S-D259P, G166Q-N185Q-N218S, G166Q-N185Q-D259P, G166Q-N218S-D259P, N185Q-N218S-D259P, T003V-T009E-P040E-N185Q, T003V-T009E-A069S-T078N, T003V-P040E-G166Q-D259P, T003V-N076D-T078N-D259P, T003V-N185Q-N218S-D259P, T009E-P040E-A069S-T078N, T009E-P040E-A069S-N185Q, T009E-P040E-A069S-D259P, T009E-P040E-N076D-T078N, T009E-P040E-T078N-N185Q, T009E-P040E-N185Q-N218S, T009E-P040E-N185Q-D259P, T009E-A069S-N076D-G166Q, T009E-A069S-N076D-N185Q, T009E-N076D-G166Q-N218S, T009E-T078N-G166Q-N185Q, P040E-A069S-T078N-N218S, P040E-A069S-G166Q-N218S, P040E-A069S-G166Q-D259P, P040E-N076D-T078N-N185Q, P040E-N076D-T078N-N218S, P040E-N076D-G166Q-D259P, P040E-T078N-N185Q-D259P, P040E-T078N-N218S-D259P, A069S-N076D-T078N-N185Q, A069S-N076D-T078N-D259P, A069S-N076D-G166Q-D259P, A069S-T078N-G166Q-N185Q, A069S-T078N-N185Q-D259P, N076D-T078N-G166Q-N218S, N076D-T078N-G166Q-D259P, N076D-T078N-N218S-D259P, and T078N-N185Q-N218S-D259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin CP474 with one or more mutations at T003, P009, S024, A040, A069, T078, T079, 5087, G118, L124, 5166, Q182, N185, P210, N218, N248, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin CP474 from the group consisting of T003V, A040E, A069S, T078N, T079I, S166Q, N185Q, T003V-5166Q, T003V-N185Q, T003V-N218S, T003V-A040E, T003V-D259P, T003V-A069S, T003V-T078N, T003V-T079I, T003V-L124I, A040E-S166Q, A040E-N185Q, A040E-N218S, A040E-D259P, A040E-A069S, A040E-T078N, A040E-T079I, A040E-L124I, A069S-N185Q, A069S-T078N, A069S-T079I, A069S-S166Q, T078N-D259P, T078N-T079I, T078N-S166Q, T078N-N185Q, T078N-N218S, T079I-L124I, T079I-S166Q, T079I-N185Q, T079I-N218S, T079I-D259P, S166Q-N218S, N185Q-D259P, N185Q-N218S, and N218S-D259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin DSM14391 with one or more mutations at T003, T009, S024, S040, A069, N076, S078, S087, N118, M124, D129, A130, G166, Q182, R185, L210, M217, P218, N248, and N259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin DSM14391 from the group consisting of T003V, T009E, S024Q, S040E, A069S, N076D, S078N, A130S, G166Q, Q182E, R185Q, P218S, N248D, N259P, T003V-R185Q, T003V-A069S, T003V-G166Q, T003V-N259P, T003V-N076D, T003V-S078N, T003V-P218S, S040E-S078N, A069S-S078N, A069S-N259P, A069S-R185Q, A069S-P218S, A069S-N076D, N076D-S078N, N076D-P218S, N076D-D129P, S078N-D129P, S078N-N259P, S078N-M124I, S078N-P218S, S078N-R185Q, M124I-G166Q, M124I-P218S, M124I-N259K, D129P-P218S, D129P-G166Q, D129P-R185Q, D129P-N259P, G166Q-R185Q, G166Q-N259P, R185Q-P218S, R185Q-N259P, T003V-T009E, T003V-S040E, T009E-S040E, T009E-A069S, T009E-N076D, T009E-S078N, T009E-R185Q, T009E-P218S, T009E-N259P, S040E-A069S, S040E-N076D, S040E-R185Q, S040E-P218S, S040E-N259P, N076D-R185Q, N076D-N259P, G166Q-P218S, P218S-N259P, T003V-S040E-P218S, T003V-G166Q-P218S, T003V-G166Q-N259P, S040E-N076D-G166Q, S040E-N076D-R185Q, S040E-S078N-D129P, S040E-R185Q-P218S, S040E-R185Q-N259P, A069S-S078N-N259P, A069S-D129P-P218S, A069S-P218S-N259P, N076D-R185Q-P218S, N076D-P218S-N259P, S078N-R185Q-P218S, S078N-P218S-N259P, T003V-T009E-S040E, T003V-T009E-A069S, T003V-T009E-N076D, T003V-T009E-S078N, T003V-T009E-R185Q, T003V-T009E-P218S, T003V-T009E-N259P, T003V-S040E-A069S, T003V-S040E-N076D, T003V-S040E-S078N, T003V-S040E-R185Q, T003V-S040E-N259P, T003V-A069S-N076D, T003V-A069S-R185Q, T003V-A069S-P218S, T003V-N076D-S078N, T003V-N076D-R185Q, T003V-N076D-P218S, T003V-N076D-N259P, T003V-S078N-R185Q, T003V-S078N-P218S, T003V-S078N-N259P, T003V-R185Q-P218S, T003V-R185Q-N259P, T003V-P218S-N259P, T009E-S040E-N076D, T009E-S040E-S078N, T009E-S040E-R185Q, T009E-S040E-P218S, T009E-S040E-N259P, T009E-A069S-S078N, T009E-A069S-P218S, T009E-A069S-N259P, T009E-N076D-S078N, T009E-N076D-R185Q, T009E-N076D-P218S, T009E-N076D-N259P, T009E-S078N-R185Q, T009E-S078N-P218S, T009E-S078N-N259P, T009E-G166Q-P218S, T009E-R185Q-P218S, T009E-R185Q-N259P, T009E-P218S-N259P, S040E-A069S-N076D, S040E-A069S-R185Q, S040E-A069S-P218S, S040E-A069S-N259P, S040E-N076D-S078N, S040E-N076D-P218S, S040E-N076D-N259P, S040E-S078N-R185Q, S040E-S078N-P218S, S040E-S078N-N259P, S040E-G166Q-P218S, S040E-P218S-N259P, A069S-N076D-S078N, A069S-N076D-R185Q, A069S-N076D-P218S, A069S-N076D-N259P, A069S-S078N-R185Q, A069S-S078N-P218S, A069S-G166Q-P218S, A069S-R185Q-P218S, A069S-R185Q-N259P, N076D-S078N-R185Q, N076D-S078N-P218S, N076D-S078N-N259P, N076D-G166Q-P218S, S078N-G166Q-P218S, S078N-R185Q-N259P, G166Q-R185Q-P218S, G166Q-P218S-N259P, R185Q-P218S-N259P, T003V-S040E-A069S-N076D, T003V-S040E-N076D-S078N, T003V-S040E-N076D-D129P, T003V-S040E-G166Q-R185Q, T003V-S040E-G166Q-N259P, T003V-A069S-R185Q-N259P, T003V-N076D-S078N-N259P, T003V-N076D-R185Q-N259P, T003V-S078N-D129P-N259P, T003V-S078N-P218S-N259P, S040E-N076D-S078N-R185Q, S040E-N076D-P218S-N259P, S040E-S078N-G166Q-P218S, S040E-R185Q-P218S-N259P, A069S-D129P-R185Q-P218S, A069S-R185Q-P218S-N259P, S078N-D129P-R185Q-P218S, D129P-R185Q-P218S-N259P, T003V-T009E-S040E-N076D, T003V-A069S-S078N-P218S, T003V-G166Q-R185Q-P218S, T003V-R185Q-P218S-N259P, T009E-S040E-A069S-P218S, T009E-A069S-N076D-R185Q, T009E-N076D-S078N-R185Q, T009E-N076D-G166Q-P218S, T009E-S078N-R185Q-P218S, S040E-A069S-S078N-P218S, S040E-A069S-G166Q-P218S, S040E-N076D-G166Q-R185Q, S040E-N076D-G166Q-P218S, A069S-N076D-S078N-N259P, A069S-S078N-R185Q-P218S, N076D-S078N-G166Q-R185Q, S078N-G166Q-P218S-N259P, and S078N-R185Q-P218S-N259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin WP_082194748 with one or more mutations at T003, P009, A024, P040, A069, T078, S087, N118, M124, G128, G166, S182, V185, P210, Y217, T218, Q248, and S259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin WP_082194748 from the group consisting of Y217L, S087D, T078N, G128S, G166Q, S182E, G128S-V185Q, A069S-G128S, T078N-V185Q, V185Q-S259P, G166Q-V185Q, T078N-S259P, T003V-G128S, T078N-G128S, G128S-S259P, G166Q-S259P, A069S-M124I, T003V-M124I, M124I-S259P, M124I-V185Q, T003V-S259P, A069S-S259P, M124I-G128S, A069S-G166Q, T078N-M124I, M124I-G166Q, T078N-G166Q, T003V-G166Q, T003V-T078N-G166Q, T003V-T078N-S259P, T003V-M124I-G128S, T003V-G128S-G166Q, T003V-G128S-S259P, T003V-G166Q-S259P, T078N-M124I-G166Q, T078N-G128S-G166Q, T078N-G166Q-S259P, M124I-G128S-G166Q, M124I-G166Q-S259P, T003V-T078N-M124I-G166Q, T003V-T078N-G166Q-S259P, T003V-M124I-G128S-S259P, T003V-M124I-G166Q-S259P, T078N-M124I-G128S-S259P, and T078N-M124I-G166Q-S259P, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin ZP-00454 with one or more mutations at P009, S024, A040, A069, N076, T078, T079, N087, M118, M124, I128, S129, T130, S145, G166, S182, N185, P210, F217, N218, N248, and D259, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

In yet another embodiment, the disclosure provides variants of subtilisin ZP-00454 from the group consisting of A040E, A069S, N076D, T078N, T079I, I128S, A040E-M124I, A040E-I128S, A040E-S129P, A040E-G166Q, A040E-A069S, A040E-N185Q, A040E-N076D, A040E-N218S, A040E-T078N, A040E-D259P, A069S-N076D, A069S-N218S, A069S-T078N, A069S-T079I, A069S-1128S, A069S-S129P, A069S-N185Q, N076D-N185Q, N076D-N218S, N076D-T078N, N076D-D259P, N076D-T079I, N076D-M124I, N076D-I128S, N076D-S129P, N076D-G166Q, T078N-N185Q, T078N-D259P, T078N-T079I, T078N-I128S, T078N-S129P, T078N-G166Q, T079I-N218S, T079I-D259P, T079I-M124I, T07914128S, T079I-S129P, T079I-G166Q, T079I-N185Q, 1128S-S129P, and S129P-G166Q, where the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′).

The subtilisin variants having at least one, two, three, four, or more features selected from the group consisting of: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, and X259P, where the amino acid positions are numbered by correspondence with SEQ ID NO: 1, include variants derived from subtilisin polypeptides of AprE (e.g. WP_003233171); WP_082194748 (formerly WP_008359041); Chemgen_164A (SEQ ID NO: 2 in U.S. Pat. No. 5,275,945); CP474 (e.g. SEQ ID NO: 3 in WO2015038792); ZP-00454 (e.g. variant of WP_010192403, SEQ ID NO:7 in WO2015/038792); DSM14391 (SEQ ID NO: 13 in WO2018118917); WP_010192403 (formerly ZP 07707657 (SEQ ID NO: 7 in WO2015038792)); BspZ00056 (SEQ ID NO:9 in WO 2016069544);) Bba02069 (SEQ ID NO: 3 in WO2016061438); Bad02409 (SEQ ID NO: 13 in WO201069557); BspAK01305 (SEQ ID NO: 6 in WO2016069569); BspZ00258 (SEQ ID NO: 9 in WO2016069552); BspAI02518 (SEQ ID NO: 3 in WO2015089441); and Bpan01744 (SEQ ID NO: 3 in WO2016069563). Other subtilisin polypeptides in which the disclosed substitutions find use include, but are not limited to, SEQ ID NO:7 in WO2016/001449; SEQ ID NO: 1 in WO2012/139964; SEQ ID NO: 7 in WO2012/163855; SEQ ID NO: 9 in WO2016/001449; SEQ ID NO: 5 in WO2016/001449; SEQ ID NO: 6 in WO2016/001449; SEQ ID NO: 6 in WO2014/177430; SEQ ID NO: 4 in WO2011/036263; SEQ ID NO: 4 in WO2016/174234; SEQ ID NO: 7 in WO2015144932; SEQ ID NO: 119 in U.S. Pat. No. 7,981,659; SEQ ID NO: 4 in WO2016/001449; SEQ ID NO: 2 in JP2004313043; SEQ ID NO: 2 in US2015/275148; SEQ ID NO: 12 in WO 201600144; SEQ ID NO: 2 in WO 2016000970; SEQ ID NO:19 in U.S. Pat. No. 8,530,218; SEQ ID NO: 8 in WO 2016000973; SEQ ID NO: 8 in WO2016001449; SEQ ID NO 21 or 22 in WO2016203064 and SEQ ID NO: 21 in U.S. Pat. No. 8,530,218. That is, in some embodiments, the substitutions provided herein can be used in any subtilisin having at least about 80% sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22. For example, subtilisins, such as SEQ ID NO 21 or 22 in WO2016203064 can be engineered to include one, two, three or more additional features with respect to SEQ ID NO: 1 selected from a T, or V at position 3; an E at position 9; a Q at position 24; an E at position 40; an S at position 69; a D at position 76; a N at position 78; an I at position 79; a D at position 87; an Rat position 118; an I at position 124; an R, or S at position 128; a P at position 129; an S at position 130; an Rat position 145; a Q at position 166; an E at position 182; a Q at position 185; an I at position 210; a P at position 211; an L at position 217; an S at position 218; a D at position 248; and a P at position 259. In one such embodiment, subtilisins, such as SEQ ID NO 21 or 22 in WO2016203064 can be engineered to include one, two, three, four, or more, substitutions with respect to SEQ ID NO: 1 selected from a T, or V at position 3; an E at position 9; a Q at position 24; an E at position 40; an S at position 69; a D at position 76; a N at position 78; an I at position 79; a D at position 87; an Rat position 118; an I at position 124; an R, or S at position 128; a P at position 129; an S at position 130; an R at position 145; a Q at position 166; an E at position 182; a Q at position 185; an I at position 210; a P at position 211; an L at position 217; an S at position 218; a D at position 248; and a P at position 259.

Still other subtilisin polypeptides in which the disclosed substitutions find use include, but are not limited to, those disclosed in WO_2012_175708_2; WO_2012_175708_4; U.S. Pat. No. 7,951,573 B22; U.S. Pat. No. 7,951,573 B24; U.S. Pat. No. 7,951,573 B26; U.S. Pat. No. 7,951,573 B237; US7727756-0001; US9365844-0001; US7262042-0002; US20090275493-0002; US7811076-0004; US8455424-0003; WO03054184-CAE48421/SEQ ID NO: 25 in WO2015089447; WO2007131657-CA591385/SEQ ID NO:24 in WO2015089447; WO2008086916-CAV33594/SEQ ID NO:26 in WO2015089447; WO2017089162-0001; WO2017089162-0002; WO2017089162-0003; WO2017089162-0004; WO2017089162-0005; WO2017089162-0006; WO2017089162-0007; WO2017089162-0008, and WO2019180111.

In an even still further embodiment, one or more subtilisin variants described herein has improved stability, for example, improved stability in a detergent composition. In another embodiment, parent subtilisin comprises an amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, or has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22. In still yet another embodiment, the stability of the one or more subtilisin variants in detergent is measured in accordance with the stability assays of Example 2.

In other embodiments, one or more subtilisin variants are more stable than a reference, or parent subtilisin lacking the one, two, three or more features. In some embodiments, such variants having increased stability are characterized by having a Performance Index (PI) greater than about 1.1 with respect to a parent, or reference, protease after 20 minutes incubation in 10% liquid detergent at 30-70 degrees Celsius. In some embodiments, the reference subtilisin refers to a subtilisin having the highest identity to the variant subtilisin, but not containing the recited features.

One or more subtilisin variants described herein can be subject to various changes, such as one or more amino acid insertion, deletion, and/or substitution, either conservative or non-conservative, including where such changes do not substantially alter the enzymatic activity of the variant. Similarly, a polynucleotide encoding the subtilisin variant of the invention can also be subject to various changes, such as one or more substitution of one or more nucleotide in one or more codon such that a particular codon encodes the same or a different amino acid, resulting in either a silent variation (e.g., when the encoded amino acid is not altered by the nucleotide mutation) or non-silent variation; one or more deletion of one or more nucleotides (or codon) in the sequence; one or more addition or insertion of one or more nucleotides (or codon) in the sequence; and/or cleavage of, or one or more truncation, of one or more nucleotides (or codon) in the sequence. Many such changes in the nucleic acid sequence may not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. A nucleic acid sequence described herein can also be modified to include one or more codon that provides for optimum expression in an expression system (e.g., bacterial expression system), while, if desired, said one or more codon still encodes the same amino acid(s).

Described herein is one or more isolated, non-naturally occurring, or recombinant polynucleotide comprising a nucleic acid sequence that encodes one or more subtilisin variants described herein, or recombinant polypeptide or active fragment thereof. One or more nucleic acid sequence described herein is useful in recombinant production (e.g., expression) of one or more subtilisin variants described herein, for example, through expression of a plasmid expression vector comprising a sequence encoding the one or more subtilisin variants described herein or fragment thereof. One embodiment provides nucleic acids encoding one or more subtilisin variants described herein, wherein the variant is a mature form having proteolytic activity. In some embodiments, one or more subtilisin variants described herein is expressed recombinantly with a homologous pro-peptide sequence. In other embodiments, one or more subtilisin variants described herein is expressed recombinantly with a heterologous pro-peptide sequence (e.g., pro-peptide sequence from B. lentus).

One or more nucleic acid sequence described herein can be generated by using any suitable synthesis, manipulation, and/or isolation techniques, or combinations thereof. For example, one or more polynucleotide described herein may be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques that are well-known to those skilled in the art. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized, then joined (e.g., by enzymatic or chemical ligation methods) to form essentially any desired continuous nucleic acid sequence. The synthesis of the one or more polynucleotide described herein can be also facilitated by any suitable method known in the art, including but not limited to chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al. Tetrahedron Letters 22:1859-69 (1981)), or the method described in Matthes et al., EMBO J. 3:801-805 (1984) as is typically practiced in automated synthetic methods. One or more polynucleotide described herein can also be produced by using an automatic DNA synthesizer. Customized nucleic acids can be ordered from a variety of commercial sources (e.g., ATUM (DNA 2.0), Newark, Calif., USA; Life Tech (GeneArt), Carlsbad, Calif., USA; GenScript, Ontario, Canada; Base Clear B. V., Leiden, Netherlands; Integrated DNA Technologies, Skokie, Ill., USA; Ginkgo Bioworks (Gen9), Boston, Mass., USA; and Twist Bioscience, San Francisco, Calif., USA). Other techniques for synthesizing nucleic acids and related principles are described by, for example, Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

Recombinant DNA techniques useful in modification of nucleic acids are well known in the art, such as, for example, restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotide described herein may also be obtained by screening cDNA libraries using one or more oligonucleotide probes that can hybridize to or PCR-amplify polynucleotides which encode one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those of skill in the art and described in standard references known to those skilled in the art. One or more polynucleotide described herein can be obtained by altering a naturally occurring polynucleotide backbone (e.g., that encodes one or more subtilisin variant described herein or reference subtilisin) by, for example, a known mutagenesis procedure (e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination). A variety of methods are known in the art that are suitable for generating modified polynucleotides described herein that encode one or more subtilisin variant described herein, including, but not limited to, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

A further embodiment is directed to one or more vector comprising one or more subtilisin variant described herein (e.g., a polynucleotide encoding one or more subtilisin variant described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequence described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequence described herein; isolated or recombinant cells comprising one or more polynucleotide sequence described herein; and compositions comprising one or more such vector, nucleic acid, expression vector, expression cassette, DNA construct, cell, cell culture, or any combination or mixtures thereof.

Some embodiments are directed to one or more recombinant cell comprising one or more vector (e.g., expression vector or DNA construct) described herein which comprises one or more nucleic acid or polynucleotide sequence described herein. Some such recombinant cells are transformed or transfected with such at least one vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells comprise bacterial cells, including, but not limited to Bacillus sp. cells, such as B. subtilis cells. Other embodiments are directed to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisin described herein.

In some embodiments, one or more vector described herein is an expression vector or expression cassette comprising one or more polynucleotide sequence described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more polynucleotide sequence described herein). A vector may include a transcription terminator and/or a selection gene (e.g., an antibiotic resistance gene) that enables continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing media.

An expression vector may be derived from plasmid or viral DNA, or in alternative embodiments, contains elements of both. Exemplary vectors include, but are not limited to pC194, pJH101, pE194, pHP13 (See, Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for B. subtilis include those listed on p. 92). (See also, Perego, “Integrational Vectors for Genetic Manipulations in Bacillus subtilis”; Sonenshein et al., [eds.]; “Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics”, American Society for Microbiology, Washington, D.C. (1993), pp. 615-624); and p2JM103BBI).

For expression and production of a protein of interest (e.g., one or more subtilisin variant described herein) in a cell, one or more expression vector comprising one or more copy of a polynucleotide encoding one or more subtilisin variant described herein, and in some instances comprising multiple copies, is transformed into the cell under conditions suitable for expression of the variant. In some embodiments, a polynucleotide sequence encoding one or more subtilisin variant described herein (as well as other sequences included in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector comprising a polynucleotide sequence encoding one or more subtilisin variant described herein remains as autonomous extra-chromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements as well as incoming nucleotide sequences that are integrated into the host cell genome. The vectors described herein are useful for production of the one or more subtilisin variant described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variant described herein is present on an integrating vector that enables the integration and optionally the amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those skilled in the art. In some embodiments, transcription of a polynucleotide encoding one or more subtilisin variant described herein is effectuated by a promoter that is the wildtype promoter for the parent subtilisin. In some other embodiments, the promoter is heterologous to the one or more subtilisin variant described herein, but is functional in the host cell. Exemplary promoters for use in bacterial host cells include, but are not limited to the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis cryIIIA; and the B. licheniformis alpha-amylase gene. Additional promoters include, but are not limited to the A4 promoter, as well as phage Lambda PR or PL promoters and the E. coli lac, trp or tac promoters.

One or more subtilisin variant described herein can be produced in host cells of any suitable microorganism, including bacteria and fungi. In some embodiments, one or more subtilisin variant described herein can be produced in Gram-positive bacteria. In some embodiments, the host cells are Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichia spp. In some embodiments, one or more subtilisin variant described herein is produced by Bacillus sp. host cells. Examples of Bacillus sp. host cells that find use in the production of the one or more subtilisin variant described herein include, but are not limited to B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus. In some embodiments, B. subtilis host cells are used to produce the variants described herein. U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more subtilisin variant described herein, although other suitable strains can be used.

Several bacterial strains that can be used to produce one or more subtilisin variant described herein include non-recombinant (i.e., wildtype) Bacillus sp. strains, as well as variants of naturally-occurring strains and/or recombinant strains. In some embodiments, the host strain is a recombinant strain, wherein a polynucleotide encoding one or more subtilisin variant described herein has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain and particularly a recombinant B. subtilis host strain. Numerous B. subtilis strains are known, including, but not limited to for example, 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211strain (See e.g., Hoch et al., Genetics 73:215-228 (1973); See also, U.S. Pat. Nos. 4,450,235; 4,302,544; and EP 0134048). The use of B. subtilis as an expression host cell is well known in the art (See e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).

In some embodiments, the Bacillus host cell is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes: degU, degS, degR and degQ. In some embodiments, the mutation is in a degU gene, and in some embodiments the mutation is degU(Hy)32 (See e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562-567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (See e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and/or oppA or other genes of the opp operon (See e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene will find use in some embodiments of the altered Bacillus strain described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, an altered Bacillus host cell strain that can be used to produce one or more subtilisin variant described herein is a Bacillus host strain that already includes a mutation in one or more of the above-mentioned genes. In addition, Bacillus sp. host cells that comprise mutation(s) and/or deletion(s) of endogenous protease genes find use. In some embodiments, the Bacillus host cell comprises a deletion of the aprE and the nprE genes. In other embodiments, the Bacillus sp. host cell comprises a deletion of 5 protease genes, while in other embodiments the Bacillus sp. host cell comprises a deletion of 9 protease genes (See e.g., US 2005/0202535).

Host cells are transformed with one or more nucleic acid sequence encoding one or more subtilisin variant described herein using any suitable method known in the art. Methods for introducing a nucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizing plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmids are subsequently isolated from E. coli cells and transformed into Bacillus cells. However, it is not essential to use intervening microorganisms such as E. coli, and in some embodiments, a DNA construct or vector is directly introduced into a Bacillus host.

Exemplary methods for introducing one or more nucleic acid sequence described herein into Bacillus cells are described in, for example, Ferrari et al., “Genetics,” in Harwood et al. [eds.], Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol. 157:718-726 (1984); Hoch et al., J. Bacteriol. 93:1925-1937 (1967); Mann et al., Current Microbiol. 13:131-135 (1986); Holubova, Folia Microbiol. 30:97 (1985); Chang et al., Mol. Gen. Genet. 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 (1980); Smith et al., Appl. Env. Microbiol. 51:634 (1986); Fisher et al., Arch. Microbiol. 139:213-217 (1981); and McDonald, J. Gen. Microbiol. 130:203 (1984)). Indeed, such methods as transformation, including protoplast transformation and transfection, transduction, and protoplast fusion are well known and suited for use herein. Methods known in the art to transform Bacillus cells include such methods as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (See, Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)). In this method, the incoming donor plasmid recombines with the homologous region of the resident “helper” plasmid in a process that mimics chromosomal transformation.

In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variant described herein (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct or vector prior to introduction into the host cell). Introduction of a DNA construct or vector described herein into the host cell includes those physical and chemical methods known in the art to introduce a nucleic acid sequence (e.g., DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, DNA constructs or vector are co-transformed with a plasmid, without being inserted into the plasmid. In further embodiments, a selective marker is deleted from the altered Bacillus strain by methods known in the art (See, Stahl et al., J. Bacteriol. 158:411-418 (1984); and Palmeros et al., Gene 247:255-264 (2000)).

In some embodiments, the transformed cells are cultured in conventional nutrient media. The suitable specific culture conditions, such as temperature, pH and the like are known to those skilled in the art and are well described in the scientific literature. Some embodiments provide a culture (e.g., cell culture) comprising one or more subtilisin variant or nucleic acid sequence described herein.

In some embodiments, host cells transformed with one or more polynucleotide sequence encoding one or more subtilisin variant described herein are cultured in a suitable nutrient medium under conditions permitting the expression of the variant, after which the resulting variant is recovered from the culture. In some embodiments, the variant produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to, for example, separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

In some embodiments, one or more subtilisin variant produced by a recombinant host cell is secreted into the culture medium. A nucleic acid sequence that encodes a purification facilitating domain may be used to facilitate purification of the variant. A vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin variant described herein may further comprise a nucleic acid sequence encoding a purification facilitating domain to facilitate purification of the variant (See e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification facilitating domains include, but are not limited to, for example, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (See, Porath, Protein Expr. Purif. 3:263-281 [1992]), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system. The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (e.g., sequences available from Invitrogen, San Diego, Calif.) between the purification domain and the heterologous protein also find use to facilitate purification.

A variety of methods can be used to determine the level of production of one or more mature subtilisin variant described herein in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See e.g., Maddox et al., J. Exp. Med. 158:1211 (1983)).

Some other embodiments provide methods for making or producing one or more mature subtilisin variant described herein. A mature subtilisin variant does not include a signal peptide or a propeptide sequence. Some methods comprise making or producing one or more subtilisin variant described herein in a recombinant bacterial host cell, such as for example, a Bacillus sp. cell (e.g., a B. subtilis cell). Other embodiments provide a method of producing one or more subtilisin variant described herein, wherein the method comprises cultivating a recombinant host cell comprising a recombinant expression vector comprising a nucleic acid sequence encoding one or more subtilisin variant described herein under conditions conducive to the production of the variant. Some such methods further comprise recovering the variant from the culture.

Further embodiments provide methods of producing one or more subtilisin variant described herein, wherein the methods comprise: (a) introducing a recombinant expression vector comprising a nucleic acid encoding the variant into a population of cells (e.g., bacterial cells, such as B. subtilis cells); and (b) culturing the cells in a culture medium under conditions conducive to produce the variant encoded by the expression vector. Some such methods further comprise: (c) isolating the variant from the cells or from the culture medium.

Unless otherwise noted, all component or composition levels provided herein are made in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. Compositions described herein include cleaning compositions, such as detergent compositions. In the exemplified detergent compositions, the enzyme levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.

The subtilisin variants provided herein may be used in the production of various compositions, such as enzyme compositions and cleaning or detergent compositions. An enzyme composition comprises a subtilisin variant as provided herein. The enzyme composition can be in any form, such as granule, liquid formulations, or enzyme slurries.

Enzyme granules may be made by, e.g., rotary atomization, wet granulation, dry granulation, spray drying, disc granulation, extrusion, pan coating, spheronization, drum granulation, fluid-bed agglomeration, high-shear granulation, fluid-bed spray coating, crystallization, precipitation, emulsion gelation, spinning disc atomization and other casting approaches, and prilling processes. The core of the granule may be the granule itself or the inner nucleus of a layered granule.

The core may comprise one or more water soluble or dispersible agent(s), including but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous material (e.g., cellulose and cellulose derivatives such as hydroxyl-propyl-methyl cellulose, carboxy-methyl cellulose, and hydroxyl-ethyl cellulose), phosphate, calcium, a protease inhibitor and combinations thereof. Suitable dispersible agents include, but are not limited to, clays, nonpareils (combinations of sugar and starch; e.g., starch-sucrose non-pareils-ASNP), talc, silicates, carboxymethyl cellulose, starch, and combinations thereof.

In some embodiments, the core comprises mainly sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In a particular embodiment, the core consists of only sodium sulfate.

In some embodiments, the core comprises a subtilisin variant as provided herein. In other embodiments, the core comprises one or more enzymes in addition to protease. In other embodiments, the core is inert and does not comprise enzymes.

In some embodiments, the core is an enzyme powder, including UFC containing an enzyme. The enzyme powder may be spray dried and may optionally be admixed with any of the water soluble or dispersible agents listed, herein. The enzyme may be, or may include, the protease to be stabilized, in which case the enzyme power should further include a stabilizer.

In some embodiments the core is coated with at least one coating layer. In a particular embodiment, the core is coated with at least two coating layers. In another particular embodiment the core is coated with at least three coating layers. The materials used in the coating layer(s) can be suitable for use in cleaning and/or detergent compositions.

In some embodiments, a coating layer comprises one of more of the following materials: an inorganic salt (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, a sugar (e.g., sucrose, lactose, glucose, and fructose), a plasticizer (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous material (e.g., cellulose and cellulose derivatives such as hydroxyl-propyl-methyl cellulose, carboxy-methyl cellulose, and hydroxyl-ethyl cellulose), clay, nonpareil (a combination of sugar and starch), silicate, carboxymethyl cellulose, phosphate, starch (e.g., corn starch), fats, oils (e.g., rapeseed oil, and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-agglomeration agents (e.g., talc, clays, amorphous silica, and titanium dioxide), anti-foam agents (such as FOAMBLAST 882® and EROL 6000K®), and talc. US20100124586, WO9932595, and U.S. Pat. No. 5,324,649 detail suitable components for the coating layers.

In some embodiments, the coating layer comprises sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose). In some embodiments, the coating layer comprises a polymer such as polyvinyl alcohol (PVA). Suitable PVA for incorporation in the coating layer(s) of the multi-layered granule include partially hydrolyzed, fully hydrolyzed and intermediately hydrolyzed having low to high degrees of viscosity. In some embodiments, the coating layer comprises an inorganic salt, such as sodium sulfate.

In some embodiments, at least one coating layer is an enzyme coating layer. In some embodiments the core is coated with at least two enzyme layers. In another embodiment the core is coated with at least three or more enzyme layers.

In some embodiments, the enzymes are protease in combination with one or more additional enzymes selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, metalloproteases, nucleases (e.g. DNases and/or RNases), oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Generally, at least one enzyme coating layer comprises at least one protease.

The above enzyme lists are examples only and are not meant to be exclusive. Any enzyme can be used in the granules described herein, including wild type, recombinant and variant enzymes of bacterial, fungal, yeast sources, and acid, neutral or alkaline enzymes.

In one embodiment, one or more subtilisin variant described herein is useful in cleaning applications, such as, for example, but not limited to, cleaning dishware or tableware items, fabrics, medical instruments and items having hard surfaces (e.g., the hard surface of a table, table top, wall, furniture item, floor, and ceiling). In other embodiments, one or more subtilisin variant described herein is useful in disinfecting applications, such as, for example, but not limited to, disinfecting an automatic dishwashing or laundry machine. In other embodiments, one or more subtilisin variant described herein and compositions comprising such variant are useful in applications to remove or prevent malodor, such as, for example, but not limited to, on laundry, hard surfaces, automatic dishwashing or laundry machines.

Another embodiment is directed to a composition comprising one or more subtilisin variant described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In yet other embodiments, the composition is selected from a laundry detergent composition, an automatic dishwashing (ADW) composition, a hand (manual) dishwashing detergent composition, a hard surface cleaning composition, an eyeglass cleaning composition, a medical instrument cleaning composition, a disinfectant (e.g., malodor or microbial) composition, and a personal care cleaning composition. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand (manual) dishwashing detergent composition. Even still further embodiments are directed to fabric cleaning compositions, while other embodiments are directed to non-fabric cleaning compositions. In some embodiments, the cleaning composition is boron-free. In other embodiments, the cleaning composition is phosphate-free. In still other embodiments, the composition comprises one or more subtilisin variant described herein and one or more of an excipient, adjunct material, and/or additional enzyme.

In yet still a further embodiment, the composition described herein contains phosphate, is phosphate-free, contains boron, is boron-free, or combinations thereof. In other embodiments, the composition is a boron-free composition. In some embodiments, a boron-free composition is a composition to which a borate stabilizer has not been added. In another embodiment, a boron-free composition is a composition that contains less than 5.5% boron. In a still further embodiment, a boron-free composition is a composition that contains less than 4.5% boron. In yet still another embodiment, a boron-free composition is a composition that contains less than 3.5% boron. In yet still a further embodiment, a boron-free composition is a composition that contains less than 2.5% boron. In even further embodiments, a boron-free composition is a composition that contains less than 1.5% boron. In another embodiment, a boron-free composition is a composition that contains less than 1.0% boron. In still further embodiments, a boron-free composition is a composition that contains less than 0.5% boron. In still further embodiments, a boron-free composition is a composition substantially free of boron. In other embodiments, the composition is a composition free or substantially free of enzyme stabilizers or peptide inhibitors.

In another embodiment, one or more composition described herein is in a form selected from gel, tablet, powder, granular, solid, liquid, unit dose, and combinations thereof. In yet another embodiment, one or more composition described herein is in a form selected from a low water compact formula, low water HDL or Unit Dose (UD), or high water formula or HDL. In some embodiments, the cleaning composition described herein is in a unit dose form. In other embodiments, the unit dose form is selected from pills, tablets, capsules, gelcaps, sachets, pouches, multi-compartment pouches, and pre-measured powders or liquids. In some embodiments, the unit dose format is designed to provide controlled release of the ingredients within a multi-compartment pouch (or other unit dose format). Suitable unit dose and controlled release formats are described, for example, in EP 2100949; WO 02/102955; U.S. Pat. Nos. 4,765,916; 4,972,017; and WO 04/111178. In some embodiments, the unit dose form is a tablet or powder contained in a water-soluble film or pouch.

Exemplary laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions. Exemplary hard surface cleaning compositions include, but are not limited to, for example, compositions used to clean the hard surface of a non-dishware item, non-tableware item, table, table top, furniture item, wall, floor, and ceiling. Exemplary hard surface cleaning compositions are described, for example, in U.S. Pat. Nos. 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions used to clean dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care composition include those described in, for example, U.S. Pat. No. 6,376,450.

In some embodiments, one or more subtilisin variant described herein cleans at low temperatures. In other embodiments, one or more composition described herein cleans at low temperatures. In other embodiments, one or more composition described herein comprises an effective amount of one or more subtilisin variant described herein as useful or effective for cleaning a surface in need of proteinaceous stain removal.

In some embodiments, adjunct materials are incorporated, for example, to assist or enhance cleaning performance; for treatment of the substrate to be cleaned; or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. One embodiment is directed to a composition comprising one or more adjunct material and one or more subtilisin variant described herein. Another embodiment is directed to a composition comprising one or more adjunct material and one or more subtilisin variant described herein, wherein the adjunct material is selected from a bleach catalyst, an additional enzyme, an enzyme stabilizer (including, for example, an enzyme stabilizing system), a chelant, an optical brightener, a soil release polymer, a dye transfer agent, a dispersant, a suds suppressor, a dye, a perfume, a colorant, a filler, a photoactivator, a fluorescer, a fabric conditioner, a hydrolyzable surfactant, a preservative, an anti-oxidant, an anti-shrinkage agent, an anti-wrinkle agent, a germicide, a fungicide, a color speckle, a silvercare agent, an anti-tarnish agent, an anti-corrosion agent, an alkalinity source, a solubilizing agent, a carrier, a processing aid, a pigment, a pH control agent, a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a deposition aid, a catalytic material, a bleach activator, a bleach booster, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a pigment, and combinations thereof. Exemplary adjunct materials and levels of use are found in U.S. Pat. Nos. 5,576,282; 6,306,812; 6,326,348; 6,610,642; 6,605,458; 5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014 and 5,646,101. In embodiments in which one or more cleaning adjunct material is not compatible with one or more subtilisin variant described herein, methods are employed to keep the adjunct material and variant(s) separated (i.e., not in contact with each other) until combination of the two components is appropriate. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapsulation, tablets, physical separation, etc.).

Some embodiments are directed to cleaning additive products comprising one or more subtilisin variant described herein. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired.

Exemplary fillers or carriers for granular compositions include, but are not limited to, for example, various salts of sulfate, carbonate and silicate; talc; and clay. Exemplary fillers or carriers for liquid compositions include, but are not limited to, for example, water or low molecular weight primary and secondary alcohols including polyols and diols (e.g., methanol, ethanol, propanol and isopropanol). In some embodiments, the compositions contain from about 5% to about 90% of such filler or carrier. Acidic fillers may be included in such compositions to reduce the pH of the resulting solution in the cleaning method or application.

In one embodiment, one or more cleaning composition described herein comprises an effective amount of one or more subtilisin variant described herein, alone or in combination with one or more additional enzyme. Typically, a cleaning composition comprises at least about 0.0001 to about 20 wt %, from about 0.0001 to about 10 wt %, from about 0.0001 to about 1 wt %, from about 0.001 to about 1 wt %, or from about 0.01 to about 0.1 wt % of one or more protease. In another embodiment, one or more cleaning composition described herein comprises from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg of one or more protease per gram of composition.

The cleaning compositions described herein are typically formulated such that during use in aqueous cleaning operations, the wash water will have a pH of from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 to about 9.0 or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 8 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to have an alkaline pH under wash conditions, such as a pH of from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention can be formulated to have a neutral pH under wash conditions, such as a pH of from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5. In some embodiments, the neutral pH conditions can be measured when the cleaning composition is dissolved 1:100 (wt:wt) in de-ionised water at 20° C., measured using a conventional pH meter. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

In some embodiments, one or more subtilisin variant described herein is encapsulated to protect it during storage from the other components in the composition and/or control the availability of the variant during cleaning. In some embodiments, encapsulation enhances the performance of the variant and/or additional enzyme. In some embodiments, the encapsulating material typically encapsulates at least part of the subtilisin variant described herein. Typically, the encapsulating material is water-soluble and/or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0° C. or higher. Exemplary encapsulating materials include, but are not limited to, carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some embodiments, the encapsulating material is a starch (See e.g., EP0922499, U.S. Pat. Nos. 4,977,252, 5,354,559, and 5,935,826). In some embodiments, the encapsulating material is a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof. Exemplary commercial microspheres include, but are not limited to EXPANCEL® (Stockviksverken, Sweden); and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, Pa.).

There are a variety of wash conditions including varying detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time to which one or more subtilisin variant described herein may be exposed. A low detergent concentration system is directed to wash water containing less than about 800 ppm detergent components. A medium detergent concentration system is directed to wash containing between about 800 ppm and about 2000 ppm detergent components. A high detergent concentration system is directed to wash water containing greater than about 2000 ppm detergent components. In some embodiments, the “cold water washing” of the present invention utilizes “cold water detergent” suitable for washing at temperatures from about 10° C. to about 40° C., from about 20° C. to about 30° C., or from about 15° C. to about 25° C., as well as all other combinations within the range of about 15° C. to about 35° C. or 10° C. to 40° C.

Different geographies have different water hardness. Hardness is a measure of the amount of calcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Water hardness is usually described in terms of the grains per gallon (gpg) mixed Ca²⁺/Mg²⁺. Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 ppm (ppm can be converted to grains per U.S. gallon by dividing ppm by 17.1) of hardness minerals.

	Water	Grains per gallon	Parts per million
	Soft	less than 1.0	less than 17
	Slightly hard	1.0 to 3.5	17 to 60
	Moderately hard	3.5 to 7.0	60 to 120
	Hard	7.0 to 10.5	120 to 180
	Very hard	greater than 10.5	greater than 180

Other embodiments are directed to one or more cleaning composition comprising from about 0.00001% to about 10% by weight composition of one or more subtilisin variant described herein and from about 99.999% to about 90.0% by weight composition of one or more adjunct material. In another embodiment, the cleaning composition comprises from about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight composition of one or more subtilisin variant and from about 99.9999% to about 90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% by weight composition of one or more adjunct material.

In other embodiments, the composition described herein comprises one or more subtilisin variant described herein and one or more additional enzyme. The one or more additional enzyme is selected from acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, metalloproteases, nucleases (e.g. DNases and RNases), oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Some embodiments are directed to a combination of enzymes (i.e., a “cocktail”) comprising conventional enzymes like amylase, lipase, cutinase, mannanase and/or cellulase in conjunction with one or more subtilisin variant described herein and/or one or more additional protease.

In another embodiment, one or more composition described herein comprises one or more subtilisin variant described herein and one or more additional protease. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. Suitable additional proteases include those of animal, plant or microbial origin. In some embodiments, the additional protease is a microbial protease. In other embodiments, the additional protease is a chemically or genetically modified mutant. In another embodiment, the additional protease is a metalloprotease, a fungal subtilisin, an alkaline microbial protease or a trypsin-like protease. Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., subtilis, lentus, amyloliquefaciens, licheniformis, gibsonii, clausii, alkalophilus, subtilisin 309, subtilisin 147 and subtilisin 168). Exemplary additional proteases include but are not limited to those described in WO92/21760, WO95/23221, WO2008/010925, WO09/149200, WO09/149144, WO09/149145, WO 10/056640, WO10/056653, WO2010/0566356, WO11/072099, WO2011/13022, WO11/140364, WO 12/151534, WO2015/038792, WO2015/089447, WO2015/089441, WO2019180111, US Publ. No. 2008/0090747, U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, RE 34,606, U.S. Pat. Nos. 5,955,340, 5,700,676 6,312,936, 6,482,628, 8,530,219, U.S. Provisional Appl Nos. 62/180,673 and 62/161,077, and PCT Appl Nos. PCT/US2015/021813, PCT/US2015/055900, PCT/US2015/057497, PCT/US2015/057492, PCT/US2015/057512, PCT/US2015/057526, PCT/US2015/057520, PCT/US2015/057502, PCT/US2016/022282, and PCT/US16/32514, as well as metalloproteases described in WO1999014341, WO1999033960, WO1999014342, WO1999034003, WO2007044993, WO2009058303, WO 2009058661, WO2014071410, WO2014194032, WO2014194034, WO 2014194054, and WO 2014/194117. Exemplary additional proteases include, but are not limited to trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO89/06270. Exemplary commercial proteases include, but are not limited to MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX™, EXCELLASE™ PREFERENZ™ proteases (e.g. P100, P110, P280, P300), EFFECTENZ™ proteases (e.g. P1000, P1050, P2000), EXCELLENZ™ proteases (e.g. P1000), ULTIMASE®, and PURAFAST™ (DuPont); ALCALASE®, BLAZE®, and BLAZE® variants, BLAZE® EVITY® 16L, CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE® EVERTS®, PRIMASE®, DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, LIQUANASE EVERTS®, NEUTRASE®, RELASE® PROGRESS®, EASYZYME®, and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel); KAP (B. alkalophilus subtilisin (Kao)); and BIOTOUCH® (AB Enzymes). Exemplary metalloproteases include nprE, the recombinant form of neutral metalloprotease expressed in B. subtilis (See e.g., WO 07/044993), and PMN, the purified neutral metalloprotease from B. amyloliquefaciens.

Another embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more lipase. In some embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight composition. An exemplary lipase can be a chemically or genetically modified mutant. Exemplary lipases include, but are not limited to, e.g., those of bacterial or fungal origin, such as, e.g., H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T. lanuginosus lipase (see, e.g., WO 2014/059360 and WO2015/010009), Rhizomucor miehei lipase (see, e.g., EP 238023), Candida lipase, such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761), Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipase (see, e.g., EP 218272), P. cepacia lipase (see, e.g., EP 331376), P. stutzeri lipase (see, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase (Dartois et al., Biochem. Biophys. Acta 1131:253-260 (1993)), B. stearothermophilus lipase (see, e.g., JP 64/744992), and B. pumilus lipase (see, e.g., WO 91/16422)). Exemplary cloned lipases include, but not limited to Penicillium camembertii lipase (See, Yamaguchi et al., Gene 103:61-67 (1991)), Geotricum candidum lipase (See, Schimada et al., J. Biochem., 106:383-388 (1989)), and various Rhizopus lipases, such as, R. delemar lipase (See, Hass et al., Gene 109:117-113 (1991)), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and R. oryzae lipase. Other lipolytic enzymes, such as cutinases, may also find use in one or more composition described herein, including, but not limited to, e.g., cutinase derived from Pseudomonas mendocina (see, WO 88/09367) and/or Fusarium solani pisi (see, WO90/09446). Exemplary commercial lipases include, but are not limited to M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (DuPont); LIPEX®, LIPOCLEAN®, LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P™ (Amano Pharmaceutical Co. Ltd).

A still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more amylase. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase by weight composition. Any amylase (e.g., alpha and/or beta) suitable for use in alkaline solutions may be useful to include in such composition. An exemplary amylase can be a chemically or genetically modified mutant. Exemplary amylases include, but are not limited to those of bacterial or fungal origin, such as, for example, amylases described in GB 1,296,839, WO9100353, WO9402597, WO94183314, WO9510603, WO9526397, WO9535382, WO9605295, WO9623873, WO9623874, WO 9630481, WO9710342, WO9741213, WO9743424, WO9813481, WO 9826078, WO9902702, WO 9909183, WO9919467, WO9923211, WO9929876, WO9942567, WO 9943793, WO9943794, WO 9946399, WO0029560, WO0060058, WO0060059, WO0060060, WO 0114532, WO0134784, WO 0164852, WO0166712, WO0188107, WO0196537, WO02092797, WO 0210355, WO0231124, WO 2004055178, WO2004113551, WO2005001064, WO2005003311, WO 2005018336, WO2005019443, WO2005066338, WO2006002643, WO2006012899, WO2006012902, WO2006031554, WO 2006063594, WO2006066594, WO2006066596, WO2006136161, WO 2008000825, WO2008088493, WO2008092919, WO2008101894, WO2008/112459, WO2009061380, WO2009061381, WO 2009100102, WO2009140504, WO2009149419, WO 2010/059413, WO 2010088447, WO2010091221, WO2010104675, WO2010115021, WO10115028, WO2010117511, WO 2011076123, WO2011076897, WO2011080352, WO2011080353, WO 2011080354, WO2011082425, WO2011082429, WO 2011087836, WO2011098531, WO2013063460, WO2013184577, WO 2014099523, WO2014164777, and WO2015077126. Exemplary commercial amylases include, but are not limited to AMPLIFY®, DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYME ULTRA® EVITY®, and BAN™ (Novozymes); EFFECTENZ™ S 1000, POWERASE™, PREFERENZ™ S 100, PREFERENZ™ S 110, PREFERENZ™ S 210, EXCELLENZ™ S 2000, RAPIDASE® and MAXAMYL® P (DuPont). In some embodiments, the subtilisin variants provided herein may be combined with one or more amylases selected from the group consisting of AA707, AA560, AAI10, BspAmy24, and CspAmyl, and variants thereof, and combinations thereof.

Yet a still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more cellulase. In one embodiment, the composition comprises from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of composition. Any suitable cellulase may find use in a composition described herein. An exemplary cellulase can be a chemically or genetically modified mutant. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in WO2005054475, WO2005056787, U.S. Pat. Nos. 7,449,318, 7,833,773, 4,435,307; EP 0495257; and U.S. Provisional Appl. No. 62/296,678. Exemplary commercial cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, ENDOLASE®, RENOZYME®, and CAREZYME® PREMIUM (Novozymes); REVITALENZ™ 100, REVITALENZ™ 200/220, and REVITALENZ® 2000 (DuPont); and KAC-500(B)™ (Kao Corporation). In some embodiments, cellulases are incorporated as portions or fragments of mature wildtype or variant cellulases, wherein a portion of the N-terminus is deleted (see, e.g., U.S. Pat. No. 5,874,276).

An even still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more mannanase. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase by weight composition. An exemplary mannanase can be a chemically or genetically modified mutant. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in WO 2016/007929; U.S. Pat. Nos. 6,566,114; 6,602,842; and 6,440,991: and U.S. Provisional Appl. Nos. 62/251,516, 62/278,383, and 62/278,387. Exemplary commercial mannanases include, but are not limited to MANNAWAY® (Novozymes) and EFFECTENZ™ M 1000, PREFERENZ® M 100, MANNASTAR®, and PURABRITE™ (DuPont).

A yet even still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more peroxidase and/or oxidase enzyme. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% peroxidase or oxidase by weight composition. A peroxidase may be used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate) and an oxidase may be used in combination with oxygen. Peroxidases and oxidases are used for “solution bleaching” (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), alone or in combination with an enhancing agent (see, e.g., WO94/12621 and WO95/01426). An exemplary peroxidase and/or oxidase can be a chemically or genetically modified mutant. Exemplary peroxidases/oxidases include, but are not limited to those of plant, bacterial, or fungal origin.

Another embodiment is directed to a composition comprising one or more subtilisin variant described herein, and one or more perhydrolase, such as, for example, those described in WO2005/056782, WO2007/106293, WO 2008/063400, WO2008/106214, and WO2008/106215.

Another embodiment is directed to a composition comprising one or more subtilisin variant described herein, and one or more pectate lyase, such as, for example, XPect®.

In yet another embodiment, the one or more subtilisin variant described herein and one or more additional enzyme contained in one or more composition described herein may each independently range to about 10%, wherein the balance of the cleaning composition is one or more adjunct material.

In some embodiments, one or more composition described herein finds use as a detergent additive, wherein said additive is in a solid or liquid form. Such additive products are intended to supplement and/or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent composition ranges from about 400 to about 1200 g/liter, while in other embodiments it ranges from about 500 to about 950 g/liter of composition measured at 20° C.

Some embodiments are directed to a laundry detergent composition comprising one or more subtilisin variant described herein and one or more adjunct material selected from surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry compositions also contain softening agents.

Further embodiments are directed to manual dishwashing composition comprising one or more subtilisin variant described herein and one or more adjunct material selected from surfactants, organic polymeric compounds, suds enhancing agents, group II metal ions, solvents, hydrotropes, and additional enzymes.

Other embodiments are directed to one or more composition described herein, wherein said composition is a compact granular fabric cleaning composition that finds use in laundering colored fabrics or provides softening through the wash capacity, or is a heavy duty liquid (HDL) fabric cleaning composition. Exemplary fabric cleaning compositions and/or processes for making such compositions are described in USPNs 6,610,642 and 6,376,450. Other exemplary cleaning compositions are described, for example, in U.S. Pat. Nos. 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303; 4,968,451; 4,597,898; 4,561,998; 4,550,862; 4,537,706; 4,515,707; and 4,515,705.

In some embodiments, the cleaning compositions comprise an acidifying particle or an amino carboxylic builder. Examples of an amino carboxylic builder include aminocarboxylic acids, salts and derivatives thereof. In some embodiment, the amino carboxylic builder is an aminopolycarboxylic builder, such as glycine-N,N-diacetic acid or derivative of general formula MOOC—CHR-N(CH₂COOM)₂ where R is C_1-12alkyl and M is alkali metal. In some embodiments, the amino carboxylic builder can be methylglycine diacetic acid (MGDA), GLDA (glutamic-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and salts and derivatives thereof, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl) glutamic acid (SEGL), IDA (iminodiacetic acid) and salts and derivatives thereof such as N-methyliminodiacetic acid (MIDA), alpha-alanine-N,N-diacetic acid (alpha-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA) and alkali metal salts and derivative thereof. In some embodiments, the acidifying particle has a weight geometric mean particle size of from about 400 microns to about 1200 microns and a bulk density of at least 550 g/L. In some embodiments, the acidifying particle comprises at least about 5% of the builder.

In some embodiments, the acidifying particle can comprise any acid, including organic acids and mineral acids. Organic acids can have one or two carboxyls and in some instances up to 15 carbons, especially up to 10 carbons, such as formic, acetic, propionic, capric, oxalic, succinic, adipic, maleic, fumaric, sebacic, malic, lactic, glycolic, tartaric and glyoxylic acids. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric and sulfuric acid. In some instances, the acidifying particle is a highly active particle comprising a high level of amino carboxylic builder. Sulfuric acid has also been found to further contribute to the stability of the final particle.

Additional embodiments are directed to a cleaning composition comprising one or more subtilisin variant and one or more surfactant and/or surfactant system, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1 to about 60%, while in alternative embodiments the level is from about 1 to about 50%, while in still further embodiments the level is from about 5 to about 40%, by weight of the cleaning composition.

In some embodiments, one or more composition described herein comprises one or more detergent builders or builder systems. In one embodiment, the composition comprises from about 1% or greater, from about 0.1% to about 80%, from about 3% to about 60%, from about 5% to about 40%, or from about 10% to about 50% builder by weight composition. Exemplary builders include, but are not limited to alkali metal; ammonium and alkanolammonium salts of polyphosphates; alkali metal silicates; alkaline earth and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxypolycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid; ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid; polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof. In some such compositions, the builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates, e.g., sodium tripolyphosphate, sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphate. Exemplary builders are described in, e.g., EP 2100949. In some embodiments, the builders include phosphate builders and non-phosphate builders. In some embodiments, the builder is a phosphate builder. In some embodiments, the builder is a non-phosphate builder. In some embodiments, the builder comprises a mixture of phosphate and non-phosphate builders. Exemplary phosphate builders include, but are not limited to mono-phosphates, di-phosphates, tri-polyphosphates or oligomeric-polyphosphates, including the alkali metal salts of these compounds, including the sodium salts. In some embodiments, a builder can be sodium tripolyphosphate (STPP). Additionally, the composition can comprise carbonate and/or citrate. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In some embodiments, salts of the above-mentioned compounds include the ammonium and/or alkali metal salts, i.e. the lithium, sodium, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, hetero-cyclic and aromatic carboxylic acids, wherein in some embodiments, they can contain at least two carboxyl groups which are in each case separated from one another by, in some instances, no more than two carbon atoms.

In some embodiments, one or more composition described herein comprises one or more chelating agent. In one embodiment, the composition comprises from about 0.1% to about 15% or about 3% to about 10% chelating agent by weight composition. Exemplary chelating agents include, but are not limited to, e.g., copper, iron, manganese, and mixtures thereof.

In some embodiments, one or more composition described herein comprises one or more deposition aid. Exemplary deposition aids include, but are not limited to, e.g., polyethylene glycol; polypropylene glycol; polycarboxylate; soil release polymers, such as, e.g., polyterephthalic acid; clays such as, e.g., kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite; and mixtures thereof.

In other embodiments, one or more composition described herein comprises one or more anti-redeposition agent or non-ionic surfactant (which can prevent the re-deposition of soils) (see, e.g., EP 2100949). For example, in ADW compositions, non-ionic surfactants find use for surface modification purposes, in particular for sheeting, to avoid filming and spotting and to improve shine. These non-ionic surfactants also find use in preventing the re-deposition of soils. In some embodiments, the non-ionic surfactant can be ethoxylated nonionic surfactants, epoxy-capped poly(oxyalkylated) alcohols and amine oxides surfactants.

In some embodiments, one or more composition described herein comprises one or more dye transfer inhibiting agent. Exemplary polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, polyvinylimidazoles, and mixtures thereof. In one embodiment, the composition comprises from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% dye transfer inhibiting agent by weight composition.

In some embodiments, one or more composition described herein comprises one or more silicate. Exemplary silicates include, but are not limited to, sodium silicates, e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, silicates are present at a level of from about 1% to about 20% or about 5% to about 15% by weight of the composition.

In some still additional embodiments, one or more composition described herein comprises one or more dispersant. Exemplary water-soluble organic materials include, but are not limited to, e.g., homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

In some further embodiments, one or more composition described herein comprises one or more inorganic enzyme stabilizer. In some embodiments, the enzyme stabilizer is water-soluble sources of calcium and/or magnesium ions. In some embodiments, the enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts, including alkaline earth metals, such as calcium salts. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II), and oxovanadium (IV)). Chlorides and sulfates also find use in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07/145964. In some embodiments, reversible protease inhibitors also find use, such as boron-containing compounds (e.g., borate, 4-formyl phenyl boronic acid, and phenyl-boronic acid derivatives (such for example, those described in WO96/41859) and/or a peptide aldehyde, such as, for example, is further described in WO2009/118375 and WO2013004636.

Peptidic inhibitors can be naturally derived or synthetically produced oligopeptides able to bind to protease and inhibit its proteolytic activity, and thus used as protease stabilizers in liquid laundry formulations. Peptide aldehydes are peptidic inhibitors and may be used as protease stabilizers in detergent formulations as previously described (WO199813458, WO2011036153, US20140228274). Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones and might be ‘N-capped’ with for instance a ureido, a carbamate, or a urea moiety, or ‘doubly N-capped’ with for instance a carbonyl, a ureido, an oxiamide, a thioureido, a dithiooxamide, or a thiooxamide moiety(EP2358857B1). The molar ratio of these inhibitors to the protease may be 0.1:1 to 100:1, e.g. 0.5:1-50:1, 1:1-25:1 or 2:1-10:1. Other examples of protease stabilizers are benzophenone or benzoic acid anilide derivatives, which might contain carboxyl groups (U.S. Pat. No. 7,968,508 B2). The molar ratio of these stabilizers to protease is preferably in the range of 1:1 to 1000:1 in particular 1:1 to 500:1 especially preferably from 1:1 to 100:1, most especially preferably from 1:1 to 20:1.

In other embodiments, the one or more compositions provided herein does not contain an enzyme stabilizer, or peptidic inhibitors, or contains a reduced amount of an enzyme stabilizer and peptide inhibitors, such as peptide aldehydes. That is, the subtilisin variants provided herein have an increased stability with respect to a reference subtilisin in compositions that lack an enzyme stabilizer or peptide inhibitors, or contain a reduced amount of an enzyme stabilizer or peptide inhibitor.

In some embodiments, one or more composition described herein comprises one or more bleach, bleach activator, and/or bleach catalyst. In some embodiments, one or more composition described herein comprises one or more inorganic and/or organic bleaching compound. Exemplary inorganic bleaches include, but are not limited to perhydrate salts, e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate salts. In some embodiments, inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts are included as the crystalline solid, without additional protection, although in some other embodiments, the salt is coated. Bleach activators are typically organic peracid precursors that enhance the bleaching action in the course of cleaning at temperatures of 60° C. and below. Exemplary bleach activators include compounds which, under perhydrolysis conditions, give aliphatic peroxoycarboxylic acids having from about 1 to about 10 carbon atoms or about 2 to about 4 carbon atoms, and/or optionally substituted perbenzoic acid. Exemplary bleach activators ae described, for example, in EP 2100949. Exemplary bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, as well as cobalt, copper, manganese, and iron complexes. Additional exemplary bleach catalysts are described, for example, in U.S. Pat. Nos. 4,246,612; 5,227,084; 4,810,410; WO 99/06521; and EP 2100949.

In some embodiments, one or more composition described herein comprises one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst finds use. In some embodiments, the metal bleach catalyst comprises a catalyst system comprising a transition metal cation of defined bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations), an auxiliary metal cation having little or no bleach catalytic activity (e.g., zinc or aluminum cations), and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof (see, e.g., U.S. Pat. No. 4,430,243). In some embodiments, one or more composition described herein is catalyzed by means of a manganese compound. Such compounds and levels of use are described, for example, in U.S. Pat. No. 5,576,282. In additional embodiments, cobalt bleach catalysts find use and are included in one or more composition described herein. Various cobalt bleach catalysts are described, for example, in U.S. Pat. Nos. 5,597,936 and 5,595,967.

In some additional embodiments, one or more composition described herein includes a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes described herein are adjusted to provide on the order of at least one part per hundred million, from about 0.005 ppm to about 25 ppm, about 0.05 ppm to about 10 ppm, or about 0.1 ppm to about 5 ppm of active MRL in the wash liquor. Exemplary MRLs include, but are not limited to special ultra-rigid ligands that are cross-bridged, such as, e.g., 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Exemplary metal MRLs are described, for example, in WO 2000/32601 and U.S. Pat. No. 6,225,464.

In another embodiment, one or more composition described herein comprises one or more metal care agent. In some embodiments, the composition comprises from about 0.1% to about 5% metal care agent by weight composition. Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Additional exemplary metal care agents are described, for example, in EP 2100949, WO 94/26860, and WO 94/26859. In some compositions, the metal care agent is a zinc salt.

In some embodiments, the cleaning composition is a high density liquid (HDL) composition comprising one or more subtilisin variant described herein. The HDL liquid laundry detergent can comprise a detersive surfactant (10-40%) comprising anionic detersive surfactant selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof; and optionally non-ionic surfactant selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, for example, a C₈-C₁₈alkyl ethoxylated alcohol and/or C₆-C₁₂alkyl phenol alkoxylates, optionally wherein the weight ratio of anionic detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersive surfactant is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from alkyl pyridinium compounds, alkyl quarternary ammonium compounds, alkyl quarternary phosphonium compounds, alkyl ternary sulphonium compounds, and/or mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants; and mixtures thereof.

In another embodiment, the cleaning composition is a liquid or gel detergent, which is not unit dosed, that may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

The composition can comprise optionally, a surfactancy boosting polymer consisting of amphiphilic alkoxylated grease cleaning polymers selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylen imines in the range of 0.05 wt %-10 wt % and/or random graft polymers typically comprising a hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C₁-C₆carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophobic side chain(s) selected from the group consisting of: C₄-C₂₅alkyl group, polypropylene, polybutylene, vinyl ester of a saturated C₂-C₆mono-carboxylic acid, C₁-C₆alkyl ester of acrylic or methacrylic acid, and mixtures thereof.

The composition can comprise additional polymers such as soil release polymers including, for example, anionically end-capped polyesters, for example SRP1; polymers comprising at least one monomer unit selected from saccharide, dicarboxylic acid, polyol and combinations thereof, in random or block configuration; ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example, Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL; anti-redeposition polymers (0.1 wt % to 10 wt %, including, for example, carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinylpyrrolidone homopolymer; and/or polyethylene glycol with a molecular weight in the range of from 500 to 100,000 Da); cellulosic polymer (including, for example, alkyl cellulose; alkyl alkoxyalkyl cellulose; carboxyalkyl cellulose; alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric carboxylate (such as, for example, maleate/acrylate random copolymer or polyacrylate homopolymer).

The composition can further comprise saturated or unsaturated fatty acid, preferably saturated or unsaturated C₁₂-C₂₄fatty acid (0-10 wt %); deposition aids (including, for example, polysaccharides, cellulosic polymers, polydiallyl dimethyl ammonium halides (DADMAC), and co-polymers of DADMAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, in random or block configuration; cationic guar gum; cationic cellulose such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacylamides; and mixtures thereof.

The composition can further comprise dye transfer inhibiting agents examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents examples of which include ethylene-diamine-tetraacetic acid (EDTA); diethylene triamine penta methylene phosphonic acid (DTPMP); hydroxy-ethane diphosphonic acid (HEDP); ethylenediamine N,N′-disuccinic acid (EDDS); methyl glycine diacetic acid (MGDA); diethylene triamine penta acetic acid (DTPA); propylene diamine tetracetic acid (PDT A); 2-hydroxypyridine-N-oxide (HPNO); or methyl glycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamic acid tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any salts thereof; N-hydroxyethylethylenediaminetri-acetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof.

The composition can further comprise silicone or fatty-acid based suds suppressors; an enzyme stabilizer; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foam (0.001 to about 4.0 wt %), and/or structurant/thickener (0.01-5 wt %) selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

In some embodiments, the cleaning composition is a high density powder (HDD) composition comprising one or more subtilisin variant described herein. The HDD powder laundry detergent can comprise a detersive surfactant including anionic detersive surfactants (selected from linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and/or mixtures thereof), non-ionic detersive surfactant (selected from 1 linear or branched or random chain, substituted or unsubstituted C₈-C₁₈ alkyl ethoxylates, and/or C₆-C₁₂ alkyl phenol alkoxylates), cationic detersive surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulphonium compounds, and mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants and mixtures thereof; builders (phosphate free builders, e,g., zeolite builders examples of which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 to less than 10 wt %); phosphate builders, e.g., sodium tri-polyphosphate in the range of 0 to less than 10 wt %; citric acid, citrate salts and nitrilotriacetic acid or salt thereof in the range of less than 15 wt %; silicate salt (sodium or potassium silicate or sodium meta-silicate in the range of 0 to less than 10 wt % or layered silicate (SKS-6)); carbonate salt (sodium carbonate and/or sodium bicarbonate in the range of 0 to less than 10 wt %); and bleaching agents (photobleaches, e.g., sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines, xanthenes dyes, and mixtures thereof); hydrophobic or hydrophilic bleach activators (e.g., dodecanoyl oxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate, tetraacetyl ethylene diamine-TAED, and nonanoyloxybenzene sulfonate-NOBS, nitrile quats, and mixtures thereof); hydrogen peroxide; sources of hydrogen peroxide (inorganic perhydrate salts, e.g., mono or tetra hydrate sodium salt of perborate, percarbonate, persulfate, perphosphate, or persilicate); preformed hydrophilic and/or hydrophobic peracids (selected from percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof); and/or bleach catalyst (e.g., imine bleach boosters, such as iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acyl imines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof), metal-containing bleach catalyst (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations along with an auxiliary metal cations such as zinc or aluminum and a sequestrate such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof).

The composition can further comprise additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, an enzyme stabilizer, hueing agents, additional polymers including fabric integrity and cationic polymers, dye lock ingredients, fabric-softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.

In some embodiments, the cleaning composition is an automatic (auto) dish washing (ADW) detergent composition comprising one or more subtilisin variant described herein. The ADW detergent composition can comprise two or more non-ionic surfactants selected from ethoxylated non-ionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols, and amine oxide surfactants present in amounts from 0-10% by wt; builders in the range of 5-60% by wt. comprising either phosphate (mono-phosphates, di-phosphates, tri-polyphosphates or oligomeric-polyphosphates), sodium tripolyphosphate-STPP or phosphate-free builders (amino acid based compounds, e.g., MGDA (methyl-glycine-diacetic acid) and salts and derivatives thereof, GLDA (glutamic-N,Ndiacetic acid) and salts and derivatives thereof, IDS (iminodisuccinic acid) and salts and derivatives thereof, carboxy methyl inulin and salts and derivatives thereof and mixtures thereof, nitrilotriacetic acid (NTA), diethylene triamine penta acetic acid (DTPA), and B-alaninediacetic acid (B-ADA) and their salts), homopolymers and copolymers of poly-carboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts in the range of 0.5-50% by wt; sulfonated/carboxylated polymers (provide dimensional stability to the product) in the range of about 0.1 to about 50% by wt; drying aids in the range of about 0.1 to about 10% by wt (selected from polyesters, especially anionic polyesters optionally together with further monomers with 3-6 functionalities which are conducive to polycondensation, specifically acid, alcohol or ester functionalities, polycarbonate-, polyurethane- and/or polyurea-polyorganosiloxane compounds or precursor compounds thereof of the reactive cyclic carbonate and urea type); silicates in the range from about 1 to about 20% by wt (sodium or potassium silicates, e.g., sodium disilicate, sodium meta-silicate and crystalline phyllosilicates); bleach-inorganic (e.g., perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts) and organic (e.g., organic peroxyacids including diacyl and tetraacylperoxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleach activator-organic peracid precursors in the range from about 0.1 to about 10% by wt; bleach catalysts (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bispyridylamine and related complexes, and pentamine acetate cobalt(III) and related complexes); metal care agents in the range from about 0.1-5% by wt (selected from benzatriazoles, metal salts and complexes, and silicates); enzymes in the range from about 0.01-5.0 mg of active enzyme per gram of ADW detergent composition (acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polyestersases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, and mixtures thereof); and enzyme stabilizer components (selected from oligosaccharides, polysaccharides and inorganic divalent metal salts).

More embodiments are directed to compositions and methods of treating fabrics (e.g., to desize a textile) using one or more subtilisin variant described herein. Fabric-treating methods are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with a variant described herein in a solution. The fabric can be treated with the solution under pressure.

One or more subtilisin variant described herein can be applied during or after weaving a textile, during the desizing stage, or one or more additional fabric processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Prior to weaving on mechanical looms, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and to prevent breaking. One or more subtilisin variant described herein can be used alone or with other desizing chemical reagents and/or desizing enzymes to desize fabrics, including cotton-containing fabrics, as detergent additives, e.g., in aqueous compositions. An amylase also can be used in compositions and methods for producing a stonewashed look on indigo-dyed denim fabric and garments. For the manufacture of clothes, the fabric can be cut and sewn into clothes or garments, which are afterwards finished. In particular, for the manufacture of denim jeans, different enzymatic finishing methods have been developed. The finishing of denim garment normally is initiated with an enzymatic desizing step, during which garments are subjected to the action of proteolytic enzymes to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing steps. One or more subtilisin variant described herein can be used in methods of finishing denim garments (e.g., a “bio-stoning process”), enzymatic desizing and providing softness to fabrics, and/or finishing process.

One or more subtilisin variant described herein finds further use in the enzyme aided bleaching of paper pulps such as chemical pulps, semi-chemical pulps, kraft pulps, mechanical pulps or pulps prepared by the sulfite method. In general terms, paper pulps are incubated with one or more subtilisin variant described herein under conditions suitable for bleaching the paper pulp.

In some embodiments, the pulps are chlorine free pulps bleached with oxygen, ozone, peroxide or peroxyacids. In some embodiments, one or more subtilisin variant described herein is used in enzyme aided bleaching of pulps produced by modified or continuous pulping methods that exhibit low lignin contents. In some other embodiments, one or more subtilisin variant described herein is applied alone or preferably in combination with xylanase and/or endoglucanase and/or alpha-galactosidase and/or cellobiohydrolase enzymes.

The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and should not be construed as limiting.

Example 1

Construction and Expression of Subtilisin Protease Variants

Variants of a series of subtilisins of bacterial origin having one, two or more substitutions in each of the parental backbones (Table 1) were generated using molecular biology techniques known in the art. Libraries of genes were generated that have various combinations of the following amino acid features: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, X259P, listed in BPN′ (SEQ ID NO:1) numbering. Libraries of constructs for each subtilisin protease (parent and variants) were transformed into a suitable Bacillus subtilis strain using methods known in the art. The transformation mixtures were plated on LA containing skim milk and the appropriate antibiotic resistance selection. Single colonies were picked and grown on Luria broth with the appropriate antibiotic resistance selection. The DNA was extracted and the sequence of each gene of interest was confirmed by PacBio sequencing method.

For recombinant protein expression experiments, transformed cells were grown in 96-well microtiter plates (MTPs) in cultivation medium (enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, containing antibiotic selection) for 3 days at 32° C., 300 rpm, with 80% humidity in shaking incubator. After centrifugation and filtration, clarified culture supernatants containing the proteases of interest were used for assays.

Table 1 below provides the names and sequences for the subtilisin parental backbones (parent) used in this study. All subtilisins, with the exception of DSM14391 and CP474, were cloned and expressed using their own (wildtype) propeptide sequences. For expression of DSM14391 and CP474, the B. lentus subtilisin propeptide (SEQ ID NO: 40) was used instead, but the naturally occurring propeptide sequences for these protease are listed on Table 1. Prior references and accession numbers for the various subtilisin parent backbones of this study are provided on Table 1. The DNA sequence encoding the expression cassette for the pro-mature polypeptide for each parental backbone is listed on Table 1, along with the pro-peptide and predicted mature protein sequences for each protease.

TABLE 1
List of subtilisin backbones used for evaluation of variants with improved stability.
Prior patent references and sequence ID NOs, or accession numbers are provided.
	SEQ ID Nos
			pro-	mature
			peptide	protein
	Prior patent references or	DNA	AA	AA
Subtilisin	accession number	sequence	sequence	sequence
AprE (subtilisin E)	WP_003233171	24	41	8
WP_082194748	WP_082194748 [formerly listed as	25	42	9
	WP_008359041]
Chemgen_164A	SEQ ID NO: 2 in U.S. Pat. No.	26	43	10
	5,275,945
CP474	A variant of LG12 SprC protease	27	40	11
	(SprC is SEQ ID NO: 3 in
	WO2015/038792)
ZP-00454	A variant of WP_010192403	28	44	12
	(previously ZP_07707657)
	(ZP_07707657 is SEQ ID NO: 7 in
	WO2015/038792)
Bpan01744	SEQ ID NO: 3 in WO2016069563	29	45	13
DSM14391	SEQ ID NO: 13 in WO2018/118917	30	46	14
BspAK01305	SEQ ID NO: 6 in WO2016/069569	31	47	15
BspAI02518	SEQ ID NO: 3 in WO2015/089441	32	48	16
BspZ00056	SEQ ID NO: 9 in WO 2016/069544	33	49	17
Bad02409	SEQ ID NO: 13 in WO2010/69557	34	23	18
Bba02069	SEQ ID NO: 3 in WO2016/061438	35	39	19
BspZ00258	SEQ ID NO: 9 in WO 2016/069552	38	36	22

Example 2

Enzyme Assays

Protein Determination Assay: Culture supernatants were diluted into 10 mM NaCl, 0.1 mM CaCl₂), 0.005% Tween® 80 to a concentration that fits within the linear range of the standard curve for loading onto column. For high resolution concentration determinations, high performance liquid chromatography (HPLC) method was performed on protein samples. An Agilent 1100 HPLC equipped with an Agilent 300SB-C8 column was used for protein quantitation. Samples were eluted from the column using a gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile. Absorbance was measured at 220 nm, and peaks were integrated using ChemStation software (Agilent Technologies, USA). The protein concentrations of the samples were calculated based on a standard curve of a purified protease.

Alternatively, the concentration of the sample proteases in culture supernatant was determined by UHPLC using a Zorbax 300 SB-C3 column and linear gradient of 0.1% Trifluoroacetic acid (Buffer A) and 0.07% Trifluoroacetic acid in Acetonitrile (Buffer B) with absorbance detection at 220 nm. The protein concentration of the samples was calculated using a standard curve of the purified protease.

Protease Activity: The protease activity of parent and variants thereof was tested by measuring the hydrolysis of N-suc-AAPF-pNA substrate. For the AAPF assay, the reagent solutions used were: 100 mM Tris pH 8.6, 10 mM CalCl₂, 0.005% Tween®-80 (Tris/Ca buffer) and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a working solution, 1 mL suc-AAPF-pNA stock solution was added to 100 mL Tris/Ca buffer and mixed. An enzyme sample was added to a microtiter plate (MTP) containing 1 mg/mL suc-AAPF-pNA working solution and assayed for activity at 405 nm over 3-5 min using a SpectraMax plate reader in kinetic mode at room temperature (RT). The protease activity was expressed as mOD/min.

Cleaning performance assays: Detergents used in these studies include: Persil Small & Mighty Non-Bio Liquid Detergent “Persil Non-Bio” (PNB, Unilever) heavy duty liquid laundry (HDL) purchased in 2014 from local supermarket; GSM-B Phosphate-free automatic dishwashing (ADW) formula purchased without enzymes from WFK Testgewebe GmbH, Brüggen, Deutschland (www.testgewebe.de), composition listed on Table 2; and ECE2 heavy duty powder detergent (HDD) from WFK Testgewebe GmbH, Brüggen, Deutschland (www.testgewebe.de), composition listed on Table 3. Table 4 lists the conditions used for the cleaning performance assays. Persil Non-Bio Small & Mighty (Persil Non-Bio, PNB), is considered boron-free since it contains≤5 mg/Kg of boron, when tested for elemental boron content.

TABLE 2
Composition of GSM-B Phosphate-
Free Detergent (GSM-B, pH 10.5)
Component                        wt %
Sodium citrate dehydrate         30
Maleic acid/acrylic acid copolymer sodium Salt 12
(SOKALAN ® CP5, BASF)
Sodium perborate monohydrate     5
TAED                             2
Sodium disilicate: Protil A (Cognis) 25
Linear fatty alcohol ethoxylate  2
Sodium carbonate anhydrous       add to 100

TABLE 3
Composition of ECE-2 powder detergent (HDD)
                                 Weight
Component                        %
Linear sodium alkyl benzene sulfonate 9.7
Ethoxylated fatty alcohol C12-18 (7 EO) 5.2
Sodium soap                      3.6
Antifoam DC2-4248S               4.5
Sodium aluminum silicate zeolite 4A 32.5
Sodium carbonate                 11.8
Sodium salt of a copolymer from acrylic and maleic acid 5.2
(Sokalan CP5)
Sodium silicate (SiO2:Na2O = 3.3:1) 3.4
Carboxymethylcellulose           1.3
Diethylene triamine penta (methylene phosphonic acid) 0.8
Sodium sulfate                   9.8
Water                            12.2

TABLE 4
Conditions for subtilisin cleaning performance evaluations
			Hardness
		Final Wash	Conc.
Detergent	Type	Conc, (g/L)	(ppm)	Buffer	pH
Persil Non-Bio	HDL	2.7	250	5 mM	8.2
(PNB)				HEPES
GSM-B	ADW	3.0	374	not buffered	10.5
ECE-2	HDD	6.5	374	not buffered	10

Subtilisin variants were tested for cleaning performance relative to each parent backbone on BMI (EMPA-116, blood/milk/ink on cotton) for laundry-based HDL and HDD applications, and on egg yolk (PAS-38, egg yolk on polyacryl fabric, aged and colored with carbon black dye) for dish-based applications in 96 well (MTP) microtiter plates. PAS-38 swatches and pre-rinsed EMPA116 were purchased from Center for Testmaterials B.V., Vlaardingen. For all stains, pre-punched swatches in MTP plates (Costar 9017 or Greiner 655101) were prepared. These microswatch-containing plates were filled with detergent solution (listed on Table 4) prior to enzyme addition. Aliquots of enzyme were added to detergent-filled MTPs containing microswatches to reach a final volume of 180 microliters for laundry and ADW assays. Laundry cleaning assays with HDL and HDD formulas were carried out at 25° C. for 20 min, while ADW assays were carried out at 40° C. for 30 min. Following incubation, 100-150 microliters of supernatant was transferred to a fresh MTP and absorbance was read at 405 nm using a SpectraMax plate reader. Absorbance results were obtained by subtracting the value for a blank control (no enzyme) from each sample value. For each condition and subtilisin variant in Example 3, a cleaning performance index (PI) was calculated by dividing the blank subtracted absorbance of the variant by that of the respective parent protease at the same concentration. The blank subtracted absorbance value for the parent protease at the corresponding concentration of the variant was determined using a standard curve of the parent protease, which was included in the test and was generated using a Langmuir fit or Hill Sigmoidal fit, as appropriate. Results for each subtilisin variant sample were compared to the results for the parent molecule in each assay plate to generate a normalized PI and mitigate plate to plate variation.

Detergent Stability Assay: Subtilisin enzymes were tested for stability in 10% PNB detergent (10-fold dilution of commercial detergent) at temperatures specified on Table 5 to determine the residual activity following incubation at elevated temperature. The elevated temperature was set to enable discrimination of residual activity of the stressed sample compared to the unstressed sample during an incubation period of 20 minutes in a range appropriate to discern differences of variant enzymes versus their parent. A diluted enzyme sample was mixed in appropriate detergent and the protease activity on AAPF substrate was measured immediately, to serve as the unstressed value. The samples were subsequently placed in a PCR plate, sealed and incubated at elevated temperature for 20 min using a thermocycler, then assayed for AAPF activity to obtain the stressed value. Assays were carried out in 384 well MTPs. The residual activity was calculated by dividing the stressed activity by the unstressed activity for each enzyme. In some instances, the relative stability of the variant enzymes is reported at a performance index (PI), and in other instances it is reported at a percent residual activity (% RA). The stability Performance Index (PI) for each variant under each assay condition was obtained by dividing the residual activity of the variant by the residual activity of the parent wild type. The percent residual activity for each variant under each assay condition was obtained by dividing the AAPF activity absorbance for stressed sample by the AAPF activity absorbance for unstressed sample and multiplying by 100.

TABLE 5
Stress Conditions for Stability Tests in 10% PNB detergent
                    20 min incubation at
Subtilisin Backbone Temperature (° C.)
AprE                41-42
WP_082194748        46
Chemgen_164A        51-56
CP474               37
ZP-00454            42
Bpan01744           44-46
DSM14391            30-36
BspAI02518          30-36
BspZ00056           63-64
Bad02409            67
Bba02069            39-40
BspAK01305          47
BspZ00258           62

Example 3

AprE & WP_082194748 Subtilisin Variants with Improved Stability in Detergent

Variants of AprE (Subtilisin E, SEQ ID NO: 8) and WP_082194748 (SEQ ID NO: 9) subtilisins were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the respective parent molecule. The AprE (Subtilisin E) and WP_082194748 subtilisin are more closely related in sequence to BPN′ subtilisin than to other known subtilisins (86.5% and 76.4% amino acid sequence identity, respectively).

Detergent stability results are reported as either performance index (PI) or % residual activity (% RA). Tables 6A and 6B show the stability results for AprE (Subtilisin E), and Table 8 for WP_082194748 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Tables 7 and 9 show the cleaning assays results for AprE variants in Table 6A and WP_082194748 variants in Table 8 respectively, having cleaning performance PI values 1.0 or greater for at least one condition when compared to the respective parent subtilisin, and also displaying stability PI values 1.1 or greater.

TABLE 6A
AprE subtilisin variants with improved stability in liquid detergent
at 41° C. (PI values ≥ 1.1) compared to AprE parent
AprE Variant	Substitutions in	Substitutions in	Stability
Sample ID	AprE numbering	BPN′ numbering	PI
AprE-00772	S003V	S003V	1.8
AprE-00795	N076D	N076D	2.0
AprE-00488	S078N	S078N	1.6
AprE-00944	G166Q	G166Q	1.9
AprE-00511	Y217L	Y217L	1.5
AprE-00924	N218S	N218S	1.7
AprE-00447	N259P	N259P	1.7
AprE-00370	S003V-N259P	S003V-N259P	2.0
AprE-00380	S003V-P040E	S003V-P040E	1.5
AprE-00413	S003V-M124I	S003V-M124I	1.8
AprE-00515	S003V-S078N	S003V-S078N	1.8
AprE-00729	S003V-N076D	S003V-N076D	2.0
AprE-00841	S003V-G166Q	S003V-G166Q	2.2
AprE-00907	S003V-G128S	S003V-G128S	1.8
AprE-00498	A069S-N076D	A069S-N076D	1.9
AprE-00594	A069S-N218S	A069S-N218S	1.6
AprE-00666	A069S-G166Q	A069S-G166Q	1.8
AprE-00758	A069S-N259P	A069S-N259P	1.5
AprE-00788	A069S-S078N	A069S-S078N	1.4
AprE-00943	A069S-G128S	A069S-G128S	1.4
AprE-00966	A069S-M124I	A069S-M124I	1.2
AprE-00416	N076D-G128S	N076D-G128S	2.5
AprE-00774	N076D-S078N	N076D-S078N	2.1
AprE-00912	N076D-N218S	N076D-N218S	2.3
AprE-00920	N076D-G166Q	N076D-G166Q	2.5
AprE-00946	N076D-M124I	N076D-M124I	2.3
AprE-00365	S078T-M124I	S078T-M124I	1.9
AprE-00655	S078N-G128S	S078N-G128S	2.0
AprE-00891	S078N-N259P	S078N-N259P	2.0
AprE-00904	S078N-N218S	S078N-N218S	2.0
AprE-00974	S078N-G166Q	S078N-G166Q	2.1
AprE-00442	M124I-G166Q	M124I-G166Q	1.9
AprE-00653	M124I-N259P	M124I-N259P	1.5
AprE-00770	M124I-G128S	M124I-G128S	1.5
AprE-00861	M124I-N218S	M124I-N218S	1.7
AprE-00732	G128S-N259P	G128S-N259P	1.6
AprE-00757	G128S-N218S	G128S-N218S	1.7
AprE-00824	G128S-G166Q	G128S-G166Q	2.0
AprE-00694	G166Q-N259P	G166Q-N259P	2.0
AprE-00698	G166Q-N218S	G166Q-N218S	2.1
AprE-00646	N218S-N259P	N218S-N259P	2.0

TABLE 6B
Additional AprE subtilisin variants with improved stability
in liquid detergent at 42° C., reported as percent
residual activity (% RA) compared to AprE parent
AprE Variant	Substitutions in	Substitutions in
Sample ID	AprE numbering	BPN′ numbering	% RA
AprE	—	—	27
AprE-01081	S009E	S009E	80
AprE-01080	P040E	P040E	100
AprE-01089	S003V-S009E	S003V-S009E	88
AprE-01078	S003V-A069S	S003V-A069S	42
AprE-01842	S003V-N218S	S003V-N218S	58
AprE-01082	S009E-P040E	S009E-P040E	82
AprE-01083	S009E-A069S	S009E-A069S	70
AprE-01025	S009E-N076D	S009E-N076D	96
AprE-01850	S009E-S078N	S009E-S078N	75
AprE-01959	S009E-G166Q	S009E-G166Q	90
AprE-01111	S009E-N218S	S009E-N218S	83
AprE-01096	S009E-N259P	S009E-N259P	84
AprE-01951	P040E-A069S	P040E-A069S	33
AprE-01108	P040E-N076D	P040E-N076D	72
AprE-01844	P040E-S078N	P040E-S078N	43
AprE-01107	P040E-G166Q	P040E-G166Q	75
AprE-01054	P040E-N218S	P040E-N218S	65
AprE-01105	P040E-N259P	P040E-N259P	48
AprE-01836	N076D-N259P	N076D-N259P	70

TABLE 7
AprE variants with cleaning performance on
par or improved compared to AprE parent
	Cleaning performance, PI
	AprE Variant	BMI stain in PNB	PAS-38 stain n GSM-
	Sample ID	detergent	B detergent
	AprE-00772	1.2	1.2
	AprE-00795	1.0	1.1
	AprE-00488	1.0	1.1
	AprE-00944	1.0	1.1
	AprE-00511	1.1	1.3
	AprE-00924	0.9	1.0
	AprE-00447	0.9	1.1
	AprE-00370	1.0	1.0
	AprE-00380	1.0	1.0
	AprE-00413	1.0	1.0
	AprE-00515	1.0	1.2
	AprE-00729	1.0	1.1
	AprE-00841	1.1	1.1
	AprE-00907	1.0	1.1
	AprE-00498	1.0	1.1
	AprE-00594	1.0	1.1
	AprE-00666	0.9	1.1
	AprE-00758	1.1	1.1
	AprE-00788	1.1	1.2
	AprE-00943	0.9	1.0
	AprE-00966	1.0	1.1
	AprE-00416	1.1	1.0
	AprE-00774	1.0	1.1
	AprE-00912	0.9	1.0
	AprE-00920	1.1	1.1
	AprE-00946	1.1	1.0
	AprE-00365	1.0	1.1
	AprE-00655	1.1	1.1
	AprE-00891	0.9	1.1
	AprE-00904	1.0	1.1
	AprE-00974	0.9	1.1
	AprE-00442	1.1	1.1
	AprE-00653	1.1	1.1
	AprE-00770	1.3	0.9
	AprE-00861	1.1	1.1
	AprE-00732	1.1	1.0
	AprE-00757	1.1	1.0
	AprE-00824	1.0	1.1
	AprE-00694	1.0	1.0
	AprE-00698	0.9	1.0
	AprE-00646	1.1	1.1

TABLE 8
WP_082194748 subtilisin variants with improved stability
in liquid detergent at 46° C. (PI values > 1.1)
compared to WP082194748 parent
	Substitutions in	Substitutions
WP_082194748	WP_082194748	in BPN′	Stability
Variant Sample ID	numbering	numbering	PI
WP_082194748-00179	Y217L	Y217L	1.2
WP_082194748-00103	S087D	S087D	1.2
WP_082194748-00040	T078N	T078N	1.4
WP_082194748-00446	G128S	G128S	1.4
WP_082194748-00466	G166Q	G166Q	2.1
WP_082194748-00047	S182E	S182E	2.5
WP_082194748-00571	G128S-V185Q	G128S-V185Q	1.1
WP_082194748-00612	A069S-G128S	A069S-G128S	1.1
WP_082194748-00380	T078N-V185Q	T078N-V185Q	1.3
WP_082194748-00025	V185Q-S259P	V185Q-S259P	1.5
WP_082194748-00461	G166Q-V185Q	G166Q-V185Q	1.6
WP_082194748-00441	T078N-S259P	T078N-S259P	1.6
WP_082194748-00099	T003V-G128S	T003V-G128S	1.7
WP_082194748-00306	T078N-G128S	T078N-G128S	1.7
WP_082194748-00582	G128S-S259P	G128S-S259P	1.8
WP_082194748-00486	G166Q-S259P	G166Q-S259P	1.9
WP_082194748-00242	A069S-M124I	A069S-M124I	1.9
WP_082194748-00458	T003V-M124I	T003V-M124I	1.9
WP_082194748-00546	M124I-S259P	M124I-S259P	1.9
WP_082194748-00275	M124I-V185Q	M124I-V185Q	2.0
WP_082194748-00299	T003V-S259P	T003V-S259P	2.1
WP_082194748-00066	A069S-S259P	A069S-S259P	2.1
WP_082194748-00415	M124I-G128S	M124I-G128S	2.1
WP_082194748-00340	A069S-G166Q	A069S-G166Q	2.1
WP_082194748-00038	T078N-M124I	T078N-M124I	2.2
WP_082194748-00060	M124I-G166Q	M124I-G166Q	2.5
WP_082194748-00259	T078N-G166Q	T078N-G166Q	2.5
WP_082194748-00297	T003V-G166Q	T003V-G166Q	2.8

TABLE 9
WP_082194748 variants with cleaning performance on
par or improved compared to WP_082194748 parent
	Cleaning performance, PI
		BMI stain	PAS-38 stain
	WP_082194748 Variant	in PNB	in GSM-B
	Sample ID	detergent	detergent
	WP_082194748-00179	1.0	1.0
	WP_082194748-00103	1.1	1.0
	WP_082194748-00040	1.0	1.0
	WP_082194748-00446	1.0	0.9
	WP_082194748-00466	1.0	1.0
	WP_082194748-00047	1.1	0.9
	WP_082194748-00571	1.1	0.9
	WP_082194748-00612	1.1	0.9
	WP_082194748-00380	1.0	1.1
	WP_082194748-00025	1.0	1.0
	WP_082194748-00461	1.0	1.0
	WP_082194748-00441	1.0	1.0
	WP_082194748-00099	0.8	1.0
	WP_082194748-00306	1.0	1.0
	WP_082194748-00582	1.1	0.9
	WP_082194748-00486	1.1	1.0
	WP_082194748-00242	1.1	0.9
	WP_082194748-00458	1.1	1.0
	WP_082194748-00546	0.9	1.0
	WP_082194748-00275	1.2	1.0
	WP_082194748-00299	0.8	1.0
	WP_082194748-00066	1.0	1.0
	WP_082194748-00340	1.0	1.0
	WP_082194748-00038	1.2	1.1
	WP_082194748-00060	1.0	1.0
	WP_082194748-00259	1.0	1.0
	WP_082194748-00297	0.9	1.0

Example 4

Chemgen_164A, CP474, & ZP-00454 Subtilisin Variants with Improved Stability in Detergent

Variants of Chemgen_164A (Chemgen, SEQ ID NO: 10), CP474 (SEQ ID NO: 11) and ZP-00454 (SEQ ID NO: 12) subtilisins were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the respective parent molecule. The Chemgen_164A, CP474, and ZP-00454 subtilisins share high sequence homology with the LG12 (SprC) protease (described in WO2015/038792), having amino acid sequence identities of 81.8%, 79.6% and 90.2%, respectively.

Detergent stability results are reported as either performance index (PI) or residual activity (% RA). Tables 10A and 10B, show the stability results for Chemgen_164A, Table 11 for CP474, and Table 12 for ZP-00454 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Tables 13, and 14 show the cleaning assay results for CP474 variants, and ZP-00454 variants respectively, having cleaning performance PI values 1.0 or greater when compared to the respective parent subtilisin, and also displaying stability PI values 1.1 or greater. Due to conditions of the assay, in some instances the PI values calculated were very large (due to parent subtilisin activity being at level of detection), therefore PI values greater than 4.0 are shown as ≥4.0.

TABLE 10A
Chemgen_164A subtilisin variants with improved
stability in liquid detergent at 56° C. (PI values ≥
1.1) compared to Chemgen_164A wildtype (parent)
	Substitutions in	Substitutions
Chemgen_164A	Chemgen_164A	in BPN′	Stability
variant Sample ID	numbering	numbering	PI
Chemgen-00272	T003V	T003V	3.1
Chemgen-00032	A069S	A069S	1.5
Chemgen-00626	N087D	N087D	3.2
Chemgen-00300	N118R	N118R	1.1
Chemgen-00173	S129P	S129P	1.8
Chemgen-00404	G166Q	G166Q	2.2
Chemgen-00591	S182E	S182E	2.0
Chemgen-00547	N218S	N218S	2.4
Chemgen-00076	T003V-N076D	T003V-N076D	3.1
Chemgen-00108	T003V-N185Q	T003V-N185Q	3.1
Chemgen-00196	T003V-G128S	T003V-G128S	2.9
Chemgen-00329	T003V-S129P	T003V-S129P	3.2
Chemgen-00378	T003V-G166Q	T003V-G166Q	3.2
Chemgen-00443	T003V-T078N	T003V-T078N	3.2
Chemgen-00481	T003V-A069S	T003V-A069S	3.2
Chemgen-00513	T003V-M124I	T003V-M124I	2.0
Chemgen-00634	T003V-N218S	T003V-N218S	3.2
Chemgen-00289	P040E-M124I	P040E-M124I	2.5
Chemgen-00262	A069S-G128S	A069S-G128S	1.2
Chemgen-00394	A069S-G166Q	A069S-G166Q	2.9
Chemgen-00436	A069S-T078N	A069S-T078N	3.2
Chemgen-00444	A069S-D259P	A069S-D259P	3.0
Chemgen-00525	A069S-S129P	A069S-S129P	2.0
Chemgen-00527	A069S-N076D	A069S-N076D	3.2
Chemgen-00569	A069S-N218S	A069S-N218S	2.6
Chemgen-00617	A069S-N185Q	A069S-N185Q	1.9
Chemgen-00132	N076D-G166Q	N076D-G166Q	3.1
Chemgen-00367	N076D-N218S	N076D-N218S	3.2
Chemgen-00369	N076D-M124I	N076D-M124I	3.2
Chemgen-00459	N076D-T078N	N076D-T078N	3.2
Chemgen-00546	N076D-D259P	N076D-D259P	3.2
Chemgen-00619	N076D-S129P	N076D-S129P	3.2
Chemgen-00621	N076D-G128S	N076D-G128S	3.2
Chemgen-00212	T078N-G128S	T078N-G128S	2.5
Chemgen-00224	T078N-N185Q	T078N-N185Q	3.1
Chemgen-00313	T078N-N218S	T078N-N218S	3.1
Chemgen-00350	T078N-D259P	T078N-D259P	3.2
Chemgen-00351	T078N-S129P	T078N-S129P	3.2
Chemgen-00413	T078N-M124I	T078N-M124I	1.5
Chemgen-00560	T078N-G166Q	T078N-G166Q	3.2
Chemgen-00115	G128S-N218S	G128S-N218S	2.1
Chemgen-00448	G128S-D259P	G128S-D259P	2.2
Chemgen-00517	G128S-N185Q	G128S-N185Q	1.1
Chemgen-00105	S129P-N185Q	S129P-N185Q	1.9
Chemgen-00375	S129P-G166Q	S129P-G166Q	3.2
Chemgen-00616	S129P-N218S	S129P-N218S	2.9
Chemgen-00010	G166Q-D259P	G166Q-D259P	3.1
Chemgen-00122	G166Q-N185Q	G166Q-N185Q	2.9
Chemgen-00335	G166Q-N218S	G166Q-N218S	3.2
Chemgen-00198	N185Q-N218S	N185Q-N218S	2.1
Chemgen-00359	N185Q-D259P	N185Q-D259P	2.3
Chemgen-00309	N218S-D259P	N218S-D259P	3.1

TABLE 10B
Chemgen_164A subtilisin variants with improved stability
in liquid detergent at 51° C. (reported as percent residual
activity, % RA) compared to Chemgen_164A wildtype (parent)
	Substitutions in	Substitutions
Chemgen_164A	Chemgen_164A	in BPN′
variant Sample ID	numbering	numbering	% RA
Chemgen_164A	—	—	31
Chemgen-00815	P040E	P040E	63
Chemgen-00718	N076D	N076D	60
Chemgen-00720	T078N	T078N	65
Chemgen-00723	N185Q	N185Q	34
Chemgen-00795	T003V-T009E	T003V-T009E	66
Chemgen-00794	T003V-P040E	T003V-P040E	100
Chemgen-00650	T003V-D259P	T003V-D259P	100
Chemgen-00788	T009E-A069S	T009E-A069S	36
Chemgen-00712	T009E-N076D	T009E-N076D	73
Chemgen-00714	T009E-T078N	T009E-T078N	57
Chemgen-00797	T009E-G166Q	T009E-G166Q	58
Chemgen-00798	T009E-N185Q	T009E-N185Q	36
Chemgen-00741	T009E-D259P	T009E-D259P	37
Chemgen-00715	P040E-T078N	P040E-T078N	84
Chemgen-01467	P040E-G166Q	P040E-G166Q	63
Chemgen-00716	P040E-N185Q	P040E-N185Q	100
Chemgen-01219	P040E-N218S	P040E-N218S	49
Chemgen-00804	N076D-N185Q	N076D-N185Q	94

TABLE 11
CP474 subtilisin variants with improved stability in liquid detergent
at 37° C. (PI values ≥ 1.1) compared to CP474 parent
		Substitutions in	Substitutions
	CP474 Variant	CP474	in BPN′	Stability
	Sample ID	numbering	numbering	PI
	CP474-00571	T003V	T003V	1.8
	CP474-00581	A040E	A040E	≥4.0
	CP474-00591	A069S	A069S	1.3
	CP474-00601	T078N	T078N	1.8
	CP474-00611	T079I	T079I	≥4.0
	CP474-00631	S162Q	S166Q	1.2
	CP474-00562	N181Q	N185Q	1.4
	CP474-00563	T003V-S162Q	T003V-S166Q	2.5
	CP474-00573	T003V-N181Q	T003V-N185Q	2.1
	CP474-00583	T003V-N214S	T003V-N218S	1.6
	CP474-00592	T003V-A040E	T003V-A040E	≥4.0
	CP474-00593	T003V-D255P	T003V-D259P	1.7
	CP474-00602	T003V-A069S	T003V-A069S	2.3
	CP474-00612	T003V-T078N	T003V-T078N	2.5
	CP474-00622	T003V-T079I	T003V-T079I	≥4.0
	CP474-00632	T003V-L124I	T003V-L124I	1.5
	CP474-00564	A040E-S162Q	A040E-S166Q	≥4.0
	CP474-00574	A040E-N181Q	A040E-N185Q	≥4.0
	CP474-00584	A040E-N214S	A040E-N218S	≥4.0
	CP474-00594	A040E-D255P	A040E-D259P	≥4.0
	CP474-00603	A040E-A069S	A040E-A069S	≥4.0
	CP474-00613	A040E-T078N	A040E-T078N	≥4.0
	CP474-00623	A040E-T079I	A040E-T079I	≥4.0
	CP474-00633	A040E-L124I	A040E-L124I	≥4.0
	CP474-00565	A069S-N181Q	A069S-N185Q	1.4
	CP474-00604	A069S-T078N	A069S-T078N	1.5
	CP474-00614	A069S-T079I	A069S-T079I	≥4.0
	CP474-00634	A069S-S162Q	A069S-S166Q	1.5
	CP474-00566	T078N-D255P	T078N-D259P	1.6
	CP474-00595	T078N-T079I	T078N-T079I	≥4.0
	CP474-00615	T078N-S162Q	T078N-S166Q	2.2
	CP474-00625	T078N-N181Q	T078N-N185Q	2.0
	CP474-00635	T078N-N214S	T078N-N218S	1.5
	CP474-00576	T079I-L124I	T079I-L124I	≥4.0
	CP474-00586	T079I-S162Q	T079I-S166Q	≥4.0
	CP474-00596	T079I-N181Q	T079I-N185Q	≥4.0
	CP474-00606	T079I-N214S	T079I-N218S	≥4.0
	CP474-00616	T079I-D255P	T079I-D259P	≥4.0
	CP474-00597	S162Q-N214S	S166Q-N218S	1.4
	CP474-00579	N181Q-D255P	N185Q-D259P	1.4
	CP474-00617	N181Q-N214S	N185Q-N218S	1.3
	CP474-00627	N214S-D255P	N218S-D259P	1.1

TABLE 12
ZP-00454 subtilisin variants with improved stability in liquid detergent
at 42° C. (PI values ≥ 1.1) compared to ZP-00454 parent
	Substitutions in	Substitutions
ZP-00454 Variant	ZP-00454	in BPN′	Stability
Sample ID	numbering	numbering	PI
ZP-00454-00011	A040E	A040E	2.1
ZP-00454-00021	A069S	A069S	1.1
ZP-00454-00031	N076D	N076D	1.7
ZP-00454-00041	T078N	T078N	1.5
ZP-00454-00051	T079I	T079I	2.1
ZP-00454-00071	I128S	I128S	1.2
ZP-00454-00013	A040E-M124I	A040E-M124I	2.0
ZP-00454-00023	A040E-I128S	A040E-I128S	1.7
ZP-00454-00033	A040E-S129P	A040E-S129P	2.1
ZP-00454-00043	A040E-G166Q	A040E-G166Q	2.1
ZP-00454-00052	A040E-A069S	A040E-A069S	2.2
ZP-00454-00053	A040E-N185Q	A040E-N185Q	2.3
ZP-00454-00062	A040E-N076D	A040E-N076D	2.2
ZP-00454-00063	A040E-N218S	A040E-N218S	2.1
ZP-00454-00072	A040E-T078N	A040E-T078N	2.1
ZP-00454-00073	A040E-D259P	A040E-D259P	2.0
ZP-00454-00004	A069S-N076D	A069S-N076D	1.7
ZP-00454-00005	A069S-N218S	A069S-N218S	1.2
ZP-00454-00014	A069S-T078N	A069S-T078N	1.7
ZP-00454-00024	A069S-T079I	A069S-T079I	2.0
ZP-00454-00044	A069S-I128S	A069S-I128S	1.2
ZP-00454-00054	A069S-S129P	A069S-S129P	1.2
ZP-00454-00074	A069S-N185Q	A069S-N185Q	1.2
ZP-00454-00006	N076D-N185Q	N076D-N185Q	1.6
ZP-00454-00016	N076D-N218S	N076D-N218S	1.6
ZP-00454-00025	N076D-T078N	N076D-T078N	1.8
ZP-00454-00026	N076D-D259P	N076D-D259P	1.5
ZP-00454-00035	N076D-T079I	N076D-T079I	2.1
ZP-00454-00045	N076D-M124I	N076D-M124I	1.3
ZP-00454-00055	N076D-I128S	N076D-I128S	1.8
ZP-00454-00065	N076D-S129P	N076D-S129P	1.6
ZP-00454-00075	N076D-G166Q	N076D-G166Q	1.8
ZP-00454-00007	T078N-N185Q	T078N-N185Q	1.5
ZP-00454-00027	T078N-D259P	T078N-D259P	1.4
ZP-00454-00036	T078N-T079I	T078N-T079I	2.0
ZP-00454-00056	T078N-I128S	T078N-I128S	1.7
ZP-00454-00066	T078N-S129P	T078N-S129P	1.5
ZP-00454-00076	T078N-G166Q	T078N-G166Q	1.6
ZP-00454-00008	T079I-N218S	T079I-N218S	2.1
ZP-00454-00018	T079I-D259P	T079I-D259P	1.9
ZP-00454-00037	T079I-M124I	T079I-M124I	2.4
ZP-00454-00047	T079I-I128S	T079I-I128S	2.1
ZP-00454-00057	T079I-S129P	T079I-S129P	2.0
ZP-00454-00067	T079I-G166Q	T079I-G166Q	2.0
ZP-00454-00077	T079I-N185Q	T079I-N185Q	2.0
ZP-00454-00009	I128S-S129P	I128S-S129P	1.1
ZP-00454-00059	S129P-G166Q	S129P-G166Q	1.1

TABLE 13
CP474 variants with cleaning performance on
par or improved compared to CP474 parent
CP474 Variant Cleaning Performance PI,
Sample ID     BMI stain PNB detergent
CP474-00591   1.1
CP474-00601   1.0
CP474-00611   1.1
CP474-00562   1.0
CP474-00573   1.2
CP474-00583   1.1
CP474-00592   1.0
CP474-00593   1.1
CP474-00602   1.2
CP474-00612   1.2
CP474-00622   1.2
CP474-00574   1.2
CP474-00584   1.1
CP474-00594   1.1
CP474-00603   1.2
CP474-00613   1.1
CP474-00623   1.2
CP474-00565   1.2
CP474-00604   1.3
CP474-00614   1.4
CP474-00634   1.0
CP474-00595   1.3
CP474-00625   1.2
CP474-00635   1.1
CP474-00586   1.2
CP474-00596   1.2
CP474-00606   1.2
CP474-00616   1.1
CP474-00597   1.1
CP474-00617   1.3
CP474-00627   1.0

TABLE 14
ZP-00454 variants with cleaning performance on
par or improved compared to ZP-00454 parent
ZP-00454 Variant Cleaning Performance PI,
Sample ID        BMI stain PNB detergent
ZP-00454-00031   1.0
ZP-00454-00041   1.1
ZP-00454-00051   2.1
ZP-00454-00033   1.0
ZP-00454-00043   1.0
ZP-00454-00052   1.1
ZP-00454-00053   1.0
ZP-00454-00024   1.1
ZP-00454-00044   1.0
ZP-00454-00054   1.1
ZP-00454-00074   1.0
ZP-00454-00006   1.0
ZP-00454-00016   1.1
ZP-00454-00025   1.1
ZP-00454-00026   1.0
ZP-00454-00035   1.8
ZP-00454-00055   1.0
ZP-00454-00065   1.0
ZP-00454-00075   1.0
ZP-00454-00027   1.0
ZP-00454-00036   1.0
ZP-00454-00056   1.0
ZP-00454-00076   1.0
ZP-00454-00018   1.5
ZP-00454-00037   1.0
ZP-00454-00057   1.7
ZP-00454-00067   2.2
ZP-00454-00077   1.4
ZP-00454-00059   1.0

Example 5

Bpan01744 Subtilisin Variants with Improved Stability in Detergent

Variants of Bpan01744 (SEQ ID NO: 13) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The Bpan01744 wildtype subtilisin was described as SEQ ID NO: 3 in patent application WO2016069563.

Detergent stability results are reported as either performance index (PI) or % residual activity (% RA). Tables 15A and 15B show the stability results for Bpan01744 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Table 16 shows the cleaning assays results for Bpan01744 variants from Table 15A having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater. Due to conditions of the assay, in some instances the PI values calculated were very large (due to parent subtilisin activity being at level of detection), therefore PI values greater than 4.0 are shown as ≥4.0.

TABLE 15A
Bpan01744 subtilisin variants with improved stability in liquid detergent
at 46° C. (PI values ≥ 1.1) compared to Bpan01744 parent
	Substitutions in	Substitutions
Bpan01744	Bpan01744	in BPN′	Stability
Variant Sample ID	numbering	numbering	PI
Bpan01744-00498	S003V	S003V	1.8
Bpan01744-00574	S076N	S078N	2.4
Bpan01744-00152	S003V-N179Q	S003V-N185Q	3.1
Bpan01744-00166	S003V-N074D	S003V-N076D	≥4.0
Bpan01744-00167	S003V-A067S	S003V-A069S	1.9
Bpan01744-00304	S003V-S076N	S003V-S078N	3.5
Bpan01744-00417	S003V-N212S	S003V-N218S	≥4.0
Bpan01744-00544	S003V-N253P	S003V-N259P	2.0
Bpan01744-00088	A067S-N074D	A069S-N076D	≥4.0
Bpan01744-00153	A067S-G160Q	A069S-G166Q	3.8
Bpan01744-00414	A067S-N179Q	A069S-N185Q	1.7
Bpan01744-00423	A067S-S076N	A069S-S078N	2.6
Bpan01744-00488	A067S-N212S	A069S-N218S	3.6
Bpan01744-00337	N074D-S076N	N076D-S078N	≥4.0
Bpan01744-00554	N074D-S127P	N076D-S129P	≥4.0
Bpan01744-00158	S076N-G160Q	S078N-G166Q	≥4.0
Bpan01744-00179	S076N-M122I	S078N-M124I	2.1
Bpan01744-00258	S076N-S127P	S078N-S129P	3.8
Bpan01744-00121	M122I-N212S	M124I-N218S	≥4.0
Bpan01744-00362	M122I-S127P	M124I-S129P	≥4.0
Bpan01744-00550	M122I-N179Q	M124I-N185Q	1.6
Bpan01744-00238	S127P-N253P	S129P-N259P	1.9
Bpan01744-00251	S127P-N179Q	S129P-N185Q	2.4
Bpan01744-00282	S127P-N212S	S129P-N218S	≥4.0
Bpan01744-00545	S127P-G160Q	S129P-G166Q	3.3
Bpan01744-00048	G160Q-N253P	G166Q-N259P	3.1
Bpan01744-00172	G160Q-N212S	G166Q-N218S	≥4.0
Bpan01744-00369	G160Q-N179Q	G166Q-N185Q	3.1
Bpan01744-00094	N179Q-N212S	N185Q-N218S	≥4.0
Bpan01744-00339	N179Q-N253P	N185Q-N259P	1.6
Bpan01744-00126	N212S-N253P	N218S-N259P	3.5

TABLE 15B
Bpan01744 subtilisin variants with improved stability
in liquid detergent at 44° C. (reported as percent
residual activity, % RA) compared to Bpan01744 parent
	Substitutions in	Substitutions
Bpan01744	Bpan01744	in BPN′
Variant Sample ID	numbering	numbering	% RA
Bpan01744	—	—	19
Bpan01744-02141	S009E	S009E	36
Bpan01744-01310	N074D	N076D	75
Bpan01744-00717	N179Q	N185Q	23
Bpan01744-00892	N212S	N218S	47
Bpan01744-01737	N253P	N259P	27
Bpan01744-00830	S003V-S009E	S003V-S009E	58
Bpan01744-01145	S003V-G160Q	S003V-G166Q	77
Bpan01744-01257	S009E-N074D	S009E-N076D	83
Bpan01744-02075	S009E-S076N	S009E-S078N	57
Bpan01744-01122	S009E-G160Q	S009E-G166Q	62
Bpan01744-01123	S009E-N179Q	S009E-N185Q	33
Bpan01744-00795	S009E-N212S	S009E-N218S	69
Bpan01744-01124	S009E-N253P	S009E-N259P	29
Bpan01744-01035	N074D-G160Q	N076D-G166Q	74
Bpan01744-01133	N074D-N179Q	N076D-N185Q	76
Bpan01744-01135	N074D-N212S	N076D-N218S	92
Bpan01744-01148	N074D-N253P	N076D-N259P	81
Bpan01744-01629	S076N-N179Q	S078N-N185Q	37
Bpan01744-00991	S076N-N212S	S078N-N218S	71
Bpan01744-01151	S076N-N253P	S078N-N259P	37

TABLE 16
Bpan01744 variants with cleaning performance on
par or improved compared to Bpan01744 parent
	Cleaning Performance PI
		BMI stain	PAS-38 stain	BMI stain
	BpaN01744 Variant	PNB	GSM-B	ECE-2
	Sample ID	detergent	detergent	detergent
	Bpan01744-00498	1.0	1.0	0.9
	Bpan01744-00574	0.9	1.1	1.0
	Bpan01744-00166	1.0	0.9	0.9
	Bpan01744-00167	1.0	1.0	0.9
	Bpan01744-00304	1.0	1.0	1.0
	Bpan01744-00417	1.0	0.9	0.7
	Bpan01744-00544	0.9	1.1	0.9
	Bpan01744-00088	1.0	0.9	0.9
	Bpan01744-00414	0.9	1.0	0.9
	Bpan01744-00423	1.0	0.9	1.0
	Bpan01744-00488	0.9	1.0	0.8
	Bpan01744-00337	1.0	1.0	0.9
	Bpan01744-00179	1.0	0.9	0.7
	Bpan01744-00258	1.0	0.9	0.9
	Bpan01744-00550	1.0	0.9	0.7
	Bpan01744-00238	1.0	0.9	0.9
	Bpan01744-00251	1.0	1.0	0.9
	Bpan01744-00339	1.0	1.1	1.0
	Bpan01744-00126	0.9	1.0	0.8

Example 6

DSM14391 Subtilisin Variants with Improved Stability in Detergent

Variants of DSM14391 (SEQ ID NO: 14) subtilisin were evaluated for stability using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The B. gibsonii subtilisin DSM14391 (previously described in SEQ ID NO:13 of patent application WO2018118917) shares high sequence homology with B. gibsonii subtilisin Bgi02446 (described previously as SEQ ID NO:11 of patent application WO2018118917) with 90% amino acid sequence identity.

Detergent stability results are reported as either performance index (PI) or % residual activity (% RA). Tables 17A and 17B show the stability results for DSM14391 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Due to conditions of the assay, in some instances the PI values calculated were very large (due to parent subtilisin activity being at level of detection), therefore PI values greater than 4.0 are shown as >4.0.

TABLE 17A
DSM14391 subtilisin variants with improved stability in liquid detergent
at 36° C. (PI values ≥ 1.1) compared to DSM14391 parent
	Substitutions in	Substitutions
DSM14391 Variant	DSM14391	in BPN′	Stability
Sample ID	numbering	numbering	PI
DSM14391-00436	T003V	T003V	1.2
DSM14391-00091	T009E	T009E	≥4.0
DSM14391-00382	S024Q	S024Q	1.2
DSM14391-00475	S039E	S040E	≥4.0
DSM14391-00098	A067S	A069S	1.5
DSM14391-00189	N074D	N076D	≥4.0
DSM14391-00257	S076N	S078N	2.6
DSM14391-00286	A128S	A130S	1.4
DSM14391-00409	G160Q	G166Q	≥4.0
DSM14391-00289	Q176E	Q182E	3.3
DSM14391-00248	R179Q	R185Q	≥4.0
DSM14391-00202	P212S	P218S	≥4.0
DSM14391-00058	N242D	N248D	1.2
DSM14391-00504	N253P	N259P	1.5
DSM14391-00016	T003V-R179Q	T003V-R185Q	≥4.0
DSM14391-00025	T003V-A067S	T003V-A069S	2.2
DSM14391-00028	T003V-G160Q	T003V-G166Q	≥4.0
DSM14391-00063	T003V-N253P	T003V-N259P	2.1
DSM14391-00340	T003V-N074D	T003V-N076D	≥4.0
DSM14391-00373	T003V-S076N	T003V-S078N	3.0
DSM14391-00492	T003V-P212S	T003V-P218S	≥4.0
DSM14391-00217	S039E-S076N	S040E-S078N	≥4.0
DSM14391-00005	A067S-S076N	A069S-S078N	3.4
DSM14391-00096	A067S-N253P	A069S-N259P	2.5
DSM14391-00136	A067S-R179Q	A069S-R185Q	≥4.0
DSM14391-00432	A067S-P212S	A069S-P218S	≥4.0
DSM14391-00494	A067S-N074D	A069S-N076D	≥4.0
DSM14391-00007	N074D-S076N	N076D-S078N	≥4.0
DSM14391-00168	N074D-P212S	N076D-P218S	≥4.0
DSM14391-00230	N074D-D127P	N076D-D129P	≥4.0
DSM14391-00104	S076N-D127P	S078N-D129P	3.0
DSM14391-00113	S076N-N253P	S078N-N259P	3.7
DSM14391-00147	S076N-M122I	S078N-M124I	≥4.0
DSM14391-00252	S076N-P212S	S078N-P218S	≥4.0
DSM14391-00302	S076N-R179Q	S078N-R185Q	≥4.0
DSM14391-00335	M122I-G160Q	M124I-G166Q	≥4.0
DSM14391-00355	M122I-P212S	M124I-P218S	≥4.0
DSM14391-00507	M122I-N253K	M124I-N259K	≥4.0
DSM14391-00021	D127P-P212S	D129P-P218S	≥4.0
DSM14391-00122	D127P-G160Q	D129P-G166Q	≥4.0
DSM14391-00367	D127P-R179Q	D129P-R185Q	1.3
DSM14391-00438	D127P-N253P	D129P-N259P	2.3
DSM14391-00171	G160Q-R179Q	G166Q-R185Q	≥4.0
DSM14391-00430	G160Q-N253P	G166Q-N259P	≥4.0
DSM14391-00161	R179Q-P212S	R185Q-P218S	≥4.0
DSM14391-00203	R179Q-N253P	R185Q-N259P	≥4.0

TABLE 17B
DSM14391 subtilisin variants with improved stability
in liquid detergent at 30° C. (reported as percent
residual activity, % RA) compared to DSM14391 parent
	Substitutions in	Substitutions
DSM14391 Variant	DSM14391	in BPN′
Sample ID	numbering	numbering	% RA
DSM14391	—	—	40
DSM14391-00986	T003V-T009E	T003V-T009E	80
DSM14391-00987	T003V-S039E	T003V-S040E	74
DSM14391-00863	T009E-S039E	T009E-S040E	98
DSM14391-00864	T009E-A067S	T009E-A069S	86
DSM14391-00839	T009E-N074D	T009E-N076D	91
DSM14391-00962	T009E-S076N	T009E-S078N	84
DSM14391-01005	T009E-R179Q	T009E-R185Q	94
DSM14391-00971	T009E-P212S	T009E-P218S	99
DSM14391-00867	T009E-N253P	T009E-N259P	76
DSM14391-00968	S039E-A067S	S040E-A069S	71
DSM14391-00969	S039E-N074D	S040E-N076D	83
DSM14391-00985	S039E-R179Q	S040E-R185Q	96
DSM14391-00977	S039E-P212S	S040E-P218S	95
DSM14391-00849	S039E-N253P	S040E-N259P	72
DSM14391-00974	N074D-R179Q	N076D-R185Q	100
DSM14391-00976	N074D-N253P	N076D-N259P	84
DSM14391-00828	G160Q-P212S	G166Q-P218S	96
DSM14391-00844	P212S-N253P	P218S-N259P	96

Example 7

BspAI02518 Subtilisin Variants with Improved Stability in Detergent

Variants of BspAI02518 (SEQ ID NO: 16) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The BspAI02518 subtilisin was previously described as SEQ ID NO: 3 in WO2015089441 patent application, and is a member of the B. akibai/clarkii clade of subtilisins.

Detergent stability results are reported as either performance index (PI) or residual activity (% RA). Tables 18A and 18B show the stability results for BspAI02518 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Table 19 shows the cleaning assays results for BspAI02518 variants from Table 18A having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater. Due to conditions of the assay, in some instances the PI values calculated were very large (due to parent subtilisin activity being at level of detection), therefore PI values greater than 4.0 are shown as ≥4.0.

TABLE 18A
BspAI02518 subtilisin variants with improved stability in liquid detergent
at 36° C. (PI values ≥ 1.1) compared to BspAI02518 parent
	Substitutions in	Substitutions
BspAI02518 Variant	BspAI02518	in BPN′	Stability
Sample ID	numbering	numbering	PI
BspAI02518-00709	S003V	S003V	2.7
BspAI02518-00637	S009E	S009E	2.3
BspAI02518-00689	N074D	N076D	≥4.0
BspAI02518-00585	S076N	S078N	2.8
BspAI02518-00621	M122I	M124I	1.3
BspAI02518-00941	S176E	S182E	2.2
BspAI02518-00764	N179Q	N185Q	1.6
BspAI02518-00819	N212S	N218S	3.5
BspAI02518-00838	N253P	N259P	1.3
BspAI02518-00506	S003V-N253P	S003V-N259P	2.6
BspAI02518-00564	S003V-N212S	S003V-N218S	≥4.0
BspAI02518-00599	S003V-A067S	S003V-A069S	1.9
BspAI02518-00624	S003V-N179Q	S003V-N185Q	3.3
BspAI02518-00664	S003V-G126S	S003V-G128S	2.2
BspAI02518-00784	S003V-M122I	S003V-M124I	2.7
BspAI02518-00897	S003V-N074D	S003V-N076D	≥4.0
BspAI02518-00959	S003V-S160Q	S003V-S166Q	≥4.0
BspAI02518-00405	A067S-N212S	A069S-N218S	3.1
BspAI02518-00412	A067S-N074D	A069S-N076D	≥4.0
BspAI02518-00590	A067S-S076N	A069S-S078N	2.2
BspAI02518-00646	A067S-N179Q	A069S-N185Q	1.3
BspAI02518-01025	A067S-S160Q	A069S-S166Q	2.7
BspAI02518-00535	N074D-N212S	N076D-N218S	≥4.0
BspAI02518-00695	N074D-N253P	N076D-N259P	≥4.0
BspAI02518-00732	N074D-G126S	N076D-G128S	≥4.0
BspAI02518-00772	N074D-M122I	N076D-M124I	≥4.0
BspAI02518-00802	N074D-N179Q	N076D-N185Q	≥4.0
BspAI02518-00814	N074D-S076N	N076D-S078N	≥4.0
BspAI02518-00625	S076N-M122I	S078N-M124I	2.8
BspAI02518-00800	S076N-N212S	S078N-N218S	≥4.0
BspAI02518-00937	S076N-N253P	S078N-N259P	2.6
BspAI02518-01001	S076N-G126S	S078N-G128S	2.7
BspAI02518-01032	S076N-N179Q	S078N-N185Q	3.4
BspAI02518-00594	M122I-G126S	M124I-G128S	1.9
BspAI02518-00749	M122I-S160Q	M124I-S166Q	3.1
BspAI02518-00758	M122I-N179Q	M124I-N185Q	1.5
BspAI02518-01002	S160Q-N212S	S166Q-N218S	≥4.0
BspAI02518-01006	S160Q-N253P	S166Q-N259P	3.3
BspAI02518-00439	N179Q-N253P	N185Q-N259P	1.7
BspAI02518-00548	N212S-N253P	N218S-N259P	4.0

TABLE 18B
BspAI02518 subtilisin variants with improved stability
in liquid detergent at 30° C. (reported as percent
residual activity, % RA) compared to BspAI02518 parent
	Substitutions in	Substitutions
BspAI02518 Variant	BspAI02518	in BPN′
Sample ID	numbering	numbering	% RA
BspAI02518	—	—	32
BspAI02518-01177	S160Q	S166Q	59
BspAI02518-01175	S003V-S009E	S003V-S009E	70
BspAI02518-01179	S003V-S076N	S003V-S078N	62
BspAI02518-01676	S009E-A067S	S009E-A069S	45
BspAI02518-01178	S009E-N074D	S009E-N076D	82
BspAI02518-01186	S009E-S076N	S009E-S078N	68
BspAI02518-01991	S009E-S160Q	S009E-S166Q	78
BspAI02518-01187	S009E-N179Q	S009E-N185Q	60
BspAI02518-01811	S009E-N212S	S009E-N218S	84
BspAI02518-02004	S009E-N253P	S009E-N259P	56
BspAI02518-01920	A067S-N253P	A069S-N259P	34
BspAI02518-01184	N074D-S160Q	N076D-S166Q	86
BspAI02518-01190	S076N-S160Q	S078N-S166Q	67
BspAI02518-02195	S160Q-N179Q	S166Q-N185Q	64
BspAI02518-01094	N179Q-N212S	N185Q-N218S	73

TABLE 19
BspAI02518 variants with cleaning performance on
par or improved compared to BspAI02518 parent
	Cleaning performance PIs
		BMI stain	PAS-38 stain	BMI stain
	BspAI02518 Variant	in PNB	in GSM-B	in ECE-2
	Sample ID	detergent	detergent	detergent
	BspAI02518-00709	1.1	1.0	1.0
	BspAI02518-00637	1.4	2.5	1.4
	BspAI02518-00689	1.0	1.0	0.8
	BspAI02518-00585	1.1	1.2	1.1
	BspAI02518-00621	1.0	1.2	1.1
	BspAI02518-00941	0.9	1.0	0.8
	BspAI02518-00764	0.9	1.2	1.1
	BspAI02518-00819	1.0	1.0	0.9
	BspAI02518-00838	1.1	1.0	1.0
	BspAI02518-00506	1.0	1.0	0.8
	BspAI02518-00564	1.0	0.9	1.0
	BspAI02518-00599	1.4	2.4	1.3
	BspAI02518-00624	1.1	1.6	1.1
	BspAI02518-00664	1.1	1.0	0.9
	BspAI02518-00784	1.0	1.0	0.9
	BspAI02518-00897	1.1	1.0	0.8
	BspAI02518-00959	1.0	1.3	1.3
	BspAI02518-00405	0.9	1.0	0.8
	BspAI02518-00412	1.1	2.5	1.4
	BspAI02518-00590	1.2	2.8	1.3
	BspAI02518-00646	1.0	2.0	1.0
	BspAI02518-01025	1.4	2.8	2.4
	BspAI02518-00695	1.0	1.0	0.8
	BspAI02518-00732	1.4	3.0	1.6
	BspAI02518-00772	1.0	1.0	0.9
	BspAI02518-00802	1.3	6.1	2.4
	BspAI02518-00814	1.1	1.1	1.0
	BspAI02518-00625	1.0	1.0	0.9
	BspAI02518-00800	0.9	1.0	0.9
	BspAI02518-01001	1.3	1.4	1.4
	BspAI02518-01032	1.3	1.7	1.3
	BspAI02518-00594	1.0	1.0	0.6
	BspAI02518-00749	1.0	1.0	0.8
	BspAI02518-00758	1.2	1.4	1.2
	BspAI02518-01002	1.1	1.0	1.0
	BspAI02518-00439	1.0	1.0	0.9

Example 8

Bad02409 Subtilisin Variants with Improved Stability in Detergent

Variants of Bad02409 (SEQ ID NO: 18) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule.

Table 20 shows the stability results for Bad02409 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Table 21 shows the cleaning assays results for Bad02409 variants having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater.

TABLE 20
Bad02409 subtilisin variants with improved stability in liquid detergent
at 67° C. (PI values ≥ 1.1) compared to Bad02409 parent
	Substitutions in
Bad02409 Variant	Bad02409	Substitutions in	Stability
Sample ID	numbering	BPN′ numbering	PI
Bad02409-00124	N078D	N076D	2.2
Bad02409-00592	S080N	S078N	1.5
Bad02409-00355	G130S	G128S	1.2
Bad02409-00369	S185E	S182E	1.5
Bad02409-00046	T003V-N221S	T003V-N218S	1.9
Bad02409-00328	T003V-A071S	T003V-A069S	1.8
Bad02409-00360	T003V-D262P	T003V-D259P	2.3
Bad02409-00398	T003V-M126I	T003V-M124I	2.4
Bad02409-00449	T003V-N078D	T003V-N076D	2.8
Bad02409-00462	T003V-N188Q	T003V-N185Q	1.4
Bad02409-00543	T003V-G169Q	T003V-G166Q	2.0
Bad02409-00583	T003V-S080N	T003V-S078N	2.0
Bad02409-00634	T003V-S131P	T003V-S129P	1.7
Bad02409-00262	A071S-S131P	A069S-S129P	1.1
Bad02409-00388	A071S-G169Q	A069S-G166Q	2.1
Bad02409-00394	A071S-N078D	A069S-N076D	2.4
Bad02409-00574	A071S-S080N	A069S-S078N	1.9
Bad02409-00077	N078D-S131P	N076D-S129P	3.0
Bad02409-00566	S080N-N188Q	S078N-N185Q	2.0
Bad02409-00564	M126I-S131P	M124I-S129P	1.2
Bad02409-00458	S131P-D262P	S129P-D259P	1.6
Bad02409-00567	S131P-G169Q	S129P-G166Q	1.7
Bad02409-00156	G169Q-N221S	G166Q-N218S	1.2

TABLE 21
Bad02409 variants with cleaning performance on
par or improved compared to Bad02409 parent
	Cleaning performance PI
	Bad02409 Variant	BMI stain in PNB	PAS-38 stain
	Sample ID	detergent	In GSM-B detergent
	Bad02409-00124	1.2	1.1
	Bad02409-00592	1.1	1.0
	Bad02409-00355	1.3	1.0
	Bad02409-00369	1.0	1.0
	Bad02409-00046	1.0	1.0
	Bad02409-00328	1.0	1.0
	Bad02409-00360	1.2	1.1
	Bad02409-00398	1.2	1.0
	Bad02409-00449	0.9	1.1
	Bad02409-00462	1.1	1.1
	Bad02409-00543	1.5	0.9
	Bad02409-00583	1.0	1.0
	Bad02409-00634	0.9	1.2
	Bad02409-00262	1.1	1.1
	Bad02409-00388	1.2	1.0
	Bad02409-00394	1.0	1.0
	Bad02409-00574	1.0	1.0
	Bad02409-00077	1.0	0.8
	Bad02409-00566	0.9	1.1
	Bad02409-00564	1.1	0.9
	Bad02409-00458	0.8	1.2
	Bad02409-00567	0.8	1.0
	Bad02409-00156	1.6	1.0

Example 9

Bba02069 Subtilisin Variants with Improved Stability in Detergent

Variants of Bba02069 (SEQ ID NO: 19) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The Bba02069 subtilisin was previously described as SEQ ID NO:3 in WO 2016/061438 patent application, and is a member of the B. agaradhaerens clade of subtilisins.

Detergent stability results are reported as either performance index (PI) or residual activity (% RA). Tables 22A and 22B show the stability results for Bba02069 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. The stability data shown on Table 22 was collected in two separate experiments, and PI values for certain variants evaluated on both occasions showed varying degree of improvements, as can be expected due to assay to assay fluctuations. Table 23 shows the cleaning assays results for Bba02069 variants from Table 22A having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater.

TABLE 22A
Bba02069 subtilisin variants with improved stability in liquid detergent
at 40° C. (PI values ≥ 1.1) compared to Bba02069 parent
	Substitutions in
Bba02069 Variant	Bba02069	Substitutions in	Stability
Sample ID	numbering	BPN′ numbering	PI
Bba02069-00641	S129P	S129P	1.3
Bba02069-00642	G118R	G118R	1.5
Bba02069-00643	Q087D	Q087D	1.5
Bba02069-00644	N076D	N076D	3.2
Bba02069-00646	M124I	M124I	2.6
Bba02069-00647	Q249D	Q248D	1.4
Bba02069-00648	A069S	A069S	1.9
Bba02069-00649	G128S	G128S	1.5
Bba02069-00650	N024Q	N024Q	1.1
Bba02069-00652	Q003V	Q003V	2.0
Bba02069-00653	P040E	P040E	1.3
Bba02069-00654	T009E	T009E	2.3
Bba02069-00655	N219S	N218S	3.1
Bba02069-00111	G167Q	G166Q	2.0
Bba02069-00217	S260P	S259P	1.7
Bba02069-00030	Q003V-A069S	Q003V-A069S	2.0
Bba02069-00121	Q003V-V186Q	Q003V-V185Q	2.0
Bba02069-00203	Q003V-S129P	Q003V-S129P	2.0
Bba02069-00291	Q003V-M124I	Q003V-M124I	2.8
Bba02069-00347	Q003V-G167Q	Q003V-G166Q	3.9
Bba02069-00400	Q003V-G128S	Q003V-G128S	2.4
Bba02069-00477	Q003V-S260P	Q003V-S259P	3.2
Bba02069-00564	Q003V-N076D	Q003V-N076D	2.4
Bba02069-00377	P040E-V186Q	P040E-V185Q	1.8
Bba02069-00499	P040E-G167Q	P040E-G166Q	3.1
Bba02069-00007	A069S-G128S	A069S-G128S	1.8
Bba02069-00114	A069S-S260P	A069S-S259P	2.1
Bba02069-00116	A069S-M124I	A069S-M124I	2.2
Bba02069-00505	A069S-G167Q	A069S-G166Q	2.5
Bba02069-00518	A069S-N076D	A069S-N076D	3.0
Bba02069-00118	N076D-G128S	N076D-G128S	2.4
Bba02069-00247	N076D-G167Q	N076D-G166Q	3.0
Bba02069-00281	N076D-M124I	N076D-M124I	3.1
Bba02069-00423	N076D-S260P	N076D-S259P	2.5
Bba02069-00473	N076D-S129P	N076D-S129P	3.2
Bba02069-00037	M124I-G128S	M124I-G128S	1.9
Bba02069-00254	M124I-V186Q	M124I-V185Q	1.8
Bba02069-00390	M124I-S129P	M124I-S129P	2.3
Bba02069-00461	M124I-S260P	M124I-S259P	3.2
Bba02069-00465	M124I-G167Q	M124I-G166Q	2.7
Bba02069-00568	M124I-N219S	M124I-N218S	3.3
Bba02069-00223	G128S-S129P	G128S-S129P	1.5
Bba02069-00399	G128S-G167Q	G128S-G166Q	2.6
Bba02069-00429	G128S-S260P	G128S-S259P	2.4
Bba02069-00483	G128S-V186Q	G128S-V185Q	1.6
Bba02069-00008	S129P-G167Q	S129P-G166Q	2.1
Bba02069-00133	S129P-V186Q	S129P-V185Q	1.4
Bba02069-00274	S129P-S260P	S129P-S259P	1.8
Bba02069-00043	G167Q-S260P	G166Q-S259P	2.6
Bba02069-00190	G167Q-V186Q	G166Q-V185Q	2.1
Bba02069-00005	V186Q-S260P	V185Q-S259P	2.1
Bba02069-00493	V186Q-N219S	V185Q-N218S	1.6
Bba02069-00051	N219S-S260P	N218S-S259P	1.9

TABLE 22B
Bba02069 subtilisin variants with improved stability
in liquid detergent at 39° C. (reported as percent
residual activity, % RA) compared to Bba02069 parent
	Substitutions in
Bba02069 Variant	Bba02069	Substitutions in
Sample ID	numbering	BPN′ numbering	% RA
Bba02069	—	—	23
Bba02069-00854	Q003V-T009E	Q003V-T009E	87
Bba02069-00855	Q003V-P040E	Q003V-P040E	56
Bba02069-00856	Q003V-N219S	Q003V-N218S	74
Bba02069-00755	T009E-P040E	T009E-P040E	72
Bba02069-00756	T009E-A069S	T009E-A069S	70
Bba02069-00757	T009E-N076D	T009E-N076D	100
Bba02069-00871	T009E-G167Q	T009E-G166Q	76
Bba02069-00866	T009E-V186Q	T009E-V185Q	67
Bba02069-00865	T009E-N219S	T009E-N218S	84
Bba02069-00758	T009E-S260P	T009E-S259P	90
Bba02069-00863	P040E-A069S	P040E-A069S	41
Bba02069-00824	P040E-N076D	P040E-N076D	77
Bba02069-01952	P040E-N219S	P040E-N218S	59
Bba02069-00845	P040E-S260P	P040E-S259P	64
Bba02069-00844	A069S-V186Q	A069S-V185Q	31
Bba02069-00736	A069S-N219S	A069S-N218S	61
Bba02069-00822	N076D-V186Q	N076D-V185Q	74
Bba02069-00815	N076D-N219S	N076D-N218S	80
Bba02069-00817	G167Q-N219S	G166Q-N218S	68

TABLE 23
Bba02069 variants with cleaning performance on
par or improved compared to Bba02069 parent
	Cleaning performance PI
		PAS-38 in
Bba02069 Variant	BMI in PNB	GSM-B	BMI in ECE-2
Sample ID	detergent	detergent	detergent
Bba02069-00111	0.8	1.0	0.9
Bba02069-00217	1.0	0.9	0.9
Bba02069-00121	0.8	1.0	1.0
Bba02069-00203	1.1	1.1	1.0
Bba02069-00291	0.7	1.4	0.8
Bba02069-00400	0.7	1.0	0.8
Bba02069-00377	1.2	1.0	0.8
Bba02069-00499	1.0	0.9	0.8
Bba02069-00114	1.1	0.9	1.1
Bba02069-00116	0.9	1.3	0.9
Bba02069-00505	1.0	0.8	0.9
Bba02069-00518	1.1	0.9	0.8
Bba02069-00118	1.1	1.0	1.0
Bba02069-00247	1.1	0.9	0.8
Bba02069-00281	1.5	1.1	0.7
Bba02069-00423	1.1	0.8	1.1
Bba02069-00473	1.2	0.9	1.2
Bba02069-00037	1.3	1.2	0.8
Bba02069-00254	1.1	1.2	0.7
Bba02069-00390	0.7	1.2	0.8
Bba02069-00461	0.9	1.1	0.9
Bba02069-00465	1.0	1.0	0.7
Bba02069-00568	0.9	1.1	0.6
Bba02069-00223	1.0	1.0	1.0
Bba02069-00399	1.0	0.9	0.8
Bba02069-00429	0.7	1.0	0.8
Bba02069-00483	0.9	0.9	1.0
Bba02069-00133	1.1	0.9	1.0
Bba02069-00274	1.2	1.0	1.2
Bba02069-00043	1.0	0.8	0.9
Bba02069-00190	0.8	1.0	0.9

Example 10

BspZ00056 Subtilisin Variants with Improved Stability in Detergent

Variants of BspZ00056 (SEQ ID NO: 17) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The BspZ00056 subtilisin was previously described as SEQ ID NO: 9 in WO 2016/069544 patent application, and is a member of the BspAP02013 clade of subtilisins.

Detergent stability results are reported as either performance index (PI) or residual activity (% RA). Tables 24A and 24B show the stability results for BspZ00056 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Table 25 shows the cleaning assays results for BspZ00056 variants from Table 24A having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater. PI values of less than 0.5 are denoted as <0.5.

TABLE 24A
BspZ00056 subtilisin variants with improved stability in liquid detergent
at 63° C. (PI values > 1.1) compared to BspZ00056 parent
	Substitutions in
BspZ00056 Variant	BspZ00056	Substitutions in	Stability
Sample ID	numbering	BPN′ numbering	PI
BspZ00056-00354	Q187E	Q182E	1.2
BspZ00056-00177	G132S	G128S	1.7
BspZ00056-00408	E149R	E145R	1.5
BspZ00056-00191	M128I	M124I	1.7
BspZ00056-00248	N223S	N218S	1.1
BspZ00056-00380	S133P	S129P	1.5
BspZ00056-00022	G132S-S133P	G128S-S129P	1.5
BspZ00056-00005	G132S-N223S	G128S-N218S	1.9
BspZ00056-00042	A073S-N190Q	A069S-N185Q	1.1
BspZ00056-00490	A073S-D082N	A069S-D078N	1.2
BspZ00056-00297	D082N-G171Q	D078N-G166Q	1.5
BspZ00056-00257	G171Q-N190Q	G166Q-N185Q	2.0
BspZ00056-00357	T003V-M128I	T003V-M124I	2.1
BspZ00056-00294	G132S-N190Q	G128S-N185Q	1.5
BspZ00056-00492	A073S-G132S	A069S-G128S	2.0
BspZ00056-00259	M128I-N190Q	M124I-N185Q	1.8
BspZ00056-00133	G171Q-G264P	G166Q-G259P	2.1
BspZ00056-00151	A073S-G264P	A069S-G259P	1.9
BspZ00056-00288	S133P-G171Q	S129P-G166Q	2.1
BspZ00056-00384	T003V-G264P	T003V-G259P	2.1
BspZ00056-00116	T003V-N190Q	T003V-N185Q	1.1
BspZ00056-00088	D082N-G264P	D078N-G259P	1.7
BspZ00056-00092	M128I-N223S	M124I-N218S	1.8
BspZ00056-00451	G132S-G171Q	G128S-G166Q	2.1
BspZ00056-00324	A073S-M128I	A069S-M124I	2.2
BspZ00056-00207	S133P-N190Q	S129P-N185Q	1.4
BspZ00056-00135	G132S-G264P	G128S-G259P	2.2
BspZ00056-00221	T003V-G171Q	T003V-G166Q	2.1
BspZ00056-00359	T003V-G132S	T003V-G128S	1.8
BspZ00056-00094	T003V-A073S	T003V-A069S	1.2
BspZ00056-00422	M128I-G171Q	M124I-G166Q	2.2
BspZ00056-00429	M128I-S133P	M124I-S129P	2.0
BspZ00056-00491	N223S-G264P	N218S-G259P	1.9
BspZ00056-00377	N190Q-G264P	N185Q-G259P	1.9
BspZ00056-00399	D082N-S133P	D078N-S129P	1.1
BspZ00056-00201	M128I-G132S	M124I-G128S	1.7
BspZ00056-00368	T003V-N223S	T003V-N218S	1.4
BspZ00056-00389	A073S-G171Q	A069S-G166Q	2.2

TABLE 24B
BspZ00056 subtilisin variants with improved stability
in liquid detergent at 64° C. (reported as percent
residual activity, % RA) compared to BspZ00056 parent
	Substitutions in
BspZ00056 Variant	BspZ00056	Substitutions in
Sample ID	numbering	BPN′ numbering	% RA
BspZ00056	—	—	30
BspZ00056-00876	T003V	T003V	35
BspZ00056-01485	P009E	P009E	33
BspZ00056-00863	A073S	A069S	42
BspZ00056-00864	G171Q	G166Q	69
BspZ00056-00663	N190Q	N185Q	38
BspZ00056-00819	G264P	G259P	80
BspZ00056-01478	P009E-G171Q	P009E-G166Q	69
BspZ00056-00875	P009E-G264P	P009E-G259P	83
BspZ00056-00873	A073S-N223S	A069S-N218S	44
BspZ00056-00837	G171Q-N223S	G166Q-N218S	80
BspZ00056-01030	N190Q-N223S	N185Q-N218S	35

TABLE 25
BspZ00056 variants with performance on par
or improved compared to BspZ00056 parent
	Cleaning performance PI
	BspZ00056 Variant	BMI stain in PNB	PAS-38 stain in
	Sample ID	detergent	GSM-B detergent
	BspZ00056-00354	1.1	0.8
	BspZ00056-00022	1.2	0.9
	BspZ00056-00005	1.2	0.9
	BspZ00056-00042	1.1	0.7
	BspZ00056-00490	1.1	0.9
	BspZ00056-00297	1.0	1.4
	BspZ00056-00257	1.0	1.3
	BspZ00056-00357	1.0	<0.5
	BspZ00056-00294	1.0	0.9
	BspZ00056-00492	1.0	0.5
	BspZ00056-00133	1.0	1.4
	BspZ00056-00288	0.9	1.2
	BspZ00056-00088	0.9	1.0
	BspZ00056-00451	0.8	1.2
	BspZ00056-00207	0.9	1.1
	BspZ00056-00221	0.9	1.4
	BspZ00056-00389	0.6	1.0

Example 11

BspAK01305 Subtilisin Variants with Improved Stability in Detergent

Variants of BspAK01305 (SEQ ID NO: 15) subtilisin were evaluated for stability and cleaning performance using methods described in Example 2, and performance index for each variant was calculated versus the parent molecule. The BspAK01305 subtilisin was previously described as SEQ ID NO:6 in WO 2016/069569 patent application, and is a member of the BspAL03279 clade of subtilisins.

Table 26 shows the stability results for BspAK01305 variants having stability PI values 1.1 and greater, where the cleaning performance index was at least 0.5 or greater (relative to parent) in at least one cleaning assay. Table 27 shows the cleaning assays results for BspAK01305 variants having cleaning performance PI values 1.0 or greater for at least one condition when compared to the parent subtilisin, and also displaying stability PI values 1.1 or greater.

TABLE 26
BspAK01305 subtilisin variants with improved stability in liquid
detergent at 47° C. (PI values ≥ 1.1) compared to BspAK01305 parent
	Substitutions in
BspAK01305 Variant	BspAK01305	Substitutions in	Stability
Sample ID	numbering	BPN′ numbering	PI
BspAK01305-00530	S003V	S003V	1.5
BspAK01305-00444	S039E	S040E	1.5
BspAK01305-00325	G160Q	G166Q	1.7
BspAK01305-00495	S179Q	S185Q	1.4
BspAK01305-00060	P204I	P210I	1.9
BspAK01305-00509	S003V-S179Q	S003V-S185Q	1.9
BspAK01305-00514	S003V-G160Q	S003V-G166Q	1.7
BspAK01305-00538	S003V-R256L	S003V-R262L	1.5
BspAK01305-00564	S003V-S039E	S003V-S040E	1.7
BspAK01305-00027	S039E-G160Q	S040E-G166Q	2.3
BspAK01305-00264	S039E-S179Q	S040E-S185Q	1.8
BspAK01305-00316	G160Q-S179Q	G166Q-S185Q	2.2

TABLE 27
BspAK01305 variants with performance on par
or improved compared to BspAK01305 parent
	Cleaning performance PI
	BspAK01305 Variant	BMI stain in PNB	PAS-38 stain in
	Sample ID	detergent	GSM-B detergent
	BspAK01305-00530	0.9	1.1
	BspAK01305-00444	0.9	1.5
	BspAK01305-00325	1.1	2.2
	BspAK01305-00060	1.1	1.1
	BspAK01305-00514	0.9	1.8
	BspAK01305-00564	1.3	1.2
	BspAK01305-00027	0.6	1.0
	BspAK01305-00264	1.0	0.9
	BspAK01305-00316	0.6	1.2

Example 12

Sequence Comparison and Structural Features of Subtilisin Sites Providing Enhanced Stability

A multiple sequence alignment of the mature (in some cases predicted) polypeptide regions of the subtilisin backbones evaluated in this study, as well as other subtilisins was generated using the following sequences: AprE (subtilisin E, SEQ ID NO:8); WP_082194748 (SEQ ID NO: 9); Chemgen_164A (SEQ ID NO:10); CP474 (SEQ ID NO:11); ZP-00454 (SEQ ID NO:12); Bpan01744 (SEQ ID NO:13); DSM14391 (SEQ ID NO:14); BspAK01305 (SEQ ID NO:15); BspAI02518 (SEQ ID NO:16); BspZ00056 (SEQ ID NO:17); Bad02409 (SEQ ID NO:18); Bba02069 (SEQ ID NO:19); BspZ00258 (SEQ ID NO:22); BPN′ (SEQ ID NO:1); B. licheniformis AprL (SEQ ID NO:2); B. lentus GG36 (SEQ ID NO:3); B. gibsonii Bgi02446 (SEQ ID NO:4); and Bacillus sp LG12 (SEQ ID NO:6). The multiple protein sequence alignments, shown in Table 28, were generated using structural (main chain) alignments from available protein crystal structures and amino acid sequence homology to guide positioning of loops in the sequences, and using BPN′ numbering (as assigned to all variants in this application, and used in the Examples above). Positions where insertions would occur using BPN′ sequence as a reference are numbered according to BPN′ and a suffix (example: position 42, 42a, 42b). The alignments shown correspond to residues 1-275 of BPN′, and additional C-terminal residues in some subtilisin backbones are not shown. An empty cell corresponds to a position where no amino acid can be assigned for that particular subtilisin sequence. The subtilisins on Table 28 with an asterisk (*) denote backbones not evaluated in this study, and shown here for reference.

TABLE 28
Structure-based multiple sequence alignment of various subtilisins.
BPN′
numbering	*BPN′	AprE	WP_082194748	*AprL	*LG12	Chemgen_164A	CP474	ZP-00454	*GG36
1	A	A	A	A	A	A	A	A	A
2	Q	Q	Q	Q	Q	Q	Q	Q	Q
3	S	S	T	T	T	T	T	V	S
4	V	V	V	V	V	T	V	V	V
5	P	P	P	P	P	P	P	P	P
6	Y	Y	Y	Y	W	W	W	W	W
7	G	G	G	G	G	G	G	G	G
8	V	I	I	I	I	I	I	I	I
9	S	S	P	P	P	T	P	P	S
10	Q	Q	Q	L	H	H	H	H	R
11	I	I	I	I	I	I	I	I	V
12	K	K	K	K	K	N	K	K	Q
13	A	A	A	A	A	A	A	A	A
14	P	P	P	D	D	H	D	D	P
15	A	A	A	K	K	K	K	K	A
16	L	L	V	V	A	A	A	A	A
17	H	H	H	Q	H	H	H	H	H
17a
18	S	S	A	A	A	S	A	A	N
19	Q	Q	Q	Q	A	S	A	S	R
20	G	G	G	G	G	S	G	G	G
21	Y	Y	Y	F	V	V	V	V	L
22	T	T	K	K	T	T	T	T	T
23	G	G	G	G	G	G	G	G	G
24	S	S	A	A	S	S	S	S	S
25	N	N	N	N	G	G	G	G	G
26	V	V	V	V	V	V	V	V	V
27	K	K	K	K	K	K	K	K	K
28	V	V	V	V	V	V	V	V	V
29	A	A	A	A	A	A	A	A	A
30	V	V	V	V	I	V	I	V	V
31	L	L	L	L	L	L	L	L	L
32	D	D	D	D	D	D	D	D	D
33	S	S	T	T	T	T	T	T	T
34	G	G	G	G	G	G	G	G	G
35	I	I	I	I	I	I	I	I	I
36	D	D	H	Q	D	D	D	D	S
37	S	S	A	A	A	A	A	A
38	S	S	A	S	N	S	N	N	T
39	H	H	H	H	H	H	H	H	H
40	P	P	P	P	A	P	A	A	P
41	D	D	D	D	D	D	D	D	D
42	L	L	L	L	L	L	L	L	L
42a
42b
43	K	N	N	N	N	N	N	N	N
44	V	V	V	V	V	V	V	V	I
45	A	R	A	V	K	K	K	K	R
46	G	G	G	G	G	G	G	G	G
47	G	G	G	G	G	G	G	G	G
48	A	A	A	A	A	A	A	A	A
49	S	S	S	S	S	S	S	S	S
50	M	F	F	F	F	F	F	F	F
51	V	V	V	V	V	I	V	V	V
52	P	P	P	A	S	S	A	S	P
53	S	S	S	G	G	G	G	G	G
54	E	E	E	E	E	E	E	E	E
55	T	T	P		P	P	P	P	P
56	N	N	N	A	N	N	N	N	S
56a
56b
57	P	P	A	Y	A	A	A	A
58	F	Y	T	N	L	L	L	L	T
59	Q	Q	Q	T	Q	V	Q	Q	Q
60	D	D	D	D	D	D	D	D	D
61	N	G	F	G	G	T	G	G	G
62	N	S	Q	N	N	N	N	N	N
63	S	S	S	G	G	G	G	G	G
64	H	H	H	H	H	H	H	H	H
65	G	G	G	G	G	G	G	G	G
66	T	T	T	T	T	T	T	T	T
67	H	H	H	H	H	H	H	H	H
68	V	V	V	V	V	V	V	V	V
69	A	A	A	A	A	A	A	A	A
70	G	G	G	G	G	G	G	G	G
71	T	T	T	T	T	T	T	T	T
72	V	I	I	V	V	V	V	V	I
73	A	A	A	A	A	A	A	A	A
74	A	A	A	A	A	A	A	A	A
75	L	L	L	L	L	L	L	L	L
76	N	N	D	D	N	N	D	N	N
77	N	N	N	N	N	N	N	N	N
78	S	S	T	T	T	T	T	T	S
79	I	I	I	T	T	I	T	T	I
80	G	G	G	G	G	G	G	G	G
81	V	V	V	V	V	V	V	V	V
82	L	L	L	L	L	V	L	L	L
83	G	G	G	G	G	G	G	G	G
84	V	V	V	V	V	V	V	V	V
85	A	A	A	A	A	A	A	A	A
86	P	P	P	P	Y	Y	P	Y	P
87	S	S	S	S	N	N	S	N	S
88	A	A	A	V	A	A	V	A	A
89	S	S	S	S	D	D	S	F	E
90	L	L	L	L	L	L	L	L	L
91	Y	Y	Y	Y	Y	Y	Y	Y	Y
92	A	A	A	A	A	A	A	A	A
93	V	V	V	V	V	V	V	V	V
94	K	K	K	K	K	K	K	K	K
95	V	V	V	V	V	V	V	V	V
96	L	L	L	L	L	L	L	L	L
97	G	D	D	N	S	S	G	G	G
98	A	S	R	S	A	A	A	A	A
99	D	T	N	S	S	S	S	S	S
100	G	G	G	G	G	G	G	G	G
101	S	S	D	S	S	S	S	S	S
102	G	G	G	G	G	G	G	G	G
103	Q	Q	Q	S	T	T	S	T	S
104	Y	Y	Y	Y	L	L	V	L	V
105	S	S	S	S	S	S	S	S	S
106	W	W	W	G	G	G	S	G	S
107	I	I	I	I	I	I	I	I	I
108	I	I	I	V	A	A	A	A	A
109	N	N	S	S	Q	Q	Q	Q	Q
110	G	G	G	G	G	G	G	G	G
111	I	I	I	I	I	V	L	I	L
112	E	E	E	E	E	E	E	E	E
113	W	W	W	W	W	W	W	W	W
114	A	A	A	A	S	A	A	S	A
115	I	I	V	T	I	I	G	I	G
116	A	S	A	T	S	A	N	A	N
117	N	N	N	N	N	N	N	N	N
118	N	N	N	G	G	N	G	D	G
119	M	M	M	M	M	M	M	M	M
120	D	D	D	D	N	D	H	D	H
121	V	V	V	V	V	V	V	V	V
122	I	I	I	I	I	I	A	I	A
123	N	N	N	N	N	N	N	N	N
124	M	M	M	M	M	M	L	M	L
125	S	S	S	S	S	S	S	S	S
126	L	L	L	L	L	L	L	L	L
127	G	G	G	G	G	G	G	G	G
128	G	G	G	G	G	G	S	I	S
129	P	P	P	A	S	S	P	S	P
130	S	T	S	S	S	S	S	T	S
131	G	G	G	G	G	G	P	G	P
132	S	S	S	S	S	S	S	S	S
133	A	T	T	T	T	T	A	T	A
134	A	A	A	A	A	A	T	A	T
135	L	L	L	M	L	L	L	L	L
136	K	K	K	K	Q	K	E	Q	E
137	A	T	N	Q	Q	Q	Q	Q	Q
138	A	V	A	A	A	A	A	A	A
139	V	V	V	V	C	V	V	C	V
140	D	D	D	D	N	D	N	N	N
141	K	K	T	N	N	N	S	N	S
142	A	A	A	A	A	A	A	A	A
143	V	V	N	Y	Y	Y	T	Y	T
144	A	S	N	A	N	A	S	A	S
145	S	S	R	R	R	S	R	S	R
146	G	G	G	G	G	G	G	G	G
147	V	I	V	V	I	I	V	I	V
148	V	V	V	V	V	V	L	V	L
149	V	V	V	V	V	V	V	V	V
150	V	A	V	V	I	V	V	V	V
151	A	A	A	A	S	A	A	A	A
152	A	A	A	A	S	A	A	A	A
153	A	A	A	A	A	A	S	A	S
154	G	G	G	G	G	G	G	G	G
155	N	N	N	N	N	N	N	N	N
156	E	E	S	S	S	S	S	S	S
157	G	G	G	G	G	G	G	G	G
158	T	S	S	S	S	T	A	S	A
158a
159	S	S	S	S	S	R		N
160	G	G	G	G	G	G	G	G	G
161	S	S	S	N	N	R		K
162	S	T	T	T	R	Q		R
163	S	S	S	N	N	N		N
164	T	T	T	T	T	T	S	T	S
165	V	V	V	I	M	M	I	M	I
166	G	G	G	G	G	G	S	G	S
167	Y	Y	Y	Y	Y	Y	Y	Y	Y
168	P	P	P	P	P	P	P	P	P
169	A	A	A	A	A	A	A	A	A
170	K	K	K	K	R	R	R	R	R
171	Y	Y	Y	Y	Y	Y	Y	Y	Y
172	P	P	D	D	S	S	A	S	A
173	S	S	S	S	S	S	N	S	N
174	V	T	T	V	V	V	A	V	A
175	I	I	I	I	I	I	M	I	M
176	A	A	A	A	A	A	A	A	A
177	V	V	V	V	V	V	V	V	V
178	G	G	A	G	G	G	G	G	G
179	A	A	N	A	A	A	A	A	A
180	V	V	V	V	V	V	T	V	T
181	D	N	N	D	S	D	D	D	D
182	S	S	S	S	S	S	Q	S	Q
183	S	S	N	N	N	N	N	S	N
184	N	N	N	S	N	N	N	N	N
185	Q	Q	V	N	T	N	N	N	N
186	R	R	R	R	R	R	R	R	R
187	A	A	N	A	A	A	A	A	A
188	S	S	S	S	S	S	S	S	S
189	F	F	S	F	F	F	F	F	F
190	S	S	S	S	S	S	S	S	S
191	S	S	S	S	S	S	Q	S	Q
192	V	A	A	V	V	V	Y	V	Y
193	G	G	G	G	G	G	G	G	G
194	P	S	P	A	S	A	A	S	A
195	E	E	E	E	E	E	G	E	G
196	L	L	L	L	L	L	L	L	L
197	D	D	D	E	E	E	D	E	D
198	V	V	V	V	V	V	I	V	I
199	M	M	S	M	M	M	V	M	V
200	A	A	A	A	A	A	A	A	A
201	P	P	P	P	P	P	P	P	P
202	G	G	G	G	G	G	G	G	G
203	V	V	T	A	V	V	V	V	V
204	S	S	S	G	N	S	N	S	N
205	I	I	I	V	I	V	I	I	V
206	Q	Q	L	Y	L	L	L	L	Q
207	S	S	S	S	S	S	S	S	S
208	T	T	T	T	T	T	T	T	T
209	L	L	V	Y	T	V	T	T	Y
210	P	P	P	P	P	P	P	P	P
211	G	G	S	T	G	G	G	G	G
212	N	G	S	N	N	G	N	N	S
213	K	T	G	T	N	G	N	N	T
214	Y	Y	Y	Y	Y	Y	Y	Y	Y
215	G	G	A	A	A	A	A	E	A
216	A	A	S	T	S	S	S	S	S
217	Y	Y	Y	L	F	Y	L	F	L
218	N	N	T	N	N	N	N	N	N
219	G	G	G	G	G	G	G	G	G
220	T	T	T	T	T	T	T	T	T
221	S	S	S	S	S	S	S	S	S
222	M	M	M	M	M	M	M	M	M
223	A	A	A	A	A	A	A	A	A
224	S	T	S	S	A	S	A	S	T
225	P	P	P	P	P	P	P	P	P
226	H	H	H	H	H	H	H	H	H
227	V	V	V	V	V	V	V	V	V
228	A	A	A	A	A	A	A	A	A
229	G	G	G	G	G	G	G	G	G
230	A	A	A	A	A	A	A	A	A
231	A	A	A	A	A	A	A	A	A
232	A	A	A	A	A	A	A	A	A
233	L	L	L	L	L	L	L	L	L
234	I	I	I	I	I	I	I	I	V
235	L	L	L	L	K	K	K	K	K
236	S	S	S	S	A	A	A	A	Q
237	K	K	K	K	K	K	K	K	K
238	H	H	H	H	Y	Y	Y	Y	N
239	P	P	P	P	P	P	P	P	P
240	N	T	N	N	S	S	S	S	S
241	W	W	L	L	M	L	M	M	W
242	T	T	T	S	T	S	T	T	S
243	N	N	N	A	N	A	N	N	N
244	T	A	T	S	V	S	V	V	V
245	Q	Q	Q	Q	Q	Q	Q	Q	Q
246	V	V	V	V	I	I	I	I	I
247	R	R	R	R	R	R	R	R	R
248	S	D	Q	N	E	D	N	N	N
249	S	R	R	R	R	R	R	K	H
250	L	L	L	L	L	L	L	L	L
251	E	E	E	S	K	R	K	K	K
252	N	S	N	S	N	N	N	N	N
253	T	T	T	T	T	T	T	T	T
254	T	A	A	A	A	A	A	A	A
255	T	T	T	T	T	T	T	T	T
256	K	Y	P	Y	N	Y	N	N	S
257	L	L	L	L	L	L	L	L	L
258	G	G	G	G	G	G	G	G	G
259	D	N	S	S	D	D	D	D	S
260	S	S	S	S	P	P	P	A	T
261	F	F	F	F	F	F	F	F	N
262	Y	Y	Y	Y	F	Y	F	Y	L
263	Y	Y	Y	Y	Y	Y	Y	Y	Y
264	G	G	G	G	G	G	G	G	G
265	K	K	K	K	K	N	K	H	S
266	G	G	G	G	G	G	G	G	G
267	L	L	L	L	V	V	V	V	L
268	I	I	I	I	I	I	I	I	V
269	N	N	N	N	N	N	N	N	N
270	V	V	V	V	V	V	V	V	A
271	Q	Q	Q	E	E	E	E	E	E
272	A	A	A	A	S	R	S	K	A
273	A	A	A	A	A	A	A	A	A
274	A	A	A	A	L	L	L	L	T
275	Q	Q	N	Q	Q	Q	Q	Q	R
BPN′
numbering	Bpan01744	*Bgi02446	DSM14391	BspAK01305	BspZ00258	BspAI02518	BspZ00056	Bad02409	Bba02069
1	A	Q	Q	A	A	A	G	A	Q
2	Q	Q	Q	Q	Q	Q	Q	Q	Q
3	S	T	T	S	E	S	T	T	Q
4	V	V	V	I	V	T	V	V	T
5	P	P	P	P	P	P	P	P	P
6	W	W	W	W	Y	W	W	W	W
7	G	G	G	G	G	G	G	G	G
8	I	I	I	I	I	I	I	V	I
9	S	T	T	E	E	S	P	P	T
10	R	R	R	R	Q	R	H	H	R
11	V	V	V	I	I	I	V	V	V
12	Q	Q	Q	G	G	N	Q	Q	Q
13	A	A	A	T	A	A	G	G	G
14	Q	P	P	P	I	P	T	T	I
15	S	A	T	A	D	A	A	D	A
16	A	V	V	A	V	V	A	A	A
17	H	H	H	H	Q	H	Q	H	Q
17a					V
18	N	N	N	A	N	S	D	A	S
19	R	R	R	S	D	T	A	A	Q
20	G	G	G	G	G	G	G	G	G
21	I	I	I	F	N	N	Y	H	Y
22	T	T	T	T	T	F	T	T	T
23	G	G	G	G	G	G	G	G	G
24	S	S	S	S	N	Q	A	S	N
25	G	G	G	G	G	G	G	G	N
26	V	V	V	V	V	V	L	V	V
27	K	R	K	S	S	R	K	K	K
28	V	V	V	V	V	V	V	V	V
29	A	A	A	A	A	A	A	A	A
30	V	I	I	V	V	V	I	I	V
31	L	L	L	L	L	L	L	L	L
32	D	D	D	D	D	D	D	D	D
33	T	S	T	T	T	S	T	T	S
34	G	G	G	G	G	G	G	G	G
35	I	I	I	I	I	V	I	I	I
36	S	S	A	D	A	A	D	D	D
37							R	R	R
38	T	A	Q	P	A	S	N	N	S
39	H	H	H	H	H	H	H	H	H
40	E	S	S	S	E	E	E	E	P
41	D	D	D	D	D	D	D	D	D
42	L	L	L	L	L	L	L	L	L
42a							F		S
42b							A		A
43	N	N	T	N	N	R	N	N	N
44	V	I	I	V	V	I	V	V	V
45	R	R	R	Q	V	A	K	R	R
46	G	G	G	G	D	G	G	G	G
47	G	G	G	G	G	G	G	G	G
48	A	A	A	V	A	V	H	H	Y
49	S	S	S	S	S	S	S	S	S
50	F	F	F	F	F	F	V	V	V
51	V	V	V	V	I	V	F	F	F
52	A	P	P	P	A	A	T	T	G
53	G	G	G	G	G	S	D	D	D
54	E	E	E	E	E	E	S	S	S
55	P	P	S	S	P	P	A	A	P
56	G	T	T	G	D	S	N	N
56a							S	R
56b							D	D
57							P	P
58	Y	T	T	A	Y	Y	F	Y	Y
59	Q	A	A	D	E	Q	Y	Y	N
60	D	D	D	D	D	D	D	D	D
61	G	L	L	G	Y	Y	A	G	G
62	N	N	N	N	N	N	D	S	N
63	G	G	G	G	G	G	G	G	G
64	H	H	H	H	H	H	H	H	H
65	G	G	G	G	G	G	G	G	G
66	T	T	T	T	T	T	T	T	T
67	H	H	H	H	H	H	H	H	H
68	V	V	V	V	V	V	V	V	V
69	A	A	A	A	A	A	A	A	A
70	G	G	G	G	G	G	G	G	G
71	T	T	T	T	T	T	T	T	T
72	I	V	V	I	V	I	V	V	V
73	A	A	A	A	A	A	A	A	G
74	A	A	A	A	A	G	A	A	A
75	L	L	L	L	L	L	V	L	V
76	N	N	N	D	D	N	D	N	N
77	N	N	N	N	N	N	N	N	N
78	S	S	S	D	D	S	D	S	N
79	I	I	I	E	L	V	L	V	I
80	G	G	G	G	D	G	G	G	G
81	V	V	V	V	V	V	V	V	V
82	L	I	I	L	L	L	V	L	I
83	G	G	G	G	G	G	G	G	G
84	V	V	V	V	V	V	V	V	V
85	A	A	A	A	S	A	A	A	A
86	P	P	P	P	P	P	S	Y	P
87	N	N	S	E	D	S	Q	N	Q
88	A	A	A	V	V	V	A	A	A
89	E	E	D	D	D	Q	E	E	D
90	L	L	L	L	L	L	L	L	V
91	L	Y	Y	F	Y	Y	Y	Y	Y
92	A	A	A	A	A	A	A	A	A
93	V	V	V	V	V	V	V	V	V
94	K	K	K	K	K	K	K	K	K
95	V	V	V	V	V	V	V	V	V
96	L	L	L	L	L	L	L	L	L
97	G	G	G	S	G	D	N	N	N
98	A	A	A	A	A	R	N	N	N
99	S	N	N	S	D	N	S	S	S
100	G	G	G	G	G	G	G	G	G
101	S	S	R	S	G	G	S	S	S
102	G	G	G	G	G	G	G	G	G
103	S	S	S	S	S	N	S	S	S
104	I	V	V	I	H	H	Y	Y	Y
105	S	S	S	S	A	S	A	A	A
106	G	G	G	S	S	D	G	G	G
107	I	I	I	I	I	I	I	I	I
108	A	A	A	A	A	A	A	A	A
109	Q	Q	Q	Q	Q	R	E	E	Q
110	G	G	G	G	G	G	G	G	G
111	L	L	L	L	I	I	I	I	I
112	Q	E	E	E	E	E	E	E	E
113	W	W	W	W	Y	W	W	W	W
114	A	A	A	T	A	S	S	A	S
115	G	A	A	A	V	V	I	V	I
116	N	T	T	E	D	N	N	N	N
117	N	N	N	N	N	N	N	N	N
118	G	N	N	N	N	G	G	G	G
119	M	M	M	I	I	M	M	M	M
120	H	H	H	D	D	H	D	D	D
121	I	I	I	V	V	V	I	I	I
122	A	A	A	A	V	V	I	I	I
123	N	N	N	N	N	N	N	N	N
124	M	M	M	L	M	M	M	M	M
125	S	S	S	S	S	S	S	S	S
126	L	L	L	L	L	L	L	L	L
127	G	G	G	G	G	G	G	G	G
128	T	S	S	S	G	G	G	G	G
129	S	D	D	P	A	P	S	S	S
130	A	F	A	S	V	T	Q	M	S
131	P	P	P	P	G	G	S	S	S
132	S	S	S	S	S	S	S	S	S
133	A	S	T	Q	T	T	S	S	S
134	T	T	T	T	T	T	I	I	I
135	L	L	L	L	L	L	L	L	L
136	E	E	E	E	E	Q	K	E	E
137	Q	R	R	Q	Q	R	Q	E	Q
138	A	A	A	A	A	A	F	W	Y
139	V	V	V	V	V	A	S	C	C
140	N	N	N	N	N	D	D	N	N
141	A	Y	Y	D	Y	N	L	I	L
142	A	A	A	A	A	A	A	A	A
143	T	T	T	T	H	Y	Y	Y	Y
144	S	S	S	D	S	N	E	N	N
145	R	R	R	S	Q	R	E	S	R
146	G	D	G	G	G	G	G	G	G
147	V	V	V	V	V	V	L	V	L
148	L	L	L	L	T	L	L	L	L
149	V	V	V	V	L	L	V	V	V
150	I	I	I	V	I	I	V	V	V
151	A	A	A	A	A	A	A	A	A
152	A	A	A	A	A	A	A	A	A
153	S	T	T	A	A	A	A	A	A
154	G	G	G	G	G	G	G	G	G
155	N	N	N	N	N	N	N	N	N
156	S	N	N	S	E	T	S	S	S
157	G	G	G	G	G	G	G	G	G
158	A	S	T	T	S	T	N	R	T
158a					L		R	T	A
159				S	I	S	G	N	A
160	G	G	G		P		G	G	G
161					G		N	R	N
162					L		N	G	T
163					N		D	D	N
164	S	S	S	S	T	G	T	T	T
165	V	V	I	L	I	V	V	V	V
166	G	G	G	G	G	S	G	G	G
167	Y	Y	Y	Y	Y	F	Y	Y	Y
168	P	P	P	P	P	P	P	P	P
169	A	A	A	A	A	A	A	A	A
170	R	R	R	R	K	R	K	K	R
171	Y	Y	Y	Y	Y	Y	Y	Y	Y
172	A	A	A	D	D	S	D	D	N
173	N	N	N	N	N	S	S	S	S
174	A	A	A	A	V	V	V	V	V
175	M	M	M	M	I	M	I	I	I
176	A	A	A	A	A	A	A	A	A
177	V	V	V	V	V	V	V	V	V
178	G	G	G	G	G	A	A	A	A
179	A	A	A	A	A	A	A	A	A
180	T	T	T	T	V	T	V	V	T
181	D	D	D	D	D	D	D	D	N
182	Q	Q	Q	Q	S	S	Q	S	S
183	N	N	N	S	N	N	N	S	N
184	N	N	N	D	N	N	N	N	N
185	N	R	R	S	N	N	N	N	V
186	R	R	R	L	R	R	R	R	R
187	A	A	A	A	A	A	A	A	G
188	S	N	S	S	S	S	T	S	N
189	F	F	F	F	F	F	F	F	F
190	S	S	S	S	S	S	S	S	S
191	Q	Q	Q	Q	S	T	S	S	S
192	Y	Y	Y	Y	V	Y	T	T	T
193	G	G	G	G	G	G	G	G	G
194	A	T	T	E	N	S	P	P	P
195	G	G	G	G	E	Q	A	A	T
196	L	I	I	L	L	I	V	V	V
197	D	D	D	D	D	E	E	E	E
198	I	I	I	L	V	I	I	I	L
199	V	V	V	V	V	S	S	A	S
200	A	A	A	A	A	A	A	A	A
201	P	P	P	P	P	P	P	P	P
202	G	G	G	G	G	G	G	G	G
203	V	V	V	V	V	V	V	V	V
204	G	N	G	G	S	G	S	N	S
205	V	V	I	V	I	I	I	I	V
206	Q	Q	Q	E	L	N	L	L	L
207	S	S	S	S	S	S	S	S	S
208	T	T	T	T	T	T	T	T	T
209	Y	Y	Y	Y	Y	Y	T	T	T
210	P	P	L	P	L	P	P	P	P
211	G	G	N	G	G	T	G	G	G
212	N	N	N	G	N	N	N	N	G
213	R	R	S	G	D	G	N	S	N
214	Y	Y	Y	Y	Y	Y	Y	Y	Y
215	A	V	A	D	A	S	A	A	A
216	S	S	S	S	A	S	A	S	S
217	L	M	M	L	L	L	F	Y	Y
218	N	N	N	P	S	N	N	N	N
219	G	G	G	G	G	G	G	G	G
220	T	T	T	T	T	T	T	T	T
221	S	S	S	S	S	S	S	S	S
222	M	M	M	M	M	M	M	M	M
223	A	A	A	A	A	A	A	A	A
224	T	T	T	A	S	S	S	S	S
225	P	P	P	P	P	P	P	P	P
226	H	H	H	H	H	H	H	H	H
227	V	V	V	V	V	V	V	V	V
228	A	A	A	A	A	A	A	A	A
229	G	G	G	G	G	G	G	G	G
230	V	A	V	A	A	V	V	V	V
231	A	A	A	A	A	A	A	A	A
232	A	A	A	A	A	A	A	A	A
233	L	L	L	L	L	L	Q	L	Q
234	V	V	V	V	L	V	V	V	V
235	K	K	K	K	L	K	W	L	W
236	Q	Q	Q	Q	A	A	Q	A	Q
237	K	R	K	K	E	R	A	A	A
238	N	Y	N	N	N	Y	K	N	R
239	P	P	P	P	P	P	P	P	P
240	S	S	S	G	G	S	E	N	N
241	W	W	W	W	L	A	L	L	L
242	S	N	N	T	T	T	S	S	S
243	N	A	A	N	N	N	N	N	N
244	V	T	T	E	D	A	V	V	A
245	Q	Q	Q	Q	Q	Q	E	E	Q
246	V	I	I	I	V	I	L	L	L
247	R	R	R	R	R	R	R	R	R
248	N	N	N	S	A	Q	N	N	Q
249	H	H	H	H	V	H	L	R	I
250	L	L	L	L	F	L	L	L	L
251	K	K	K	N	N	R	N	N	N
252	N	N	N	D	E	S	E	D	A
253	T	T	T	T	T	T	T	T	S
254	A	A	A	A	A	S	A	A	A
255	T	T	T	N	V	T	V	Q	Q
256	N	N	N	D	P	Y	N	N	N
257	L	L	L	L	L	L	L	L	L
258	G	G	G	G	G	G	G	G	G
259	N	N	N	D	D	N	G	D	S
260	T	S	S	S	H	S	S	A	S
261	N	S	S	F	F	T	N	N	Y
262	L	Q	Q	R	Y	Y	Q	H	Q
263	Y	F	F	F	Y	Y	F	F	Y
264	G	G	G	G	G	G	G	G	G
265	S	S	S	S	N	S	H	N	N
266	G	G	G	G	G	G	G	G	G
267	L	L	L	L	L	L	L	L	L
268	V	V	V	L	I	V	V	V	V
269	N	N	N	N	D	D	Q	R	R
270	A	A	A	A	V	A	S	A	S
271	E	E	D	E	R	Q	L	V	L
272	A	A	A	N	A	R	D	D	N
273	A	A	A	A	A	A	A	A	A
274	T	T	T	V	I	T	I	I	I
275	R	R	R	Q	D	N	Q	N	Q

The percent identity for the mature (in some cases predicted) amino acid sequences of the subtilisins (corresponding to residues 1-275 of BPN′) was calculated based on the alignment shown on Table 28, using the MUSCLE (Geneious version 10.2.6) software, and results are shown on Table 29.

TABLE 29
Percent identity over the mature amino acid sequence of multiple subtilisins.
	BPN′	AprE	WP_082194748	AprL	LG12	Chemgen′164A	CP474	ZP-00454	GG36	Bpan01744
BPN′	100	86.5	76.4	69.5	65.1	68	60.7	68	60	58.2
AprE	86.5	100	77.5	69.8	65.8	66.5	58.9	66.5	60.4	59.3
WP_082194748	76.4	77.5	100	73.8	65.8	66.2	61.5	64.7	58.5	55.3
AprL	69.5	69.8	73.8	100	70.9	72	66.5	70.2	61.1	60.4
LG12	65.1	65.8	65.8	70.9	100	81.8	79.6	90.2	63.3	64.4
Chemgen_164A	68	66.5	66.2	72	81.8	100	69.8	82.9	62.5	62.2
CP474	60.7	58.9	61.5	66.5	79.6	69.8	100	76	81.2	76.8
ZP-00454	68	66.5	64.7	70.2	90.2	82.9	76	100	63.3	64.4
GG36	60	60.4	58.5	61.1	63.3	62.5	81.2	63.3	100	89.6
Bpan01744	58.2	59.3	55.3	60.4	64.4	62.2	76.8	64.4	89.6	100
Bgi02446	56	56	54.2	54.9	60	58.5	69.7	58.9	79.9	80.3
DSM14391	55.6	54.9	54.5	55.6	58.9	57.8	69.4	57.5	78.4	79.2
BspAK01305	56.7	56	54.2	57.1	59.3	60	69.5	60	72.2	69.3
BspZ00258	60.6	59.2	60.6	64.6	61	61.7	61	62.1	58.2	59.6
BspAI02518	57.1	58.5	58.5	57.1	58.9	58.2	58.8	58.5	63.3	63.3
BspZ00056	53.6	52.1	53.6	57.1	60	57.5	56.1	60	53.2	56.1
Bad02409	57.6	56.5	56.8	58.6	64.7	61.5	60.4	65.1	58.6	62.2
Bba02069	55.4	56.1	56.5	57.9	60.1	61.5	53.6	58.6	57	58.5
	Bgi02446	DSM14391	BspAK01305	BspZ00258	BspAI02518	BspZ00056	Bad02409	Bba02069
BPN′	56	55.6	56.7	60.6	57.1	53.6	57.6	55.4
AprE	56	54.9	56	59.2	58.5	52.1	56.5	56.1
WP_082194748	54.2	54.5	54.2	60.6	58.5	53.6	56.8	56.5
AprL	54.9	55.6	57.1	64.6	57.1	57.1	58.6	57.9
LG12	60	58.9	59.3	61	58.9	60	64.7	60.1
Chemgen_164A	58.5	57.8	60	61.7	58.2	57.5	61.5	61.5
CP474	69.7	69.4	69.5	61	58.8	56.1	60.4	53.6
ZP-00454	58.9	57.5	60	62.1	58.5	60	65.1	58.6
GG36	79.9	78.4	72.2	58.2	63.3	53.2	58.6	57
Bpan01744	80.3	79.2	69.3	59.6	63.3	56.1	62.2	58.5
Bgi02446	100	90	65.6	55.6	61.1	52.5	56.1	56
DSM14391	90	100	65.9	57.1	61.1	51.4	55.8	53.8
BspAK01305	65.6	65.9	100	58.9	57.2	49.3	53.6	53.1
BspZ00258	55.6	57.1	58.9	100	57.1	53.2	56.2	52.7
BspAI02518	61.1	61.1	57.2	57.1	100	49.3	54	54.2
BspZ00056	52.5	51.4	49.3	53.2	49.3	100	77.2	72.2
Bad02409	56.1	55.8	53.6	56.2	54	77.2	100	69
Bba02069	56	53.8	53.1	52.7	54.2	72.2	69	100

An analysis of available crystal structures and homology models of several of the subtilisin proteases evaluated in this study or their close homologs was performed. The three-dimensional structures or homology models of six subtilisins: the B. subtilis (strain 168) subtilisin E (AprE) Protein Data Bank (PDB) entry 1SCJ; the Bacillus sp. subtilisin LG12 SprC (LG12) homology model described in WO2015038792; the B. amyloliquefaciens (BPN′) PDB entry 2ST1; the B. licheniformis (AprL) PDB entry 1CSE; B. lentus (GG36) PDB entry 1JEA; and the B. gibsonii DSM14391 subtilisin homology model (based on B. gibsonii-clade BSP-00801 structure described in WO2016205755) were used to examine sites where globally beneficial substitutions were evaluated and identified. The superposition of the main chain fold of these subtilisins (image not shown) indicates that the structures overlap along the bulk of the sequences, having a common catalytic triad, corresponding to residues Asp 32, His 64, Ser 221 (numbered with respect to subtilisin BPN′ sequence, SEQ ID NO:1) and minor differences, mostly in loops and surface exposed regions.

FIGS. 1-6 illustrate the spatial positions of a subset of the beneficial sites evaluated in this study, wherein the residues are numbered according to the BPN′ sequence (as shown in Table 28 above). FIG. 1 shows B. subtilis (strain 168) subtilisin E (AprE) PDB entry 1SCJ (mature subtilisin region only, excluding the propeptide segment); FIG. 2 shows Bacillus sp. subtilisin LG12 SprC (LG12) homology model described in WO2015038792; FIG. 3 shows B. gibsonii DSM14391 subtilisin homology model (prepared based on the BSP-00801 B. gibsonii-clade subtilisin structure described in WO2016205755); FIG. 4 shows B. amyloliquefaciens subtilisin BPN′ PDB entry 2ST1; FIG. 5 shows B. licheniformis subtilisin Carlsberg (AprL) PDB entry 1CSE; and FIG. 6 shows B. lentus subtilisin GG36 PDB entry 1JEA. In each figure, the main chain fold of each subtilisin is schematically represented in light gray and the following sites are depicted as black sticks: 3, 24, 40, 76, 78, 79, 87, 118, 128, 129, 130, 145, 166, 182, 185, 210, 211, 217, 218, 259 (BPN′ numbering). These sites are all surface exposed and are situated in loops, outside of secondary structure motifs.

Furthermore, a subset of the sites highlighted in FIGS. 1-6 were observed to be distributed among the loops that together form an extended surface. In particular, sites 76, 78 and 79 (which are part of the same loop) are situated in spatial proximity to sites 3 and 40, which are located on distinct loops. Moreover, site 76 is also situated in spatial proximity to site 24, which, in turn, is spatially close to site 87 (belonging to a different loop). Site 40 resides on a loop that is located in spatial proximity to sites 210 and 211. Thus, sites 3, 24, 40, 76, 78, 79, 87, 210 and 211 (BPN′ numbering) are situated along a surface formed by a series of loops in which these sites reside. Sites 128, 129 and 130 (BPN′ numbering) are in spatial proximity to site 166, as the loop containing sites 128, 129 and 130 comes close to the loop where site 166 is situated. Sites 182 and 185 are also located in spatial proximity to each other—these sites form part of a turn in a loop where they reside. While site 259 is located on a different loop, it appears to form part of the same surface as the loop containing sites 182 and 185.

Together, the surface exposed sites 3, 9, 24, 40, 76, 78, 79, 87, 118, 128, 129, 130, 145, 166, 182, 185, 210, 211, 217, 218, 248 and 259 (BPN′ numbering) account for twenty-two of the twenty-four sites evaluated in this study, and the substitutions explored were: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, X259P. The location of these sites on the surface of the molecules, and mostly in loop regions outside of secondary structure motifs, suggests an underlying structural commonality for the improvements in protein stability provided by the amino acid substitutions evaluated in this study.

Example 13

Additional AprE & WP_082194748 Subtilisin Variants with Improved Stability in Detergent

Variants of AprE (Subtilisin E, SEQ ID NO: 8) and WP_082194748 (SEQ ID NO: 9) subtilisins containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability (% residual activity) and cleaning performance (PI) using methods described in Example 2. Results for AprE (Subtilisin E) variants are shown on Tables 30A and 30B, and results for WP_082194748 variants are shown on Table 31.

TABLE 30A
AprE subtilisin variants with improved stability in liquid detergent at 41°
C. (reported as percent residual activity, % RA) compared to AprE parent
	Cleaning
	performance, PI
				BMI	PAS-38
AprE				stain in	stain in
Variant	Substitutions in	Substitutions in		PNB	GSM-B
Sample ID	AprE numbering	BPN′ numbering	% RA	detergent	detergent
AprE	—	—	40	1.0	1.0
AprE-00685	S003V-N076D-S078N	S003V-N076D-S078N	81	1.1	1.1
AprE-00353	S003V-S078N-N218S	S003V-S078N-N218S	87	1.1	1.1
AprE-00806	S003V-M124I-N259P	S003V-M124I-N259P	71	1.1	1.2
AprE-00696	S003V-G128S-G166Q	S003V-G128S-G166Q	79	1.1	1.0
AprE-00773	S003V-G166Q-N218S	S003V-G166Q-N218S	91	1.2	1.1
AprE-00753	S003V-N218S-N259P	S003V-N218S-N259P	84	1.0	1.1
AprE-00540	P040E-N076D-S078N	P040E-N076D-S078N	86	1.1	1.0
AprE-00635	G128S-G166Q-N259P	G128S-G166Q-N259P	83	1.1	1.0
AprE-00452	S003V-P040E-N076D-	S003V-P040E-N076D-	83	1.1	0.9
	S078N	S078N
AprE-00850	S003V-P040E-S078N-	S003V-P040E-S078N-	80	1.3	1.1
	M124I	M124I
AprE-00756	S003V-P040E-M124I-	S003V-P040E-M124I-	85	1.2	1.1
	N218S	N218S
AprE-00613	S003V-N076D-S078N-	S003V-N076D-S078N-	100	1.2	1.0
	G128S	G128S
AprE-00462	S003V-N076D-M124I-	S003V-N076D-M124I-	100	1.0	0.9
	G166Q	G166Q
AprE-00665	S003V-N076D-G128S-	S003V-N076D-G128S-	100	1.2	1.0
	N259P	N259P
AprE-00705	S003V-N076D-G166Q-	S003V-N076D-	91	1.1	1.1
	N259P	G166Q-N259P
AprE-00796	S003V-N076D-N218S-	S003V-N076D-N218S-	96	1.1	1.1
	N259P	N259P
AprE-00551	S003V-M124I-G128S-	S003V-M124I-G128S-	81	1.1	0.9
	N218S	N218S
AprE-00617	S003V-G128S-G166Q-	S003V-G128S-G166Q-	99	1.3	1.0
	N218S	N218S
AprE-00510	P040E-N076D-S078N-	P040E-N076D-S078N-	100	1.1	1.0
	G166Q	G166Q
AprE-00403	P040E-N076D-M124I-	P040E-N076D-M124I-	93	1.2	1.0
	N218S	N218S
AprE-00764	P040E-N076D-G166Q-	P040E-N076D-G166Q-	98	1.2	1.0
	N218S	N218S
AprE-00547	P040E-N076D-G166Q-	P040E-N076D-G166Q-	98	1.1	1.0
	N259P	N259P
AprE-00769	N076D-S078N-M124I-	N076D-S078N-M124I-	89	1.3	1.1
	N218S	N218S
AprE-00684	N076D-S078N-G128S-	N076D-S078N-G128S-	94	1.2	0.9
	N259P	N259P
AprE-00507	N076D-S078N-G166Q-	N076D-S078N-	97	1.0	1.0
	N259P	G166Q-N259P
AprE-00591	N076D-M124I-G128S-	N076D-M124I-G128S-	100	1.1	0.8
	G166Q	G166Q
AprE-00583	N076D-M124I-G128S-	N076D-M124I-G128S-	100	1.3	0.8
	N259P	N259P
AprE-00709	N076D-G128S-G166Q-	N076D-G128S-	95	1.2	0.9
	N218S	G166Q-N218S
AprE-00518	S078N-G128S-G166Q-	S078N-G128S-G166Q-	100	1.2	0.9
	N218S	N218S
AprE-00589	S078N-G128S-N218S-	S078N-G128S-N218S-	96	1.1	1.0
	N259P	N259P
AprE-00366	M124I-G166Q-N218S-	M124I-G166Q-N218S-	90	1.1	1.0
	N259P	N259P

TABLE 30B
AprE subtilisin variants with improved stability in liquid detergent at 42°
C. (reported as percent residual activity, % RA) compared to AprE parent
AprE
Variant	Substitutions in	Substitutions in
Sample ID	AprE numbering	BPN′ numbering	% RA
AprE	—	—	27
AprE-01102	S003V-S009E-P040E	S003V-S009E-P040E	85
AprE-01101	S003V-S009E-A069S	S003V-S009E-A069S	89
AprE-01051	S003V-S009E-N076D	S003V-S009E-N076D	94
AprE-01099	S003V-S009E-S078N	S003V-S009E-S078N	90
AprE-01045	S003V-S009E-G166Q	S003V-S009E-G166Q	95
AprE-01077	S003V-S009E-N218S	S003V-S009E-N218S	91
AprE-01047	S003V-S009E-N259P	S003V-S009E-N259P	86
AprE-01009	S003V-P040E-A069S	S003V-P040E-A069S	61
AprE-01048	S003V-P040E-N076D	S003V-P040E-N076D	69
AprE-01915	S003V-P040E-N218S	S003V-P040E-N218S	77
AprE-01039	S003V-P040E-N259P	S003V-P040E-N259P	67
AprE-01864	S003V-A069S-N076D	S003V-A069S-N076D	54
AprE-01056	S003V-A069S-S078N	S003V-A069S-S078N	48
AprE-00991	S003V-A069S-G166Q	S003V-A069S-G166Q	75
AprE-01060	S003V-A069S-N218S	S003V-A069S-N218S	70
AprE-01064	S003V-A069S-N259P	S003V-A069S-N259P	48
AprE-01340	S003V-N076D-G166Q	S003V-N076D-G166Q	100
AprE-01867	S003V-N076D-N218S	S003V-N076D-N218S	82
AprE-01062	S003V-N076D-N259P	S003V-N076D-N259P	84
AprE-01068	S003V-S078N-N259P	S003V-S078N-N259P	73
AprE-01855	S003V-G166Q-N259P	S003V-G166Q-N259P	68
AprE-01674	S009E-P040E-A069S	S009E-P040E-A069S	73
AprE-01069	S009E-P040E-N076D	S009E-P040E-N076D	94
AprE-01997	S009E-P040E-S078N	S009E-P040E-S078N	82
AprE-01984	S009E-P040E-G166Q	S009E-P040E-G166Q	88
AprE-01529	S009E-P040E-N218S	S009E-P040E-N218S	97
AprE-01125	S009E-P040E-N259P	S009E-P040E-N259P	88
AprE-01071	S009E-A069S-N076D	S009E-A069S-N076D	91
AprE-01091	S009E-A069S-S078N	S009E-A069S-S078N	84
AprE-01871	S009E-A069S-G166Q	S009E-A069S-G166Q	83
AprE-01087	S009E-A069S-N218S	S009E-A069S-N218S	87
AprE-01857	S009E-A069S-N259P	S009E-A069S-N259P	73
AprE-01892	S009E-N076D-S078N	S009E-N076D-S078N	92
AprE-01093	S009E-N076D-G166Q	S009E-N076D-G166Q	100
AprE-01052	S009E-N076D-N218S	S009E-N076D-N218S	98
AprE-01860	S009E-N076D-N259P	S009E-N076D-N259P	100
AprE-01095	S009E-S078N-G166Q	S009E-S078N-G166Q	94
AprE-01019	S009E-S078N-N218S	S009E-S078N-N218S	97
AprE-01014	S009E-S078N-N259P	S009E-S078N-N259P	88
AprE-01029	S009E-G166Q-N218S	S009E-G166Q-N218S	94
AprE-01028	S009E-G166Q-N259P	S009E-G166Q-N259P	97
AprE-01026	S009E-N218S-N259P	S009E-N218S-N259P	94
AprE-01024	P040E-A069S-S078N	P040E-A069S-S078N	44
AprE-01865	P040E-A069S-G166Q	P040E-A069S-G166Q	56
AprE-01823	P040E-A069S-N218S	P040E-A069S-N218S	61
AprE-01036	P040E-A069S-N259P	P040E-A069S-N259P	48
AprE-01032	P040E-N076D-G166Q	P040E-N076D-G166Q	96
AprE-01824	P040E-N076D-N218S	P040E-N076D-N218S	78
AprE-01031	P040E-N076D-N259P	P040E-N076D-N259P	81
AprE-01825	P040E-S078N-G166Q	P040E-S078N-G166Q	81
AprE-01030	P040E-S078N-N218S	P040E-S078N-N218S	82
AprE-01035	P040E-S078N-N259P	P040E-S078N-N259P	59
AprE-01820	P040E-G166Q-N218S	P040E-G166Q-N218S	80
AprE-01831	P040E-G166Q-N259P	P040E-G166Q-N259P	71
AprE-01826	P040E-N218S-N259P	P040E-N218S-N259P	67
AprE-01810	A069S-N076D-S078N	A069S-N076D-S078N	58
AprE-01812	A069S-N076D-G166Q	A069S-N076D-G166Q	86
AprE-01403	A069S-N076D-N218S	A069S-N076D-N218S	81
AprE-01829	A069S-N076D-N259P	A069S-N076D-N259P	73
AprE-01830	A069S-S078N-G166Q	A069S-S078N-G166Q	69
AprE-01693	A069S-S078N-N218S	A069S-S078N-N218S	59
AprE-01873	A069S-S078N-N259P	A069S-S078N-N259P	47
AprE-00996	A069S-G166Q-N218S	A069S-G166Q-N218S	68
AprE-01117	A069S-G166Q-N259P	A069S-G166Q-N259P	54
AprE-01807	A069S-N218S-N259P	A069S-N218S-N259P	48
AprE-01805	N076D-S078N-G166Q	N076D-S078N-G166Q	85
AprE-01407	N076D-S078N-N218S	N076D-S078N-N218S	85
AprE-01118	N076D-S078N-N259P	N076D-S078N-N259P	96
AprE-01802	N076D-G166Q-N218S	N076D-G166Q-N218S	100
AprE-01819	N076D-G166Q-N259P	N076D-G166Q-N259P	92
AprE-01817	N076D-N218S-N259P	N076D-N218S-N259P	87
AprE-01722	S078N-G166Q-N218S	S078N-G166Q-N218S	83
AprE-01816	S078N-G166Q-N259P	S078N-G166Q-N259P	69
AprE-01112	S078N-N218S-N259P	S078N-N218S-N259P	70
AprE-01815	G166Q-N218S-N259P	G166Q-N218S-N259P	67
AprE-02047	S003V-P040E-A069S-G166Q	S003V-P040E-A069S-G166Q	76
AprE-01188	S003V-P040E-N076D-N218S	S003V-P040E-N076D-N218S	81
AprE-02059	S003V-A069S-G166Q-N218S	S003V-A069S-G166Q-N218S	87
AprE-01547	S009E-P040E-A069S-N076D	S009E-P040E-A069S-N076D	84
AprE-01712	S009E-P040E-A069S-N259P	S009E-P040E-A069S-N259P	71
AprE-01975	S009E-P040E-S078N-N259P	S009E-P040E-S078N-N259P	84
AprE-01553	S009E-P040E-G166Q-N218S	S009E-P040E-G166Q-N218S	93
AprE-01917	S009E-P040E-G166Q-N259P	S009E-P040E-G166Q-N259P	88
AprE-01772	S009E-P040E-N218S-N259P	S009E-P040E-N218S-N259P	93
AprE-01551	S009E-A069S-S078N-G166Q	S009E-A069S-S078N-G166Q	88
AprE-02023	S009E-S078N-N218S-N259P	S009E-S078N-N218S-N259P	94
AprE-01666	S009E-G166Q-N218S-N259P	S009E-G166Q-N218S-N259P	94
AprE-00988	P040E-A069S-N076D-S078N	P040E-A069S-N076D-S078N	50
AprE-01794	P040E-A069S-S078N-G166Q	P040E-A069S-S078N-G166Q	75
AprE-01879	P040E-A069S-S078N-N259P	P040E-A069S-S078N-N259P	56
AprE-01953	P040E-A069S-G166Q-N218S	P040E-A069S-G166Q-N218S	83
AprE-01990	N076D-S078N-G166Q-N218S	N076D-S078N-G166Q-N218S	93
AprE-01979	N076D-G166Q-N218S-N259P	N076D-G166Q-N218S-N259P	91

TABLE 31
WP_082194748 subtilisin variants with improved stability in liquid detergent at
46° C. (reported as percent residual activity, % RA) compared to WP_082194748 parent
	Cleaning
	performance, PI
					PAS-38
WP_082194748	Substitutions in			BMI stain	stain in
Variant Sample	WP_082194748	Substitutions in		in PNB	GSM-B
ID	numbering	BPN′ numbering	% RA	detergent	detergent
WP_082194748	—	—	36	1.0	1.0
WP_082194748-	T003V-T078N-G166Q	T003V-T078N-	93	1.2	1.5
00562		G166Q
WP_082194748-	T003V-T078N-S259P	T003V-T078N-	70	1.4	1.3
00205		S259P
WP_082194748-	T003V-M124I-G128S	T003V-M124I-	88	1.3	0.4
00037		G128S
WP_082194748-	T003V-G128S-G166Q	T003V-G128S-	100	1.4	1.2
00219		G166Q
WP_082194748-	T003V-G128S-S259P	T003V-G128S-	93	1.2	1.1
00101		S259P
WP_082194748-	T003V-G166Q-S259P	T003V-G166Q-	98	1.3	1.3
00511		S259P
WP_082194748-	T078N-M124I-G166Q	T078N-M124I-	80	1.4	1.3
00185		G166Q
WP_082194748-	T078N-G128S-G166Q	T078N-G128S-	98	1.4	1.2
00433		G166Q
WP_082194748-	T078N-G166Q-S259P	T078N-G166Q-	100	1.3	1.3
00339		S259P
WP_082194748-	M124I-G128S-G166Q	M124I-G128S-	90	1.3	0.8
00127		G166Q
WP_082194748-	M124I-G166Q-S259P	M124I-G166Q-	99	1.3	1.3
00218		S259P
WP_082194748-	T003V-T078N-M124I-	T003V-T078N-	100	0.9	1.3
00547	G166Q	M124I-G166Q
WP_082194748-	T003V-T078N-G166Q-	T003V-T078N-	73	1.1	1.2
00442	S259P	G166Q-S259P
WP_082194748-	T003V-M124I-G128S-	T003V-M124I-	100	1.5	0.5
00555	S259P	G128S-S259P
WP_082194748-	T003V-M124I-G166Q-	T003V-M124I-	100	1.1	1.2
00465	S259P	G166Q-S259P
WP_082194748-	T078N-M124I-G128S-	T078N-M124I-	100	1.2	1.0
00149	S259P	G128S-S259P
WP_082194748-	T078N-M124I-G166Q-	T078N-M124I-	100	1.1	1.2
00448	S259P	G166Q-S259P

Example 14

Chemgen_164A Subtilisin Variants with Improved Stability in Detergent

Variants of Chemgen_164A (Chemgen, SEQ ID NO: 10) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability (% residual activity) using methods described in Example 2. Table 32 shows the detergent stability results (% RA) for Chemgen_164A variants.

TABLE 32
Chemgen_164A subtilisin variants with improved stability in liquid detergent at
51° C. (reported as percent residual activity, % RA) compared to Chemgen_164A parent
Chemgen_164A	Substitutions in
Variant Sample	Chemgen_164A	Substitutions in
ID	numbering	BPN′ numbering	% RA
Chemgen_164A	—	—	31
Chemgen-00801	T003V-T009E-P040E	T003V-T009E-P040E	89
Chemgen-01562	T003V-T009E-A069S	T003V-T009E-A069S	66
Chemgen-00792	T003V-T009E-N185Q	T003V-T009E-N185Q	70
Chemgen-01640	T003V-P040E-N076D	T003V-P040E-N076D	100
Chemgen-00793	T003V-P040E-G166Q	T003V-P040E-G166Q	100
Chemgen-01651	T003V-P040E-N185Q	T003V-P040E-N185Q	100
Chemgen-00724	T003V-P040E-D259P	T003V-P040E-D259P	100
Chemgen-00816	T003V-A069S-N076D	T003V-A069S-N076D	100
Chemgen-00808	T003V-A069S-T078N	T003V-A069S-T078N	100
Chemgen-00807	T003V-A069S-G166Q	T003V-A069S-G166Q	100
Chemgen-01642	T003V-A069S-N185Q	T003V-A069S-N185Q	94
Chemgen-00727	T003V-A069S-N218S	T003V-A069S-N218S	100
Chemgen-01658	T003V-A069S-D259P	T003V-A069S-D259P	100
Chemgen-00729	T003V-N076D-T078N	T003V-N076D-T078N	100
Chemgen-00694	T003V-N076D-G166Q	T003V-N076D-G166Q	100
Chemgen-00776	T003V-N076D-N185Q	T003V-N076D-N185Q	100
Chemgen-00775	T003V-N076D-N218S	T003V-N076D-N218S	100
Chemgen-00832	T003V-N076D-D259P	T003V-N076D-D259P	100
Chemgen-00774	T003V-T078N-G166Q	T003V-T078N-G166Q	100
Chemgen-00696	T003V-T078N-N185Q	T003V-T078N-N185Q	100
Chemgen-00834	T003V-T078N-D259P	T003V-T078N-D259P	100
Chemgen-00738	T003V-G166Q-N185Q	T003V-G166Q-N185Q	100
Chemgen-00739	T003V-G166Q-D259P	T003V-G166Q-D259P	79
Chemgen-00785	T003V-N185Q-N218S	T003V-N185Q-N218S	91
Chemgen-00698	T003V-N185Q-D259P	T003V-N185Q-D259P	100
Chemgen-00780	T003V-N218S-D259P	T003V-N218S-D259P	100
Chemgen-01553	T009E-P040E-A069S	T009E-P040E-A069S	53
Chemgen-00703	T009E-P040E-N076D	T009E-P040E-N076D	93
Chemgen-00683	T009E-P040E-T078N	T009E-P040E-T078N	81
Chemgen-00702	T009E-P040E-G166Q	T009E-P040E-G166Q	78
Chemgen-00822	T009E-P040E-N218S	T009E-P040E-N218S	62
Chemgen-01604	T009E-P040E-D259P	T009E-P040E-D259P	68
Chemgen-00684	T009E-A069S-N076D	T009E-A069S-N076D	79
Chemgen-00700	T009E-A069S-T078N	T009E-A069S-T078N	71
Chemgen-00823	T009E-A069S-G166Q	T009E-A069S-G166Q	66
Chemgen-00681	T009E-A069S-N218S	T009E-A069S-N218S	86
Chemgen-00829	T009E-A069S-D259P	T009E-A069S-D259P	46
Chemgen-00825	T009E-N076D-T078N	T009E-N076D-T078N	87
Chemgen-01315	T009E-N076D-G166Q	T009E-N076D-G166Q	100
Chemgen-00737	T009E-N076D-N185Q	T009E-N076D-N185Q	78
Chemgen-00690	T009E-N076D-N218S	T009E-N076D-N218S	88
Chemgen-01420	T009E-N076D-D259P	T009E-N076D-D259P	74
Chemgen-00826	T009E-T078N-G166Q	T009E-T078N-G166Q	86
Chemgen-01162	T009E-T078N-N185Q	T009E-T078N-N185Q	60
Chemgen-00688	T009E-T078N-N218S	T009E-T078N-N218S	79
Chemgen-00818	T009E-T078N-D259P	T009E-T078N-D259P	80
Chemgen-00687	T009E-G166Q-N218S	T009E-G166Q-N218S	95
Chemgen-00686	T009E-G166Q-D259P	T009E-G166Q-D259P	100
Chemgen-00705	T009E-N185Q-N218S	T009E-N185Q-N218S	60
Chemgen-00685	T009E-N185Q-D259P	T009E-N185Q-D259P	51
Chemgen-00878	P040E-A069S-N076D	P040E-A069S-N076D	85
Chemgen-00876	P040E-A069S-T078N	P040E-A069S-T078N	83
Chemgen-00868	P040E-A069S-G166Q	P040E-A069S-G166Q	63
Chemgen-01310	P040E-A069S-D259P	P040E-A069S-D259P	64
Chemgen-00761	P040E-N076D-T078N	P040E-N076D-T078N	100
Chemgen-00642	P040E-N076D-G166Q	P040E-N076D-G166Q	100
Chemgen-00660	P040E-N076D-N185Q	P040E-N076D-N185Q	100
Chemgen-00641	P040E-N076D-N218S	P040E-N076D-N218S	95
Chemgen-01465	P040E-T078N-N185Q	P040E-T078N-N185Q	90
Chemgen-00758	P040E-T078N-D259P	P040E-T078N-D259P	100
Chemgen-00872	P040E-G166Q-N185Q	P040E-G166Q-N185Q	88
Chemgen-00873	P040E-G166Q-N218S	P040E-G166Q-N218S	79
Chemgen-00752	P040E-G166Q-D259P	P040E-G166Q-D259P	84
Chemgen-00750	P040E-N185Q-N218S	P040E-N185Q-N218S	66
Chemgen-00851	P040E-N185Q-D259P	P040E-N185Q-D259P	87
Chemgen-00845	P040E-N218S-D259P	P040E-N218S-D259P	83
Chemgen-00844	A069S-N076D-T078N	A069S-N076D-T078N	100
Chemgen-00746	A069S-N076D-G166Q	A069S-N076D-G166Q	100
Chemgen-00748	A069S-N076D-N185Q	A069S-N076D-N185Q	79
Chemgen-01054	A069S-N076D-N218S	A069S-N076D-N218S	94
Chemgen-00744	A069S-N076D-D259P	A069S-N076D-D259P	100
Chemgen-00861	A069S-T078N-G166Q	A069S-T078N-G166Q	100
Chemgen-00860	A069S-T078N-N185Q	A069S-T078N-N185Q	78
Chemgen-00859	A069S-T078N-N218S	A069S-T078N-N218S	86
Chemgen-00756	A069S-T078N-D259P	A069S-T078N-D259P	78
Chemgen-00862	A069S-G166Q-N218S	A069S-G166Q-N218S	100
Chemgen-00753	A069S-G166Q-D259P	A069S-G166Q-D259P	91
Chemgen-00659	A069S-N185Q-N218S	A069S-N185Q-N218S	60
Chemgen-00863	A069S-N185Q-D259P	A069S-N185Q-D259P	58
Chemgen-00864	A069S-N218S-D259P	A069S-N218S-D259P	79
Chemgen-00645	N076D-T078N-G166Q	N076D-T078N-G166Q	100
Chemgen-00865	N076D-T078N-N218S	N076D-T078N-N218S	90
Chemgen-00866	N076D-T078N-D259P	N076D-T078N-D259P	89
Chemgen-00770	N076D-G166Q-N218S	N076D-G166Q-N218S	100
Chemgen-01542	N076D-G166Q-D259P	N076D-G166Q-D259P	100
Chemgen-00771	N076D-N185Q-N218S	N076D-N185Q-N218S	95
Chemgen-00846	N076D-N218S-D259P	N076D-N218S-D259P	100
Chemgen-00847	T078N-G166Q-N185Q	T078N-G166Q-N185Q	91
Chemgen-00763	T078N-G166Q-N218S	T078N-G166Q-N218S	100
Chemgen-00849	T078N-N185Q-D259P	T078N-N185Q-D259P	100
Chemgen-00768	T078N-N218S-D259P	T078N-N218S-D259P	100
Chemgen-00765	G166Q-N185Q-N218S	G166Q-N185Q-N218S	87
Chemgen-00870	G166Q-N185Q-D259P	G166Q-N185Q-D259P	94
Chemgen-00880	G166Q-N218S-D259P	G166Q-N218S-D259P	100
Chemgen-00767	N185Q-N218S-D259P	N185Q-N218S-D259P	86
Chemgen-01489	T003V-T009E-P040E-N185Q	T003V-T009E-P040E-N185Q	65
Chemgen-01037	T003V-T009E-A069S-T078N	T003V-T009E-A069S-T078N	98
Chemgen-01217	T003V-P040E-G166Q-D259P	T003V-P040E-G166Q-D259P	100
Chemgen-01076	T003V-N076D-T078N-D259P	T003V-N076D-T078N-D259P	100
Chemgen-01516	T003V-N185Q-N218S-D259P	T003V-N185Q-N218S-D259P	100
Chemgen-01083	T009E-P040E-A069S-T078N	T009E-P040E-A069S-T078N	79
Chemgen-01336	T009E-P040E-A069S-N185Q	T009E-P040E-A069S-N185Q	61
Chemgen-00796	T009E-P040E-A069S-D259P	T009E-P040E-A069S-D259P	61
Chemgen-01279	T009E-P040E-N076D-T078N	T009E-P040E-N076D-T078N	91
Chemgen-01153	T009E-P040E-T078N-N185Q	T009E-P040E-T078N-N185Q	77
Chemgen-01326	T009E-P040E-N185Q-N218S	T009E-P040E-N185Q-N218S	68
Chemgen-01040	T009E-P040E-N185Q-D259P	T009E-P040E-N185Q-D259P	73
Chemgen-01521	T009E-A069S-N076D-G166Q	T009E-A069S-N076D-G166Q	100
Chemgen-01261	T009E-A069S-N076D-N185Q	T009E-A069S-N076D-N185Q	76
Chemgen-01074	T009E-N076D-G166Q-N218S	T009E-N076D-G166Q-N218S	98
Chemgen-01290	T009E-T078N-G166Q-N185Q	T009E-T078N-G166Q-N185Q	100
Chemgen-01624	P040E-A069S-T078N-N218S	P040E-A069S-T078N-N218S	100
Chemgen-01317	P040E-A069S-G166Q-N218S	P040E-A069S-G166Q-N218S	88
Chemgen-01466	P040E-A069S-G166Q-D259P	P040E-A069S-G166Q-D259P	89
Chemgen-01147	P040E-N076D-T078N-N185Q	P040E-N076D-T078N-N185Q	100
Chemgen-01000	P040E-N076D-T078N-N218S	P040E-N076D-T078N-N218S	100
Chemgen-01238	P040E-N076D-G166Q-D259P	P040E-N076D-G166Q-D259P	100
Chemgen-01578	P040E-T078N-N185Q-D259P	P040E-T078N-N185Q-D259P	96
Chemgen-01527	P040E-T078N-N218S-D259P	P040E-T078N-N218S-D259P	79
Chemgen-01548	A069S-N076D-T078N-N185Q	A069S-N076D-T078N-N185Q	75
Chemgen-01080	A069S-N076D-T078N-D259P	A069S-N076D-T078N-D259P	100
Chemgen-01510	A069S-N076D-G166Q-D259P	A069S-N076D-G166Q-D259P	100
Chemgen-01158	A069S-T078N-G166Q-N185Q	A069S-T078N-G166Q-N185Q	100
Chemgen-01278	A069S-T078N-N185Q-D259P	A069S-T078N-N185Q-D259P	80
Chemgen-01073	N076D-T078N-G166Q-N218S	N076D-T078N-G166Q-N218S	100
Chemgen-01184	N076D-T078N-G166Q-D259P	N076D-T078N-G166Q-D259P	100
Chemgen-01273	N076D-T078N-N218S-D259P	N076D-T078N-N218S-D259P	100
Chemgen-01595	T078N-N185Q-N218S-D259P	T078N-N185Q-N218S-D259P	100

Example 15

Bpan01744 Subtilisin Variants with Improved Stability in Detergent

Variants of Bpan01744 (SEQ ID NO: 13) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Tables 33A and 33B show the results for Bpan01744 variants.

TABLE 33A
Bpan01744 subtilisin variants with improved stability in liquid detergent at 46°
C. (reported as percent residual activity, % RA) compared to Bpan01744 parent
	Cleaning performance, PI
Bpan01744	Substitutions in	Substitutions		BMI stain	PAS-38 stain	BMI stain
Variant	Bpan01744	in BPN′		in PNB	in GSM-B	in ECE-2
Sample ID	numbering	numbering	% RA	detergent	detergent	detergent
Bpan01744	—	—	11	1.0	1.0	1.0
Bpan01744-	S003V-A067S-	S003V-A069S-	72	1.4	1.2	1.2
00169	N074D	N076D
Bpan01744-	S003V-A067S-	S003V-A069S-	48	1.2	1.4	1.1
00248	S127P	S129P
Bpan01744-	S003V-A067S-	S003V-A069S-	29	1.2	1.2	1.2
00150	N179Q	N185Q
Bpan01744-	S003V-N074D-	S003V-N076D-	81	1.3	1.3	1.0
00270	S127P	S129P
Bpan01744-	S003V-N074D-	S003V-N076D-	74	1.5	1.2	1.2
00170	N179Q	N185Q
Bpan01744-	S003V-S076N-	S003V-S078N-	66	1.0	1.1	1.0
00059	G160Q	G166Q
Bpan01744-	S003V-S127P-	S003V-S129P-	40	1.2	1.1	1.3
00518	N253P	N259P
Bpan01744-	S003V-G160Q-	S003V-G166Q-	85	1.1	1.0	1.0
00547	N212S	N218S
Bpan01744-	A067S-N074D-	A069S-N076D-	73	1.2	1.5	1.2
00291	N179Q	N185Q
Bpan01744-	A067S-G160Q-	A069S-G166Q-	53	1.1	1.1	0.8
00174	N179Q	N185Q
Bpan01744-	N074D-S076N-	N076D-S078N-	100	1.2	1.1	0.8
00228	G160Q	G166Q
Bpan01744-	N074D-S127P-	N076D-S129P-	77	1.2	1.2	0.9
00478	N212S	N218S
Bpan01744-	S076N-S127P-	S078N-S129P-	43	1.3	1.1	1.2
00040	N253P	N259P
Bpan01744-	S076N-N179Q-	S078N-N185Q-	37	1.5	1.2	1.3
00034	N253P	N259P
Bpan01744-	S003V-A067S-	S003V-A069S-	79	1.2	1.1	1.0
00224	N074D-S076N	N076D-S078N
Bpan01744-	S003V-A067S-	S003V-A069S-	73	1.5	1.5	0.9
00437	N074D-N179Q	N076D-N185Q
Bpan01744-	S003V-A067S-	S003V-A069S-	75	1.2	1.2	1.3
00104	N074D-N253P	N076D-N259P
Bpan01744-	S003V-A067S-	S003V-A069S-	51	1.2	1.1	1.1
00130	S076N-S127P	S078N-S129P
Bpan01744-	S003V-A067S-	S003V-A069S-	50	1.2	1.2	1.0
00468	S076N-N179Q	S078N-N185Q
Bpan01744-	S003V-A067S-	S003V-A069S-	67	1.1	1.1	0.8
00456	S127P-N212S	S129P-N218S
Bpan01744-	S003V-A067S-	S003V-A069S-	32	1.1	0.2	1.1
00474	N179Q-N253P	N185Q-N259P
Bpan01744-	S003V-N074D-	S003V-N076D-	93	1.1	1.1	0.9
00010	S127P-G160Q	S129P-G166Q
Bpan01744-	S003V-N074D-	S003V-N076D-	84	1.3	1.1	1.2
00155	S127P-N179Q	S129P-N185Q
Bpan01744-	S003V-N074D-	S003V-N076D-	81	1.3	1.3	0.9
00470	S127P-N253P	S129P-N259P
Bpan01744-	S003V-S076N-	S003V-S078N-	78	2.2	1.4	1.4
00562	N179Q-N212S	N185Q-N218S
Bpan01744-	S003V-S076N-	S003V-S078N-	56	1.3	1.3	1.5
00164	N179Q-N253P	N185Q-N259P
Bpan01744-	S003V-G160Q-	S003V-G166Q-	57	1.1	1.3	1.0
00435	N179Q-N253P	N185Q-N259P
Bpan01744-	S003V-N179Q-	S003V-N185Q-	60	1.2	1.5	1.0
00269	N212S-N253P	N218S-N259P
Bpan01744-	A067S-N074D-	A069S-N076D-	84	1.3	1.2	0.9
00259	S076N-N212S	S078N-N218S
Bpan01744-	A067S-N074D-	A069S-N076D-	76	1.2	1.1	1.1
00515	N179Q-N253P	N185Q-N259P
Bpan01744-	A067S-S076N-	A069S-S078N-	74	1.2	0.3	0.8
00272	N179Q-N212S	N185Q-N218S
Bpan01744-	A067S-S127P-	A069S-S129P-	88	0.9	1.2	0.9
00301	G160Q-N212S	G166Q-N218S
Bpan01744-	A067S-S127P-	A069S-S129P-	57	1.6	1.3	1.4
00573	N179Q-N212S	N185Q-N218S
Bpan01744-	A067S-G160Q-	A069S-G166Q-	80	1.1	1.0	0.9
00035	N212S-N253P	N218S-N259P
Bpan01744-	A067S-N179Q-	A069S-N185Q-	50	1.3	1.3	0.9
00479	N212S-N253P	N218S-N259P
Bpan01744-	N074D-S076N-	N076D-S078N-	87	1.2	1.3	1.0
00415	S127P-N179Q	S129P-N185Q
Bpan01744-	N074D-S076N-	N076D-S078N-	85	1.2	1.4	1.0
00452	N179Q-N212S	N185Q-N218S
Bpan01744-	N074D-S127P-	N076D-S129P-	83	1.3	1.1	1.1
00183	N179Q-N212S	N185Q-N218S
Bpan01744-	N074D-G160Q-	N076D-G166Q-	100	1.1	1.3	0.9
00261	N179Q-N253P	N185Q-N259P
Bpan01744-	S076N-S127P-	S078N-S129P-	70	1.0	1.1	1.1
00143	G160Q-N253P	G166Q-N259P
Bpan01744-	S076N-S127P-	S078N-S129P-	50	1.3	1.3	1.1
00246	N179Q-N253P	N185Q-N259P
Bpan01744-	S076N-G160Q-	S078N-G166Q-	83	1.1	1.2	1.0
00432	N212S-N253P	N218S-N259P
Bpan01744-	S127P-G160Q-	S129P-G166Q-	77	1.1	1.1	0.8
00300	N212S-N253P	N218S-N259P
Bpan01744-	S127P-N179Q-	S129P-N185Q-	53	1.2	1.3	1.1
00292	N212S-N253P	N218S-N259P

TABLE 33B
Bpan01744 subtilisin variants with improved stability in liquid detergent at 44°
C. (reported as percent residual activity, % RA) compared to Bpan01744 parent
Bpan01744	Substitutions in
Variant Sample	Bpan01744	Substitutions in
ID	numbering	BPN′ numbering	% RA
Bpan01744	—	—	19
Bpan01744-01709	S003V-S009E-A067S	S003V-S009E-A069S	52
Bpan01744-01195	S003V-S009E-N074D	S003V-S009E-N076D	91
Bpan01744-01267	S003V-S009E-S076N	S003V-S009E-S078N	67
Bpan01744-01140	S003V-S009E-N179Q	S003V-S009E-N185Q	62
Bpan01744-01141	S003V-S009E-N212S	S003V-S009E-N218S	86
Bpan01744-01315	S003V-S009E-N253P	S003V-S009E-N259P	59
Bpan01744-01142	S003V-A067S-S076N	S003V-A069S-S078N	50
Bpan01744-00677	S003V-A067S-N212S	S003V-A069S-N218S	62
Bpan01744-01265	S003V-N074D-S076N	S003V-N076D-S078N	81
Bpan01744-01585	S003V-N074D-G160Q	S003V-N076D-G166Q	100
Bpan01744-01252	S003V-N074D-N212S	S003V-N076D-N218S	98
Bpan01744-01253	S003V-S076N-N179Q	S003V-S078N-N185Q	52
Bpan01744-01254	S003V-S076N-N212S	S003V-S078N-N218S	78
Bpan01744-01260	S003V-S076N-N253P	S003V-S078N-N259P	49
Bpan01744-01274	S003V-N179Q-N212S	S003V-N185Q-N218S	67
Bpan01744-00685	S003V-N179Q-N253P	S003V-N185Q-N259P	39
Bpan01744-01275	S003V-N212S-N253P	S003V-N218S-N259P	60
Bpan01744-00693	S009E-A067S-N074D	S009E-A069S-N076D	77
Bpan01744-00777	S009E-A067S-S076N	S009E-A069S-S078N	44
Bpan01744-00898	S009E-A067S-G160Q	S009E-A069S-G166Q	100
Bpan01744-01231	S009E-A067S-N253P	S009E-A069S-N259P	33
Bpan01744-01236	S009E-N074D-S076N	S009E-N076D-S078N	92
Bpan01744-01237	S009E-N074D-G160Q	S009E-N076D-G166Q	65
Bpan01744-01238	S009E-N074D-N179Q	S009E-N076D-N185Q	90
Bpan01744-01196	S009E-N074D-N212S	S009E-N076D-N218S	99
Bpan01744-01307	S009E-N074D-N253P	S009E-N076D-N259P	73
Bpan01744-01248	S009E-S076N-N179Q	S009E-S078N-N185Q	59
Bpan01744-01241	S009E-S076N-N212S	S009E-S078N-N218S	85
Bpan01744-01242	S009E-S076N-N253P	S009E-S078N-N259P	50
Bpan01744-01165	S009E-G160Q-N179Q	S009E-G166Q-N185Q	65
Bpan01744-01244	S009E-G160Q-N212S	S009E-G166Q-N218S	99
Bpan01744-00976	S009E-N179Q-N212S	S009E-N185Q-N218S	78
Bpan01744-01351	S009E-N179Q-N253P	S009E-N185Q-N259P	31
Bpan01744-01157	S009E-N212S-N253P	S009E-N218S-N259P	68
Bpan01744-00810	A067S-N074D-S076N	A069S-N076D-S078N	91
Bpan01744-01159	A067S-N074D-G160Q	A069S-N076D-G166Q	74
Bpan01744-00736	A067S-N074D-N212S	A069S-N076D-N218S	85
Bpan01744-00888	A067S-N074D-N253P	A069S-N076D-N259P	71
Bpan01744-01154	A067S-S076N-N179Q	A069S-S078N-N185Q	40
Bpan01744-01162	A067S-S076N-N212S	A069S-S078N-N218S	70
Bpan01744-01779	A067S-S076N-N253P	A069S-S078N-N259P	38
Bpan01744-01885	A067S-G160Q-N212S	A069S-G166Q-N218S	100
Bpan01744-00963	A067S-N179Q-N212S	A069S-N185Q-N218S	50
Bpan01744-01174	A067S-N212S-N253P	A069S-N218S-N259P	44
Bpan01744-01182	N074D-S076N-N179Q	N076D-S078N-N185Q	86
Bpan01744-01173	N074D-S076N-N212S	N076D-S078N-N218S	95
Bpan01744-01957	N074D-S076N-N253P	N076D-S078N-N259P	75
Bpan01744-01170	N074D-G160Q-N212S	N076D-G166Q-N218S	100
Bpan01744-01292	N074D-N179Q-N212S	N076D-N185Q-N218S	95
Bpan01744-01289	N074D-N179Q-N253P	N076D-N185Q-N259P	76
Bpan01744-01294	S076N-G160Q-N212S	S078N-G166Q-N218S	86
Bpan01744-01717	S076N-N179Q-N212S	S078N-N185Q-N218S	73
Bpan01744-01952	S076N-N212S-N253P	S078N-N218S-N259P	61
Bpan01744-00899	G160Q-N179Q-N212S	G166Q-N185Q-N218S	96
Bpan01744-01867	N179Q-N212S-N253P	N185Q-N218S-N259P	48
Bpan01744-00749	S003V-S009E-A067S-N179Q	S003V-S009E-A069S-N185Q	58
Bpan01744-02012	S003V-S009E-N212S-N253P	S003V-S009E-N218S-N259P	77
Bpan01744-00879	S003V-A067S-N074D-N212S	S003V-A069S-N076D-N218S	94
Bpan01744-02146	S003V-A067S-N179Q-N212S	S003V-A069S-N185Q-N218S	71
Bpan01744-01874	S003V-A067S-N212S-N253P	S003V-A069S-N218S-N259P	59
Bpan01744-01995	S003V-N074D-N212S-N253P	S003V-N076D-N218S-N259P	84
Bpan01744-01812	S003V-G160Q-N179Q-N212S	S003V-G166Q-N185Q-N218S	87
Bpan01744-00832	S009E-A067S-N074D-S076N	S009E-A069S-N076D-S078N	95
Bpan01744-01838	S009E-A067S-S076N-G160Q	S009E-A069S-S078N-G166Q	44
Bpan01744-01675	S009E-A067S-S076N-N212S	S009E-A069S-S078N-N218S	87
Bpan01744-01985	S009E-A067S-G160Q-N212S	S009E-A069S-G166Q-N218S	59
Bpan01744-00765	S009E-A067S-N212S-N253P	S009E-A069S-N218S-N259P	67
Bpan01744-01907	S009E-N074D-S076N-G160Q	S009E-N076D-S078N-G166Q	77
Bpan01744-02114	S009E-N074D-S076N-N179Q	S009E-N076D-S078N-N185Q	90
Bpan01744-00845	S009E-N074D-N212S-N253P	S009E-N076D-N218S-N259P	89
Bpan01744-01813	S009E-S076N-N179Q-N212S	S009E-S078N-N185Q-N218S	87
Bpan01744-01847	S009E-S076N-N212S-N253P	S009E-S078N-N218S-N259P	80
Bpan01744-00974	S009E-G160Q-N179Q-N212S	S009E-G166Q-N185Q-N218S	100
Bpan01744-01036	A067S-N074D-S076N-N253P	A069S-N076D-S078N-N259P	81
Bpan01744-00791	A067S-S076N-G160Q-N212S	A069S-S078N-G166Q-N218S	83
Bpan01744-01856	A067S-S076N-N179Q-N253P	A069S-S078N-N185Q-N259P	36
Bpan01744-00743	A067S-S076N-N212S-N253P	A069S-S078N-N218S-N259P	67
Bpan01744-01873	N074D-S076N-G160Q-N179Q	N076D-S078N-G166Q-N185Q	100
Bpan01744-00813	N074D-S076N-G160Q-N212S	N076D-S078N-G166Q-N218S	95
Bpan01744-02034	N074D-S076N-N212S-N253P	N076D-S078N-N218S-N259P	86
Bpan01744-02019	N074D-G160Q-N179Q-N212S	N076D-G166Q-N185Q-N218S	100
Bpan01744-01690	N074D-G160Q-N212S-N253P	N076D-G166Q-N218S-N259P	86
Bpan01744-00970	N074D-N179Q-N212S-N253P	N076D-N185Q-N218S-N259P	97
Bpan01744-00912	S076N-G160Q-N179Q-N212S	S078N-G166Q-N185Q-N218S	95
Bpan01744-01078	S076N-G160Q-N179Q-N253P	S078N-G166Q-N185Q-N259P	100

Example 16

DSM14391 Subtilisin Variants with Improved Stability in Detergent

Variants of DSM14391 (SEQ ID NO: 14) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Tables 34A and 34B show the results for DSM14391 variants.

TABLE 34A
DSM14391 subtilisin variants with improved stability in liquid detergent at 36°
C. (reported as percent residual activity, % RA) compared to DSM14391 parent
DSM14391 Variant	Substitutions in	Substitutions in		BMI stain in	PAS-38 stain in
Sample ID	DSM14391 numbering	BPN′ numbering	% RA	PNB detergent	GSM-B detergent
DSM14391	—	—	11	1.0	1.0
DSM14391-00421	T003V-S039E-P212S	T003V-S040E-P218S	93	1.2	1.1
DSM14391-00214	T003V-G160Q-P212S	T003V-G166Q-P218S	100	0.9	1.1
DSM14391-00359	T003V-G160Q-N253P	T003V-G166Q-N259P	75	1.0	1.2
DSM14391-00272	S039E-N074D-G160Q	S040E-N076D-G166Q	100	1.3	1.2
DSM14391-00469	S039E-N074D-R179Q	S040E-N076D-R185Q	96	1.5	1.3
DSM14391-00251	S039E-S076N-D127P	S040E-S078N-D129P	59	1.0	1.1
DSM14391-00281	S039E-R179Q-P212S	S040E-R185Q-P218S	100	1.4	1.1
DSM14391-00293	S039E-R179Q-N253P	S040E-R185Q-N259P	100	1.6	1.2
DSM14391-00474	A067S-S076N-N253P	A069S-S078N-N259P	32	1.1	1.2
DSM14391-00357	A067S-D127P-P212S	A069S-D129P-P218S	91	1.0	0.8
DSM14391-00264	A067S-P212S-N253P	A069S-P218S-N259P	100	1.1	0.9
DSM14391-00473	N074D-R179Q-P212S	N076D-R185Q-P218S	95	1.5	1.1
DSM14391-00162	N074D-P212S-N253P	N076D-P218S-N259P	100	1.0	1.1
DSM14391-00170	S076N-R179Q-P212S	S078N-R185Q-P218S	100	1.7	1.0
DSM14391-00263	S076N-P212S-N253P	S078N-P218S-N259P	100	1.0	1.0
DSM14391-00169	T003V-S039E-	T003V-S040E-	61	1.6	1.3
	A067S-N074D	A069S-N076D
DSM14391-00292	T003V-S039E-	T003V-S040E-	79	1.4	1.3
	N074D-S076N	N076D-S078N
DSM14391-00463	T003V-S039E-	T003V-S040E-	61	1.0	1.1
	N074D-D127P	N076D-D129P
DSM14391-00221	T003V-S039E-	T003V-S040E-	100	1.3	1.3
	G160Q-R179Q	G166Q-R185Q
DSM14391-00457	T003V-S039E-	T003V-S040E-	93	1.0	1.2
	G160Q-N253P	G166Q-N259P
DSM14391-00454	T003V-A067S-	T003V-A069S-	49	1.6	1.2
	R179Q-N253P	R185Q-N259P
DSM14391-00386	T003V-N074D-	T003V-N076D-	66	1.3	1.3
	S076N-N253P	S078N-N259P
DSM14391-00328	T003V-N074D-	T003V-N076D-	92	1.5	1.3
	R179Q-N253P	R185Q-N259P
DSM14391-00179	T003V-S076N-	T003V-S078N-	37	0.9	1.0
	D127P-N253P	D129P-N259P
DSM14391-00273	T003V-S076N-	T003V-S078N-	100	1.1	1.0
	P212S-N253P	P218S-N259P
DSM14391-00306	S039E-N074D-	S040E-N076D-	100	1.6	1.2
	S076N-R179Q	S078N-R185Q
DSM14391-00178	S039E-N074D-	S040E-N076D-	100	1.3	1.1
	P212S-N253P	P218S-N259P
DSM14391-00405	S039E-S076N-	S040E-S078N-	100	1.2	1.1
	G160Q-P212S	G166Q-P218S
DSM14391-00402	S039E-R179Q-	S040E-R185Q-	100	1.4	1.1
	P212S-N253P	P218S-N259P
DSM14391-00226	A067S-D127P-	A069S-D129P-	100	1.0	0.9
	R179Q-P212S	R185Q-P218S
DSM14391-00267	A067S-R179Q-	A069S-R185Q-	100	1.2	0.9
	P212S-N253P	P218S-N259P
DSM14391-00345	S076N-D127P-	S078N-D129P-	100	1.1	1.0
	R179Q-P212S	R185Q-P218S
DSM14391-00439	D127P-R179Q-	D129P-R185Q-	100	1.0	0.9
	P212S-N253P	P218S-N259P

TABLE 34B
DSM14391 subtilisin variants with improved stability in liquid detergent at 30°
C. (reported as percent residual activity, % RA) compared to DSM14391 parent
DSM14391 Variant	Substitutions in	Substitutions in
Sample ID	DSM14391 numbering	BPN′ numbering	% RA
DSM14391	—	—	40
DSM14391-00824	T003V-T009E-S039E	T003V-T009E-S040E	98
DSM14391-00996	T003V-T009E-A067S	T003V-T009E-A069S	89
DSM14391-00995	T003V-T009E-N074D	T003V-T009E-N076D	98
DSM14391-00823	T003V-T009E-S076N	T003V-T009E-S078N	100
DSM14391-00868	T003V-T009E-R179Q	T003V-T009E-R185Q	82
DSM14391-00822	T003V-T009E-P212S	T003V-T009E-P218S	100
DSM14391-00817	T003V-T009E-N253P	T003V-T009E-N259P	86
DSM14391-00836	T003V-S039E-A067S	T003V-S040E-A069S	77
DSM14391-00819	T003V-S039E-N074D	T003V-S040E-N076D	88
DSM14391-00837	T003V-S039E-S076N	T003V-S040E-S078N	76
DSM14391-01004	T003V-S039E-R179Q	T003V-S040E-R185Q	94
DSM14391-00829	T003V-S039E-N253P	T003V-S040E-N259P	70
DSM14391-00831	T003V-A067S-N074D	T003V-A069S-N076D	100
DSM14391-00858	T003V-A067S-R179Q	T003V-A069S-R185Q	76
DSM14391-00992	T003V-A067S-P212S	T003V-A069S-P218S	97
DSM14391-00833	T003V-N074D-S076N	T003V-N076D-S078N	86
DSM14391-01080	T003V-N074D-R179Q	T003V-N076D-R185Q	92
DSM14391-01084	T003V-N074D-P212S	T003V-N076D-P218S	98
DSM14391-01058	T003V-N074D-N253P	T003V-N076D-N259P	87
DSM14391-00922	T003V-S076N-R179Q	T003V-S078N-R185Q	90
DSM14391-01083	T003V-S076N-P212S	T003V-S078N-P218S	91
DSM14391-01054	T003V-S076N-N253P	T003V-S078N-N259P	68
DSM14391-00929	T003V-R179Q-P212S	T003V-R185Q-P218S	98
DSM14391-01078	T003V-R179Q-N253P	T003V-R185Q-N259P	79
DSM14391-00919	T003V-P212S-N253P	T003V-P218S-N259P	97
DSM14391-00931	T009E-S039E-N074D	T009E-S040E-N076D	100
DSM14391-00928	T009E-S039E-S076N	T009E-S040E-S078N	85
DSM14391-01025	T009E-S039E-R179Q	T009E-S040E-R185Q	91
DSM14391-01034	T009E-S039E-P212S	T009E-S040E-P218S	97
DSM14391-00899	T009E-S039E-N253P	T009E-S040E-N259P	93
DSM14391-01035	T009E-A067S-S076N	T009E-A069S-S078N	92
DSM14391-01030	T009E-A067S-P212S	T009E-A069S-P218S	88
DSM14391-01042	T009E-A067S-N253P	T009E-A069S-N259P	89
DSM14391-01040	T009E-N074D-S076N	T009E-N076D-S078N	89
DSM14391-00908	T009E-N074D-R179Q	T009E-N076D-R185Q	95
DSM14391-01038	T009E-N074D-P212S	T009E-N076D-P218S	95
DSM14391-01049	T009E-N074D-N253P	T009E-N076D-N259P	82
DSM14391-00915	T009E-S076N-R179Q	T009E-S078N-R185Q	99
DSM14391-01047	T009E-S076N-P212S	T009E-S078N-P218S	97
DSM14391-01053	T009E-S076N-N253P	T009E-S078N-N259P	98
DSM14391-01043	T009E-G160Q-P212S	T009E-G166Q-P218S	100
DSM14391-00911	T009E-R179Q-P212S	T009E-R185Q-P218S	92
DSM14391-00912	T009E-R179Q-N253P	T009E-R185Q-N259P	99
DSM14391-01045	T009E-P212S-N253P	T009E-P218S-N259P	98
DSM14391-01074	S039E-A067S-N074D	S040E-A069S-N076D	86
DSM14391-00689	S039E-A067S-R179Q	S040E-A069S-R185Q	88
DSM14391-01032	S039E-A067S-P212S	S040E-A069S-P218S	97
DSM14391-01033	S039E-A067S-N253P	S040E-A069S-N259P	88
DSM14391-01352	S039E-N074D-S076N	S040E-N076D-S078N	84
DSM14391-00953	S039E-N074D-P212S	S040E-N076D-P218S	95
DSM14391-00686	S039E-N074D-N253P	S040E-N076D-N259P	91
DSM14391-01066	S039E-S076N-R179Q	S040E-S078N-R185Q	89
DSM14391-01065	S039E-S076N-P212S	S040E-S078N-P218S	97
DSM14391-00659	S039E-S076N-N253P	S040E-S078N-N259P	79
DSM14391-00947	S039E-G160Q-P212S	S040E-G166Q-P218S	85
DSM14391-00960	S039E-P212S-N253P	S040E-P218S-N259P	97
DSM14391-00945	A067S-N074D-S076N	A069S-N076D-S078N	81
DSM14391-01016	A067S-N074D-R179Q	A069S-N076D-R185Q	91
DSM14391-01013	A067S-N074D-P212S	A069S-N076D-P218S	100
DSM14391-01012	A067S-N074D-N253P	A069S-N076D-N259P	98
DSM14391-00883	A067S-S076N-R179Q	A069S-S078N-R185Q	89
DSM14391-01014	A067S-S076N-P212S	A069S-S078N-P218S	96
DSM14391-01007	A067S-G160Q-P212S	A069S-G166Q-P218S	98
DSM14391-01105	A067S-R179Q-P212S	A069S-R185Q-P218S	88
DSM14391-00882	A067S-R179Q-N253P	A069S-R185Q-N259P	98
DSM14391-01018	N074D-S076N-R179Q	N076D-S078N-R185Q	91
DSM14391-01021	N074D-S076N-P212S	N076D-S078N-P218S	100
DSM14391-01019	N074D-S076N-N253P	N076D-S078N-N259P	89
DSM14391-01100	N074D-G160Q-P212S	N076D-G166Q-P218S	74
DSM14391-01090	S076N-G160Q-P212S	S078N-G166Q-P218S	76
DSM14391-01086	S076N-R179Q-N253P	S078N-R185Q-N259P	89
DSM14391-00893	G160Q-R179Q-P212S	G166Q-R185Q-P218S	100
DSM14391-01445	G160Q-P212S-N253P	G166Q-P218S-N259P	100
DSM14391-00872	R179Q-P212S-N253P	R185Q-P218S-N259P	100
DSM14391-01556	T003V-T009E-S039E-N074D	T003V-T009E-S040E-N076D	99
DSM14391-01330	T003V-A067S-S076N-P212S	T003V-A069S-S078N-P218S	96
DSM14391-01481	T003V-G160Q-R179Q-P212S	T003V-G166Q-R185Q-P218S	100
DSM14391-01588	T003V-R179Q-P212S-N253P	T003V-R185Q-P218S-N259P	99
DSM14391-01843	T009E-S039E-A067S-P212S	T009E-S040E-A069S-P218S	100
DSM14391-01801	T009E-A067S-N074D-R179Q	T009E-A069S-N076D-R185Q	100
DSM14391-01467	T009E-N074D-S076N-R179Q	T009E-N076D-S078N-R185Q	93
DSM14391-01565	T009E-N074D-G160Q-P212S	T009E-N076D-G166Q-P218S	97
DSM14391-01293	T009E-S076N-R179Q-P212S	T009E-S078N-R185Q-P218S	100
DSM14391-01322	S039E-A067S-S076N-P212S	S040E-A069S-S078N-P218S	100
DSM14391-01634	S039E-A067S-G160Q-P212S	S040E-A069S-G166Q-P218S	92
DSM14391-01764	S039E-N074D-G160Q-R179Q	S040E-N076D-G166Q-R185Q	100
DSM14391-01881	S039E-N074D-G160Q-P212S	S040E-N076D-G166Q-P218S	100
DSM14391-01649	A067S-N074D-S076N-N253P	A069S-N076D-S078N-N259P	88
DSM14391-01617	A067S-S076N-R179Q-P212S	A069S-S078N-R185Q-P218S	100
DSM14391-01888	N074D-S076N-G160Q-R179Q	N076D-S078N-G166Q-R185Q	81
DSM14391-01490	S076N-G160Q-P212S-N253P	S078N-G166Q-P218S-N259P	83
DSM14391-01092	S076N-R179Q-P212S-N253P	S078N-R185Q-P218S-N259P	94

Example 17

BspAI02518 Subtilisin Variants with Improved Stability in Detergent

Variants of BspAI02518 (SEQ ID NO: 16) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Table 35A and 35B show the results for BspAI02518 variants.

TABLE 35A
BspAI02518 subtilisin variants with improved stability in liquid detergent at 36°
C. (reported as percent residual activity, % RA) compared to BspAI02518 parent
	Cleaning performance, PI
BspAI02518	Substitutions in	Substitutions		BMI stain	PAS-38 stain	BMI stain
Variant	BspAI02518	in BPN′		in PNB	in GSM-B	in ECE-2
Sample ID	numbering	numbering	% RA	detergent	detergent	detergent
BspAI02518	—	—	12	1.0	1.0	1.0
BspAI02518-	S003V-A067S-	S003V-A069S-	29	1.1	1.9	1.2
00534	N179Q	N185Q
BspAI02518-	S003V-S076N-	S003V-S078N-	38	1.1	1.2	1.0
00467	G126S	G128S
BspAI02518-	S003V-N179Q-	S003V-N185Q-	40	0.8	1.0	0.8
00539	N253P	N259P
BspAI02518-	A067S-N074D-	A069S-N076D-	57	1.6	5.4	1.9
00442	G126S	G128S
BspAI02518-	A067S-N074D-	A069S-N076D-	73	1.2	1.2	0.8
00870	N212S	N218S
BspAI02518-	A067S-S076N-	A069S-S078N-	23	1.1	1.3	0.9
00868	M122I	M124I
BspAI02518-	N074D-S076N-	N076D-S078N-	77	1.1	2.1	1.5
00999	N179Q	N185Q
BspAI02518-	N074D-S076N-	N076D-S078N-	86	1.0	1.0	0.9
00507	N212S	N218S
BspAI02518-	N074D-G126S-	N076D-G128S-	69	1.5	2.8	1.5
00939	S160Q	S166Q
BspAI02518-	M122I-G126S-	M124I-G128S-	21	1.2	1.1	0.6
00708	N179Q	N185Q
BspAI02518-	G126S-S160Q-	G128S-S166Q-	40	1.3	1.8	1.0
00607	N179Q	N185Q
BspAI02518-	S003V-A067S-	S003V-A069S-	62	1.5	4.8	1.8
01017	N074D-G126S	N076D-G128S
BspAI02518-	S003V-A067S-	S003V-A069S-	32	1.1	1.7	1.2
00435	S076N-G126S	S078N-G128S
BspAI02518-	S003V-A067S-	S003V-A069S-	60	0.9	1.9	1.4
00768	S076N-S160Q	S078N-S166Q
BspAI02518-	S003V-A067S-	S003V-A069S-	56	1.0	1.0	1.0
00518	M122I-N212S	M124I-N218S
BspAI02518-	S003V-A067S-	S003V-A069S-	47	1.4	2.3	1.8
00555	G126S-S160Q	G128S-S166Q
BspAI02518-	S003V-A067S-	S003V-A069S-	18	1.3	1.0	0.8
00887	G126S-N253P	G128S-N259P
BspAI02518-	S003V-N074D-	S003V-N076D-	88	1.2	1.6	1.0
00658	S160Q-N179Q	S166Q-N185Q
BspAI02518-	S003V-S076N-	S003V-S078N-	70	1.0	1.0	0.8
01005	M122I-S160Q	M124I-S166Q
BspAI02518-	S003V-S076N-	S003V-S078N-	50	1.0	1.1	1.0
00556	G126S-N179Q	G128S-N185Q
BspAI02518-	S003V-S076N-	S003V-S078N-	75	1.0	1.0	0.9
00509	G126S-N212S	G128S-N218S
BspAI02518-	S003V-M122I-	S003V-M124I-	60	0.9	1.2	0.8
00648	S160Q-N253P	S166Q-N259P
BspAI02518-	S003V-M122I-	S003V-M124I-	36	1.1	1.2	0.8
00840	N179Q-N253P	N185Q-N259P
BspAI02518-	S003V-G126S-	S003V-G128S-	60	1.2	1.1	0.8
00877	N179Q-N212S	N185Q-N218S
BspAI02518-	S003V-G126S-	S003V-G128S-	60	1.0	1.0	0.7
00567	N212S-N253P	N218S-N259P
BspAI02518-	S003V-S160Q-	S003V-S166Q-	83	0.8	1.0	0.8
00615	N212S-N253P	N218S-N259P
BspAI02518-	A067S-N074D-	A069S-N076D-	66	1.7	10.9	2.8
00738	S076N-G126S	S078N-G128S
BspAI02518-	A067S-N074D-	A069S-N076D-	70	1.5	2.6	0.8
00888	M122I-G126S	M124I-G128S
BspAI02518-	A067S-N074D-	A069S-N076D-	89	1.2	2.4	1.8
00581	S160Q-N212S	S166Q-N218S
BspAI02518-	A067S-N074D-	A069S-N076D-	57	1.4	2.2	1.4
00919	N179Q-N253P	N185Q-N259P
BspAI02518-	A067S-N074D-	A069S-N076D-	76	1.3	1.3	1.1
00530	N212S-N253P	N218S-N259P
BspAI02518-	A067S-S076N-	A069S-S078N-	57	0.9	1.0	0.9
00745	G126S-N212S	G128S-N218S
BspAI02518-	A067S-S076N-	A069S-S078N-	34	0.9	1.3	1.1
00978	N179Q-N253P	N185Q-N259P
BspAI02518-	A067S-M122I-	A069S-M124I-	39	1.1	1.2	0.3
00508	G126S-N179Q	G128S-N185Q
BspAI02518-	A067S-M122I-	A069S-M124I-	36	1.2	1.1	0.7
00921	G126S-N212S	G128S-N218S
BspAI02518-	A067S-G126S-	A069S-G128S-	71	1.4	1.6	1.2
00699	S160Q-N212S	S166Q-N218S
BspAI02518-	A067S-G126S-	A069S-G128S-	43	1.0	0.8	0.7
01013	N212S-N253P	N218S-N259P
BspAI02518-	N074D-S160Q-	N076D-S166Q-	78	0.9	1.2	0.7
00815	N179Q-N253P	N185Q-N259P
BspAI02518-	S076N-M122I-	S078N-M124I-	56	1.1	1.1	0.5
00867	G126S-N212S	G128S-N218S
BspAI02518-	S076N-N179Q-	S078N-N185Q-	75	1.1	1.2	1.0
00702	N212S-N253P	N218S-N259P
BspAI02518-	M122I-G126S-	M124I-G128S-	43	1.0	1.1	0.4
00866	S160Q-N253P	S166Q-N259P
BspAI02518-	M122I-S160Q-	M124I-S166Q-	78	1.0	1.0	0.8
00885	N212S-N253P	N218S-N259P
BspAI02518-	G126S-S160Q-	G128S-S166Q-	38	1.2	1.1	0.9
00504	N179Q-N253P	N185Q-N259P

TABLE 35B
BspAI02518 subtilisin variants with improved stability in liquid detergent at 30°
C. (reported as percent residual activity, % RA) compared to BspAI02518 parent
BspAI02518 Variant	Substitutions in	Substitutions in
Sample ID	BspAI02518 numbering	BPN′ numbering	% RA
BspAI02518	—	—	32
BspAI02518-01092	S003V-S009E-A067S	S003V-S009E-A069S	68
BspAI02518-01095	S003V-S009E-N074D	S003V-S009E-N076D	93
BspAI02518-01270	S003V-S009E-S076N	S003V-S009E-S078N	74
BspAI02518-01096	S003V-S009E-S160Q	S003V-S009E-S166Q	87
BspAI02518-01276	S003V-S009E-N179Q	S003V-S009E-N185Q	75
BspAI02518-01097	S003V-S009E-N212S	S003V-S009E-N218S	90
BspAI02518-01282	S003V-S009E-N253P	S003V-S009E-N259P	100
BspAI02518-01085	S003V-A067S-N074D	S003V-A069S-N076D	84
BspAI02518-01086	S003V-A067S-S076N	S003V-A069S-S078N	61
BspAI02518-01087	S003V-A067S-N212S	S003V-A069S-N218S	71
BspAI02518-01088	S003V-N074D-S076N	S003V-N076D-S078N	83
BspAI02518-01250	S003V-N074D-S160Q	S003V-N076D-S166Q	88
BspAI02518-01084	S003V-N074D-N179Q	S003V-N076D-N185Q	89
BspAI02518-01220	S003V-N074D-N212S	S003V-N076D-N218S	83
BspAI02518-01230	S003V-N074D-N253P	S003V-N076D-N259P	73
BspAI02518-01222	S003V-S076N-S160Q	S003V-S078N-S166Q	80
BspAI02518-01895	S003V-S076N-N179Q	S003V-S078N-N185Q	68
BspAI02518-01231	S003V-S076N-N212S	S003V-S078N-N218S	83
BspAI02518-01992	S003V-S076N-N253P	S003V-S078N-N259P	65
BspAI02518-01110	S003V-S160Q-N179Q	S003V-S166Q-N185Q	70
BspAI02518-02118	S003V-S160Q-N253P	S003V-S166Q-N259P	76
BspAI02518-01099	S003V-N179Q-N212S	S003V-N185Q-N218S	83
BspAI02518-01960	S003V-N212S-N253P	S003V-N218S-N259P	74
BspAI02518-01238	S009E-A067S-N074D	S009E-A069S-N076D	81
BspAI02518-01102	S009E-A067S-N179Q	S009E-A069S-N185Q	70
BspAI02518-01165	S009E-A067S-N212S	S009E-A069S-N218S	83
BspAI02518-01105	S009E-N074D-S076N	S009E-N076D-S078N	89
BspAI02518-01235	S009E-N074D-S160Q	S009E-N076D-S166Q	94
BspAI02518-01153	S009E-N074D-N179Q	S009E-N076D-N185Q	87
BspAI02518-02121	S009E-N074D-N212S	S009E-N076D-N218S	99
BspAI02518-01107	S009E-N074D-N253P	S009E-N076D-N259P	80
BspAI02518-02083	S009E-S076N-S160Q	S009E-S078N-S166Q	80
BspAI02518-01268	S009E-S076N-N179Q	S009E-S078N-N185Q	67
BspAI02518-01140	S009E-S076N-N212S	S009E-S078N-N218S	88
BspAI02518-01142	S009E-S076N-N253P	S009E-S078N-N259P	72
BspAI02518-01167	S009E-S160Q-N179Q	S009E-S166Q-N185Q	81
BspAI02518-01138	S009E-S160Q-N212S	S009E-S166Q-N218S	90
BspAI02518-01226	S009E-S160Q-N253P	S009E-S166Q-N259P	82
BspAI02518-01227	S009E-N179Q-N212S	S009E-N185Q-N218S	80
BspAI02518-01148	S009E-N179Q-N253P	S009E-N185Q-N259P	56
BspAI02518-01251	S009E-N212S-N253P	S009E-N218S-N259P	87
BspAI02518-01243	A067S-N074D-S076N	A069S-N076D-S078N	86
BspAI02518-01245	A067S-N074D-S160Q	A069S-N076D-S166Q	86
BspAI02518-01139	A067S-N074D-N179Q	A069S-N076D-N185Q	75
BspAI02518-01219	A067S-N074D-N253P	A069S-N076D-N259P	70
BspAI02518-01205	A067S-S076N-S160Q	A069S-S078N-S166Q	74
BspAI02518-01207	A067S-S076N-N212S	A069S-S078N-N218S	80
BspAI02518-01166	A067S-S160Q-N179Q	A069S-S166Q-N185Q	66
BspAI02518-01201	A067S-S160Q-N212S	A069S-S166Q-N218S	84
BspAI02518-01151	A067S-S160Q-N253P	A069S-S166Q-N259P	64
BspAI02518-01215	A067S-N179Q-N212S	A069S-N185Q-N218S	73
BspAI02518-01202	A067S-N212S-N253P	A069S-N218S-N259P	69
BspAI02518-01199	N074D-S076N-S160Q	N076D-S078N-S166Q	93
BspAI02518-01218	N074D-S076N-N253P	N076D-S078N-N259P	76
BspAI02518-01210	N074D-S160Q-N179Q	N076D-S166Q-N185Q	85
BspAI02518-01211	N074D-S160Q-N212S	N076D-S166Q-N218S	96
BspAI02518-01212	N074D-S160Q-N253P	N076D-S166Q-N259P	91
BspAI02518-01124	N074D-N179Q-N212S	N076D-N185Q-N218S	92
BspAI02518-01132	N074D-N179Q-N253P	N076D-N185Q-N259P	80
BspAI02518-01122	N074D-N212S-N253P	N076D-N218S-N259P	88
BspAI02518-02142	S076N-S160Q-N179Q	S078N-S166Q-N185Q	89
BspAI02518-01128	S076N-S160Q-N212S	S078N-S166Q-N218S	89
BspAI02518-01119	S076N-S160Q-N253P	S078N-S166Q-N259P	67
BspAI02518-01114	S076N-N179Q-N212S	S078N-N185Q-N218S	83
BspAI02518-01263	S076N-N179Q-N253P	S078N-N185Q-N259P	62
BspAI02518-01115	S076N-N212S-N253P	S078N-N218S-N259P	79
BspAI02518-01265	S160Q-N179Q-N212S	S166Q-N185Q-N218S	86
BspAI02518-01117	S160Q-N179Q-N253P	S166Q-N185Q-N259P	65
BspAI02518-01116	S160Q-N212S-N253P	S166Q-N218S-N259P	84
BspAI02518-01621	S003V-S009E-N074D-S160Q	S003V-S009E-N076D-S166Q	100
BspAI02518-02172	S003V-A067S-S076N-N253P	S003V-A069S-S078N-N259P	64
BspAI02518-02095	S003V-A067S-S160Q-N212S	S003V-A069S-S166Q-N218S	93
BspAI02518-01687	S003V-N074D-S076N-S160Q	S003V-N076D-S078N-S166Q	96
BspAI02518-01786	S003V-N074D-S076N-N253P	S003V-N076D-S078N-N259P	82
BspAI02518-01818	S003V-N074D-S160Q-N253P	S003V-N076D-S166Q-N259P	95
BspAI02518-01782	S003V-S160Q-N179Q-N253P	S003V-S166Q-N185Q-N259P	82
BspAI02518-02090	S009E-A067S-N074D-S160Q	S009E-A069S-N076D-S166Q	85
BspAI02518-01666	S009E-A067S-N074D-N212S	S009E-A069S-N076D-N218S	87
BspAI02518-01743	S009E-A067S-N074D-N253P	S009E-A069S-N076D-N259P	86
BspAI02518-01859	S009E-A067S-S076N-S160Q	S009E-A069S-S078N-S166Q	84
BspAI02518-01646	S009E-A067S-S076N-N212S	S009E-A069S-S078N-N218S	81
BspAI02518-01924	S009E-A067S-S160Q-N253P	S009E-A069S-S166Q-N259P	88
BspAI02518-01696	S009E-N074D-S076N-N253P	S009E-N076D-S078N-N259P	92
BspAI02518-02000	S009E-N074D-S160Q-N179Q	S009E-N076D-S166Q-N185Q	89
BspAI02518-01753	S009E-N074D-S160Q-N253P	S009E-N076D-S166Q-N259P	94
BspAI02518-01299	S009E-N074D-N179Q-N212S	S009E-N076D-N185Q-N218S	93
BspAI02518-01620	S009E-N074D-N179Q-N253P	S009E-N076D-N185Q-N259P	75
BspAI02518-01959	S009E-S076N-S160Q-N212S	S009E-S078N-S166Q-N218S	96
BspAI02518-01703	S009E-S076N-N179Q-N253P	S009E-S078N-N185Q-N259P	77
BspAI02518-01628	S009E-S076N-N212S-N253P	S009E-S078N-N218S-N259P	94
BspAI02518-01708	A067S-N074D-S076N-N212S	A069S-N076D-S078N-N218S	97
BspAI02518-01144	A067S-N074D-S160Q-N179Q	A069S-N076D-S166Q-N185Q	93
BspAI02518-01638	A067S-N074D-N179Q-N212S	A069S-N076D-N185Q-N218S	85
BspAI02518-01256	A067S-S076N-S160Q-N212S	A069S-S078N-S166Q-N218S	94
BspAI02518-01627	A067S-S076N-N179Q-N212S	A069S-S078N-N185Q-N218S	86
BspAI02518-01693	A067S-S076N-N212S-N253P	A069S-S078N-N218S-N259P	87
BspAI02518-01677	A067S-S160Q-N179Q-N212S	A069S-S166Q-N185Q-N218S	81
BspAI02518-01730	A067S-S160Q-N179Q-N253P	A069S-S166Q-N185Q-N259P	60
BspAI02518-01887	N074D-S076N-S160Q-N212S	N076D-S078N-S166Q-N218S	100
BspAI02518-01778	N074D-S076N-N179Q-N212S	N076D-S078N-N185Q-N218S	98
BspAI02518-01581	N074D-S076N-N179Q-N253P	N076D-S078N-N185Q-N259P	76
BspAI02518-01720	N074D-S076N-N212S-N253P	N076D-S078N-N218S-N259P	96
BspAI02518-01842	N074D-S160Q-N179Q-N212S	N076D-S166Q-N185Q-N218S	100
BspAI02518-02079	N074D-S160Q-N212S-N253P	N076D-S166Q-N218S-N259P	86
BspAI02518-02016	N074D-N179Q-N212S-N253P	N076D-N185Q-N218S-N259P	93
BspAI02518-01792	S076N-S160Q-N179Q-N212S	S078N-S166Q-N185Q-N218S	93
BspAI02518-02157	S076N-S160Q-N179Q-N253P	S078N-S166Q-N185Q-N259P	93
BspAI02518-01716	S160Q-N179Q-N212S-N253P	S166Q-N185Q-N218S-N259P	88

Example 18

Bad02409 Subtilisin Variants with Improved Stability in Detergent

Variants of Bad02409 (SEQ ID NO: 18) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Table 36 shows the results for Bad02409 variants.

TABLE 36
Bad02409 subtilisin variants with improved stability in liquid detergent at 67°
C. (reported as percent residual activity, % RA) compared to Bad02409 parent
	Cleaning
	performance, PI
				BMI stain	PAS-38 stain
Bad02409 Variant	Substitutions in	Substitutions in		in PNB	in GSM-B
Sample ID	Bad02409 numbering	BPN′ numbering	% RA	detergent	detergent
Bad02409	—	—	21	1.0	1.0
Bad02409-00464	T003V-A071S-G169Q	T003V-A069S-G166Q	47	1.4	1.1
Bad02409-00516	T003V-N078D-D262P	T003V-N076D-D259P	76	1.1	0.9
Bad02409-00380	T003V-S080N-N188Q	T003V-S078N-N185Q	51	1.0	1.2
Bad02409-00327	T003V-S131P-N188Q	T003V-S129P-N185Q	40	1.0	1.2
Bad02409-00323	T003V-G169Q-D262P	T003V-G166Q-D259P	43	1.2	1.1
Bad02409-00167	T003V-N188Q-D262P	T003V-N185Q-D259P	45	0.9	1.1
Bad02409-00548	A071S-N078D-G169Q	A069S-N076D-G166Q	73	1.1	0.9
Bad02409-00344	N078D-G169Q-N188Q	N076D-G166Q-N185Q	64	1.4	1.1
Bad02409-00417	T003V-A071S-	T003V-A069S-	55	1.0	1.0
	S080N-S131P	S078N-S129P
Bad02409-00014	T003V-N078D-	T003V-N076D-	55	1.0	1.2
	N188Q-D262P	N185Q-D259P
Bad02409-00195	T003V-S080N-	T003V-S078N-	52	1.4	1.2
	S131P-N188Q	S129P-N185Q
Bad02409-00098	T003V-S080N-	T003V-S078N-	77	1.3	1.1
	G169Q-D262P	G166Q-D259P
Bad02409-00455	T003V-G169Q-	T003V-G166Q-	46	1.2	1.2
	N188Q-D262P	N185Q-D259P
Bad02409-00209	A071S-N078D-	A069S-N076D-	82	0.9	1.1
	S131P-D262P	S129P-D259P
Bad02409-00465	A071S-S080N-	A069S-S078N-	47	1.3	1.0
	S131P-N188Q	S129P-N185Q
Bad02409-00130	A071S-S131P-	A069S-S129P-	38	1.5	1.0
	G169Q-N188Q	G166Q-N185Q
Bad02409-00349	N078D-S080N-	N076D-S078N-	71	0.9	1.0
	S131P-N188Q	S129P-N185Q
Bad02409-00469	N078D-S131P-	N076D-S129P-	80	1.0	1.0
	G169Q-D262P	G166Q-D259P
Bad02409-00444	N078D-G169Q-	N076D-G166Q-	66	1.3	1.2
	N188Q-D262P	N185Q-D259P

Example 19

Bba02069 Subtilisin Variants with Improved Stability in Detergent

Variants of Bba02069 (SEQ ID NO: 19) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Table 37A and 37B show the results for Bba02069 variants.

TABLE 37A
Bba02069 subtilisin variants with improved stability in liquid detergent at 40°
C. (reported as percent residual activity, % RA) compared to Bba02069 parent
	Cleaning performance, PI
Bba02069	Substitutions in	Substitutions		BMI stain	PAS-38 stain	BMI stain
Variant	Bba02069	in BPN′		in PNB	in GSM-B	in ECE-2
Sample ID	numbering	numbering	% RA	detergent	detergent	detergent
Bba02069	—	—	30	1.0	1.0	1.0
Bba02069-	Q003V-P040E-	Q003V-P040E-	85	0.9	1.2	0.9
00013	G167Q	G166Q
Bba02069-	Q003V-G167Q-	Q003V-G166Q-	84	0.8	1.0	1.2
00471	S260P	S259P
Bba02069-	A069S-S129P-	A069S-S129P-	68	0.9	1.0	0.9
00211	G167Q	G166Q
Bba02069-	A069S-S129P-	A069S-S129P-	59	1.0	0.5	0.7
00305	V186Q	V185Q
Bba02069-	G128S-S129P-	G128S-S129P-	54	0.9	1.0	0.9
00443	N219S	N218S
Bba02069-	G128S-V186Q-	G128S-V185Q-	74	0.7	1.1	0.9
00510	N219S	N218S
Bba02069-	Q003V-A069S-	Q003V-A069S-	84	0.7	1.0	1.0
00170	S129P-S260P	S129P-S259P
Bba02069-	Q003V-M124I-	Q003V-M124I-	100	0.6	1.3	0.7
00558	G128S-G167Q	G128S-G166Q
Bba02069-	Q003V-G128S-	Q003V-G128S-	73	0.8	1.2	0.9
00196	S129P-V186Q	S129P-V185Q
Bba02069-	P040E-A069S-	P040E-A069S-	82	1.1	1.0	0.9
00183	S129P-N219S	S129P-N218S
Bba02069-	P040E-M124I-	P040E-M124I-	87	0.9	1.2	0.6
00248	N219S-S260P	N218S-S259P
Bba02069-	A069S-N076D-	A069S-N076D-	96	1.0	1.0	1.1
00543	S129P-G167Q	S129P-G166Q
Bba02069-	A069S-M124I-	A069S-M124I-	77	0.9	1.3	0.7
00437	S129P-S260P	S129P-S259P
Bba02069-	A069S-M124I-	A069S-M124I-	83	0.6	1.2	0.5
00469	V186Q-N219S	V185Q-N218S
Bba02069-	G128S-G167Q-	G128S-G166Q-	76	0.9	0.9	1.0
00022	V186Q-N219S	V185Q-N218S
Bba02069-	S129P-G167Q-	S129P-G166Q-	62	0.8	1.2	1.1
00484	V186Q-S260P	V185Q-S259P

TABLE 37B
Bba02069 subtilisin variants with improved stability in liquid detergent at 39°
C. (reported as percent residual activity, % RA) compared to Bba02069 parent
Bba02069 Variant	Substitutions in	Substitutions in
Sample ID	Bba02069 numbering	BPN′ numbering	% RA
Bba02069	—	—	23
Bba02069-00812	Q003V-T009E-P040E	Q003V-T009E-P040E	88
Bba02069-00811	Q003V-T009E-A069S	Q003V-T009E-A069S	77
Bba02069-00809	Q003V-T009E-N076D	Q003V-T009E-N076D	100
Bba02069-00744	Q003V-T009E-G167Q	Q003V-T009E-G166Q	95
Bba02069-00808	Q003V-T009E-V186Q	Q003V-T009E-V185Q	83
Bba02069-00829	Q003V-T009E-N219S	Q003V-T009E-N218S	98
Bba02069-00843	Q003V-T009E-S260P	Q003V-T009E-S259P	86
Bba02069-00747	Q003V-P040E-A069S	Q003V-P040E-A069S	89
Bba02069-00833	Q003V-P040E-N076D	Q003V-P040E-N076D	88
Bba02069-00664	Q003V-P040E-V186Q	Q003V-P040E-V185Q	74
Bba02069-00781	Q003V-P040E-N219S	Q003V-P040E-N218S	74
Bba02069-00834	Q003V-P040E-S260P	Q003V-P040E-S259P	84
Bba02069-00779	Q003V-A069S-N076D	Q003V-A069S-N076D	78
Bba02069-00778	Q003V-A069S-G167Q	Q003V-A069S-G166Q	95
Bba02069-00679	Q003V-A069S-N219S	Q003V-A069S-N218S	81
Bba02069-00776	Q003V-A069S-S260P	Q003V-A069S-S259P	81
Bba02069-00836	Q003V-N076D-G167Q	Q003V-N076D-G166Q	89
Bba02069-00827	Q003V-N076D-V186Q	Q003V-N076D-V185Q	86
Bba02069-00825	Q003V-N076D-N219S	Q003V-N076D-N218S	91
Bba02069-00842	Q003V-N076D-S260P	Q003V-N076D-S259P	91
Bba02069-00760	Q003V-G167Q-V186Q	Q003V-G166Q-V185Q	82
Bba02069-00840	Q003V-G167Q-N219S	Q003V-G166Q-N218S	98
Bba02069-00838	Q003V-V186Q-S260P	Q003V-V185Q-S259P	73
Bba02069-00764	Q003V-N219S-S260P	Q003V-N218S-S259P	88
Bba02069-00846	T009E-P040E-A069S	T009E-P040E-A069S	64
Bba02069-00886	T009E-P040E-N076D	T009E-P040E-N076D	97
Bba02069-00847	T009E-P040E-G167Q	T009E-P040E-G166Q	92
Bba02069-01933	T009E-P040E-V186Q	T009E-P040E-V185Q	70
Bba02069-01674	T009E-P040E-N219S	T009E-P040E-N218S	94
Bba02069-00763	T009E-P040E-S260P	T009E-P040E-S259P	81
Bba02069-00848	T009E-A069S-N076D	T009E-A069S-N076D	90
Bba02069-00849	T009E-A069S-G167Q	T009E-A069S-G166Q	94
Bba02069-00791	T009E-A069S-V186Q	T009E-A069S-V185Q	67
Bba02069-00722	T009E-A069S-N219S	T009E-A069S-N218S	87
Bba02069-00795	T009E-A069S-S260P	T009E-A069S-S259P	88
Bba02069-00796	T009E-N076D-G167Q	T009E-N076D-G166Q	94
Bba02069-00725	T009E-N076D-V186Q	T009E-N076D-V185Q	93
Bba02069-01905	T009E-N076D-N219S	T009E-N076D-N218S	99
Bba02069-00793	T009E-N076D-S260P	T009E-N076D-S259P	98
Bba02069-00794	T009E-G167Q-V186Q	T009E-G166Q-V185Q	83
Bba02069-00797	T009E-G167Q-N219S	T009E-G166Q-N218S	91
Bba02069-00715	T009E-G167Q-S260P	T009E-G166Q-S259P	84
Bba02069-00799	T009E-V186Q-N219S	T009E-V185Q-N218S	88
Bba02069-00718	T009E-V186Q-S260P	T009E-V185Q-S259P	78
Bba02069-00801	T009E-N219S-S260P	T009E-N218S-S259P	87
Bba02069-00720	P040E-A069S-N076D	P040E-A069S-N076D	100
Bba02069-00802	P040E-A069S-G167Q	P040E-A069S-G166Q	65
Bba02069-01826	P040E-A069S-V186Q	P040E-A069S-V185Q	48
Bba02069-00882	P040E-A069S-N219S	P040E-A069S-N218S	70
Bba02069-00730	P040E-A069S-S260P	P040E-A069S-S259P	66
Bba02069-00693	P040E-N076D-G167Q	P040E-N076D-G166Q	98
Bba02069-00729	P040E-N076D-V186Q	P040E-N076D-V185Q	79
Bba02069-00695	P040E-N076D-N219S	P040E-N076D-N218S	84
Bba02069-00696	P040E-N076D-S260P	P040E-N076D-S259P	94
Bba02069-00692	P040E-G167Q-V186Q	P040E-G166Q-V185Q	52
Bba02069-00691	P040E-G167Q-N219S	P040E-G166Q-N218S	87
Bba02069-00705	P040E-G167Q-S260P	P040E-G166Q-S259P	69
Bba02069-00706	P040E-V186Q-N219S	P040E-V185Q-N218S	76
Bba02069-00686	P040E-V186Q-S260P	P040E-V185Q-S259P	54
Bba02069-00685	P040E-N219S-S260P	P040E-N218S-S259P	77
Bba02069-00708	A069S-N076D-G167Q	A069S-N076D-G166Q	92
Bba02069-00883	A069S-N076D-V186Q	A069S-N076D-V185Q	75
Bba02069-00684	A069S-N076D-N219S	A069S-N076D-N218S	88
Bba02069-00683	A069S-N076D-S260P	A069S-N076D-S259P	92
Bba02069-00703	A069S-G167Q-V186Q	A069S-G166Q-V185Q	51
Bba02069-01832	A069S-G167Q-N219S	A069S-G166Q-N218S	85
Bba02069-00704	A069S-G167Q-S260P	A069S-G166Q-S259P	73
Bba02069-01679	A069S-V186Q-N219S	A069S-V185Q-N218S	65
Bba02069-00930	A069S-V186Q-S260P	A069S-V185Q-S259P	52
Bba02069-00700	A069S-N219S-S260P	A069S-N218S-S259P	72
Bba02069-00699	N076D-G167Q-V186Q	N076D-G166Q-V185Q	95
Bba02069-00698	N076D-G167Q-N219S	N076D-G166Q-N218S	100
Bba02069-01319	N076D-G167Q-S260P	N076D-G166Q-S259P	95
Bba02069-01333	N076D-V186Q-N219S	N076D-V185Q-N218S	94
Bba02069-01321	N076D-V186Q-S260P	N076D-V185Q-S259P	89
Bba02069-01323	N076D-N219S-S260P	N076D-N218S-S259P	97
Bba02069-01324	G167Q-V186Q-N219S	G166Q-V185Q-N218S	64
Bba02069-01335	G167Q-V186Q-S260P	G166Q-V185Q-S259P	52
Bba02069-01328	G167Q-N219S-S260P	G166Q-N218S-S259P	73
Bba02069-01329	V186Q-N219S-S260P	V185Q-N218S-S259P	60
Bba02069-01318	Q003V-T009E-P040E-G167Q	Q003V-T009E-P040E-G166Q	97
Bba02069-01136	Q003V-T009E-P040E-V186Q	Q003V-T009E-P040E-V185Q	82
Bba02069-01264	Q003V-T009E-N076D-N219S	Q003V-T009E-N076D-N218S	82
Bba02069-01585	Q003V-T009E-G167Q-V186Q	Q003V-T009E-G166Q-V185Q	95
Bba02069-01764	Q003V-P040E-N076D-V186Q	Q003V-P040E-N076D-V185Q	73
Bba02069-01124	T009E-P040E-A069S-V186Q	T009E-P040E-A069S-V185Q	80
Bba02069-01219	T009E-P040E-A069S-N219S	T009E-P040E-A069S-N218S	100
Bba02069-01040	T009E-P040E-N076D-V186Q	T009E-P040E-N076D-V185Q	99
Bba02069-01182	T009E-A069S-N076D-N219S	T009E-A069S-N076D-N218S	98
Bba02069-01203	T009E-A069S-G167Q-V186Q	T009E-A069S-G166Q-V185Q	83
Bba02069-01007	T009E-A069S-G167Q-N219S	T009E-A069S-G166Q-N218S	100
Bba02069-00924	T009E-A069S-V186Q-N219S	T009E-A069S-V185Q-N218S	82
Bba02069-00904	T009E-N076D-V186Q-S260P	T009E-N076D-V185Q-S259P	98
Bba02069-00908	T009E-V186Q-N219S-S260P	T009E-V185Q-N218S-S259P	83
Bba02069-01165	P040E-A069S-N076D-V186Q	P040E-A069S-N076D-V185Q	89
Bba02069-01900	P040E-A069S-G167Q-N219S	P040E-A069S-G166Q-N218S	94
Bba02069-01768	P040E-A069S-V186Q-N219S	P040E-A069S-V185Q-N218S	67
Bba02069-01072	P040E-A069S-V186Q-S260P	P040E-A069S-V185Q-S259P	72
Bba02069-01470	P040E-A069S-N219S-S260P	P040E-A069S-N218S-S259P	82
Bba02069-01003	P040E-N076D-G167Q-V186Q	P040E-N076D-G166Q-V185Q	94
Bba02069-00893	P040E-N076D-V186Q-N219S	P040E-N076D-V185Q-N218S	86
Bba02069-00996	P040E-N076D-V186Q-S260P	P040E-N076D-V185Q-S259P	92
Bba02069-01478	P040E-G167Q-V186Q-N219S	P040E-G166Q-V185Q-N218S	79
Bba02069-01856	P040E-V186Q-N219S-S260P	P040E-V185Q-N218S-S259P	78
Bba02069-00954	A069S-N076D-G167Q-V186Q	A069S-N076D-G166Q-V185Q	95
Bba02069-01024	A069S-N076D-G167Q-N219S	A069S-N076D-G166Q-N218S	96
Bba02069-01011	A069S-N076D-G167Q-S260P	A069S-N076D-G166Q-S259P	97
Bba02069-01803	A069S-N076D-V186Q-N219S	A069S-N076D-V185Q-N218S	94
Bba02069-01919	A069S-N076D-N219S-S260P	A069S-N076D-N218S-S259P	93
Bba02069-01842	A069S-G167Q-V186Q-N219S	A069S-G166Q-V185Q-N218S	62
Bba02069-01958	N076D-G167Q-V186Q-S260P	N076D-G166Q-V185Q-S259P	93
Bba02069-01312	N076D-G167Q-N219S-S260P	N076D-G166Q-N218S-S259P	99
Bba02069-01570	G167Q-V186Q-N219S-S260P	G166Q-V185Q-N218S-S259P	68

Example 20

BspZ00258 Subtilisin Variants with Improved Stability in Detergent

Variants of BspZ00258 (SEQ ID NO: 22) subtilisin containing one, two, three or four amino acid substitutions at positions of interest to increase enzyme stability, were evaluated for detergent stability and cleaning performance using methods described in Example 2. The BspZ00258 subtilisin was previously described as SEQ ID NO:9 in WO 2016069552 patent application, and is a member of the BspM04284-clade of subtilisins. Table 38 shows the results for BspZ00258 variants.

TABLE 38
BspZ00258 subtilisin variants with improved stability in liquid detergent at 62°
C. (reported as percent residual activity, % RA) compared to BspZ00258 parent
	Cleaning
	performance, PI
BspZ00258 Variant	Substitutions in	Substitutions in		BMI stain in	PAS-38 stain in
Sample ID	BspZ00258 numbering	BPN′ numbering	% RA	PNB detergent	GSM-B detergent
BspZ00258	—	—	57	1.0	1.0
BspZ00258-00124	E003V	E003V	84	0.6	1.1
BspZ00258-00027	G166Q	G166Q	78	0.5	1.1
BspZ00258-00045	D259P	D259P	66	0.9	1.6
BspZ00258-00098	A068S-N185Q	A069S-N185Q	67	0.8	1.3
BspZ00258-00429	A068S-N218S	A069S-N218S	61	1.0	1.6
BspZ00258-00244	A128P-D259P	A129P-D259P	76	0.9	1.4
BspZ00258-00338	G166Q-N185Q	G166Q-N185Q	78	0.7	1.1
BspZ00258-00106	G166Q-D259P	G166Q-D259P	72	0.8	1.3
BspZ00258-00476	E003V-A068S-G127S	E003V-A069S-G128S	90	0.9	1.6
BspZ00258-00565	E003V-G127S-D259P	E003V-G128S-D259P	88	2.3	3.4
BspZ00258-00337	E003V-A128P-N185Q	E003V-A129P-N185Q	88	0.7	1.4
BspZ00258-00538	A068S-A128P-G166Q	A069S-A129P-G166Q	79	0.8	1.2
BspZ00258-00095	E003V-A068S-	E003V-A069S-	90	1.2	2.6
	G127S-N185Q	G128S-N185Q
BspZ00258-00020	E003V-A068S-	E003V-A069S-	88	0.8	2.0
	A128P-N185Q	A129P-N185Q
BspZ00258-00145	E003V-G127S-	E003V-G128S-	93	1.5	2.7
	N185Q-D259P	N185Q-D259P
BspZ00258-00096	E003V-A128P-	E003V-A129P-	86	0.7	1.6
	N185Q-N218S	N185Q-N218S

Example 21

BspZ00056 Subtilisin Variants with Improved Stability in Detergent

Variants of BspZ00056 (SEQ ID NO:17) subtilisin containing three or four amino acid substitutions at positions of interest to increase enzyme stability, were generated using methods similar to the ones described in Example 1. These variant samples were evaluated for detergent stability and cleaning performance using methods described in Example 2. Table 39A and 39B show the results for BspZ00056 variants.

TABLE 39A
BspZ00056 subtilisin variants with improved stability in liquid detergent at 64°
C. (reported as percent residual activity, % RA) compared to BspZ00056 parent
	Cleaning
	performance, PI
BspZ00056 Variant	Substitutions in	Substitutions in		BMI stain in	PAS-38 stain in
Sample ID	BspZ00056 numbering	BPN′ numbering	% RA	PNB detergent	GSM-B detergent
BspZ00056	—	—	23	1.0	1.0
BspZ00056-00318	T003V-S133P-G171Q	T003V-S129P-G166Q	84	1.0	0.6
BspZ00056-00428	T003V-S133P-N190Q	T003V-S129P-N185Q	61	1.2	0.7
BspZ00056-00131	T003V-S133P-G264P	T003V-S129P-G259P	80	1.3	0.8
BspZ00056-00223	T003V-N190Q-N223S	T003V-N185Q-N218S	46	1.3	0.2
BspZ00056-00474	A073S-G132S-N190Q	A069S-G128S-N185Q	74	1.1	0.2
BspZ00056-00104	A073S-S133P-G264P	A069S-S129P-G259P	86	1.3	0.4
BspZ00056-00110	A073S-G171Q-N190Q	A069S-G166Q-N185Q	80	1.3	0.7
BspZ00056-00500	A073S-G171Q-N223S	A069S-G166Q-N218S	82	1.5	0.7
BspZ00056-00113	M128I-S133P-N190Q	M124I-S129P-N185Q	67	1.3	0.3
BspZ00056-00198	M128I-S133P-G264P	M124I-S129P-G259P	88	1.3	0.2
BspZ00056-00303	M128I-N190Q-G264P	M124I-N185Q-G259P	85	1.2	0.2
BspZ00056-00264	M128I-N223S-G264P	M124I-N218S-G259P	89	1.3	0.1
BspZ00056-00302	G132S-G171Q-G264P	G128S-G166Q-G259P	85	1.2	0.6
BspZ00056-00483	G132S-N190Q-G264P	G128S-N185Q-G259P	100	1.4	0.6
BspZ00056-00238	G171Q-N223S-G264P	G166Q-N218S-G259P	87	1.1	0.7
BspZ00056-00334	T003V-A073S-	T003V-A069S-	75	1.6	0.2
	G132S-N190Q	G128S-N185Q
BspZ00056-00482	T003V-A073S-	T003V-A069S-	81	1.8	0.6
	N190Q-G264P	N185Q-G259P
BspZ00056-00498	T003V-A073S-	T003V-A069S-	75	1.5	0.3
	N223S-G264P	N218S-G259P
BspZ00056-00021	T003V-M128I-	T003V-M124I-	89	1.3	0.3
	G171Q-G264P	G166Q-G259P
BspZ00056-00041	T003V-N190Q-	T003V-N185Q-	78	1.5	0.3
	N223S-G264P	N218S-G259P
BspZ00056-00376	A073S-S133P-	A069S-S129P-	85	1.0	0.5
	G171Q-G264P	G166Q-G259P
BspZ00056-00308	M128I-S133P-	M124I-S129P-	91	1.0	0.2
	G171Q-G264P	G166Q-G259P
BspZ00056-00158	M128I-G171Q-	M124I-G166Q-	90	1.1	0.3
	N223S-G264P	N218S-G259P
BspZ00056-00409	M128I-N190Q-	M124I-N185Q-	95	1.0	0.2
	N223S-G264P	N218S-G259P
BspZ00056-00155	G132S-S133P-	G128S-S129P-	90	1.2	0.5
	G171Q-G264P	G166Q-G259P
BspZ00056-00012	S133P-G171Q-	S129P-G166Q-	84	1.5	0.8
	N190Q-G264P	N185Q-G259P

TABLE 39B
BspZ00056 subtilisin variants with improved stability in liquid detergent at 64°
C. (reported as percent residual activity, % RA) compared to BspZ00056 parent
BspZ00056 Variant	Substitutions in	Substitutions in
Sample ID	Backbone Numbering	BPN′ Numbering	% RA
BspZ00056	—	—	30
BspZ00056-00909	T003V-P009E-G171Q	T003V-P009E-G166Q	74
BspZ00056-00859	T003V-A073S-G171Q	T003V-A069S-G166Q	72
BspZ00056-00848	T003V-A073S-G264P	T003V-A069S-G259P	92
BspZ00056-00808	T003V-G171Q-N190Q	T003V-G166Q-N185Q	68
BspZ00056-00785	T003V-G171Q-N223S	T003V-G166Q-N218S	78
BspZ00056-00786	T003V-G171Q-G264P	T003V-G166Q-G259P	93
BspZ00056-00891	T003V-N190Q-G264P	T003V-N185Q-G259P	75
BspZ00056-00787	T003V-N223S-G264P	T003V-N218S-G259P	79
BspZ00056-00810	P009E-A073S-G171Q	P009E-A069S-G166Q	67
BspZ00056-01468	P009E-A073S-G264P	P009E-A069S-G259P	87
BspZ00056-00783	P009E-G171Q-N190Q	P009E-G166Q-N185Q	73
BspZ00056-00913	P009E-G171Q-N223S	P009E-G166Q-N218S	75
BspZ00056-01238	P009E-G171Q-G264P	P009E-G166Q-G259P	91
BspZ00056-00884	P009E-N223S-G264P	P009E-N218S-G259P	82
BspZ00056-00704	A073S-G171Q-G264P	A069S-G166Q-G259P	87
BspZ00056-00693	A073S-N190Q-G264P	A069S-N185Q-G259P	78
BspZ00056-00705	A073S-N223S-G264P	A069S-N218S-G259P	90
BspZ00056-00710	G171Q-N190Q-G264P	G166Q-N185Q-G259P	69
BspZ00056-01490	N190Q-N223S-G264P	N185Q-N218S-G259P	98
BspZ00056-01432	T003V-P009E-A073S-G171Q	T003V-P009E-A069S-G166Q	86
BspZ00056-01158	T003V-P009E-G171Q-G264P	T003V-P009E-G166Q-G259P	100
BspZ00056-01153	T003V-A073S-G171Q-N190Q	T003V-A069S-G166Q-N185Q	90
BspZ00056-01078	T003V-G171Q-N223S-G264P	T003V-G166Q-N218S-G259P	97
BspZ00056-01084	P009E-A073S-G171Q-N223S	P009E-A069S-G166Q-N218S	79
BspZ00056-01115	P009E-G171Q-N190Q-N223S	P009E-G166Q-N185Q-N218S	82
BspZ00056-01032	P009E-G171Q-N190Q-G264P	P009E-G166Q-N185Q-G259P	100
BspZ00056-01179	P009E-G171Q-N223S-G264P	P009E-G166Q-N218S-G259P	87
BspZ00056-01233	P009E-N190Q-N223S-G264P	P009E-N185Q-N218S-G259P	79
BspZ00056-01152	A073S-G171Q-N190Q-N223S	A069S-G166Q-N185Q-N218S	82
BspZ00056-01379	A073S-G171Q-N190Q-G264P	A069S-G166Q-N185Q-G259P	83
BspZ00056-01194	A073S-G171Q-N223S-G264P	A069S-G166Q-N218S-G259P	92
BspZ00056-01234	G171Q-N190Q-N223S-G264P	G166Q-N185Q-N218S-G259P	100

Example 22

Globally Beneficial Stability Mutations in Subtilisins

Analysis of improved detergent stability results across the following backbones: AprE (subtilisin E, SEQ ID NO:8); Chemgen_164A (SEQ ID NO:10); Bpan01744 (SEQ ID NO:13); DSM14391 (SEQ ID NO:14); BspAI02518 (SEQ ID NO:16); BspZ00056 (SEQ ID NO:17); Bba02069 (SEQ ID NO:19); CP474 (SEQ ID NO:11) and ZP-00454 (SEQ ID NO:12) was performed. Variants comprising 1 or 2 substitutions on the wild-type backbone are shown in Table 40. At least half of all possible double combinations of the following features: X003V, X009E, X040E, X069S, X076D, X078N, X166Q, X185Q, X218S, and X259P introduced into the 9 backbones listed above showed increased stability for singly or doubly substituted variants. Variants comprising 3 substitutions on the wild-type backbone are shown on Table 41. At least half of all possible triple combinations of the following features: X003V, X009E, X040E, X069S, X076D, X078N, X166Q, X185Q, X218S, and X259P introduced into the following 7 backbones AprE (subtilisin E, SEQ ID NO:8); Chemgen_164A (SEQ ID NO:10); Bpan01744 (SEQ ID NO:13); DSM14391 (SEQ ID NO:14); BspAI02518 (SEQ ID NO:16); BspZ00056 (SEQ ID NO:17); Bba02069 (SEQ ID NO:19) showed increased stability for triply substituted variants.

In Tables 40 and 41, the term “shared feature” corresponds to amino acid position of interest where a substitution was introduced, or in some cases the amino acid of interest is naturally occurring. Sample IDs on Table 40 are as follows: variants of AprE have an AprE suffix, variants of Chemgen_164A have a Chemgen suffix, variants of DSM14391 have a DSM14391 suffix, variants of BspZ00056 have a BspZ00056 suffix, variants of Bba02069 have a Bba02069 suffix, variants of BspAI02518 have a BspAI02518 suffix, variants of Bpan01744 have a Bpan01744 suffix, variants of CP474 have a CP474 suffix, and variants of ZP-00454 have a ZP-00454 suffix. All substitutions are listed based on corresponding positions in BPN′ numbering (SEQ ID NO: 1) according to multiple protein sequence alignment shown in Table 28.

TABLE 40
Variants of AprE, Chemgen, DSM14391, BspZ00056, Bba02069, BspAI02518,
Bpan01744, CP474, and ZP-00454 subtilisins with shared features
Shared feature in
BPN′ numbering Variants with shared feature
X003V-X009E    AprE-01089, Chemgen-00795, Bpan01744-00830, DSM14391-00986,
               BspAI02518-01175, Bba02069-00854
X003V-X040E    AprE-00380, Chemgen-00794, CP474-00592, ZP-00454-00011,
               Bpan01744-00498, DSM14391-00987, BspAI02518-00709, Bba02069-
               00855, BspZ00056-00876
X003V-X069S    AprE-01078, Chemgen-00481, CP474-00602, ZP-00454-00021,
               Bpan01744-00167, DSM14391-00025, BspAI02518-00599, Bba02069-
               00030, BspZ00056-00094
X003V-X076D    AprE-00729, Chemgen-00076, CP474-00571, ZP-00454-00031,
               Bpan01744-00166, DSM14391-00340, BspAI02518-00897, Bba02069-
               00564, BspZ00056-00876
X003V-X078N    AprE-00515, Chemgen-00443, CP474-00612, ZP-00454-00041,
               Bpan01744-00304, DSM14391-00373, BspAI02518-01179, Bba02069-00652
X003V-X166Q    AprE-00841, Chemgen-00378, CP474-00563, Bpan01744-01145,
               DSM14391-00028, BspAI02518-00959, Bba02069-00347, BspZ00056-00221
X003V-X185Q    AprE-00772, Chemgen-00108, CP474-00573, Bpan01744-00152,
               DSM14391-00016, BspAI02518-00624, Bba2069-00121, BspZ00056-00116
X003V-X218S    AprE-01842, Chemgen-00634, CP474-00583, Bpan01744-00417,
               DSM14391-00492, BspAI02518-00564, Bba02069-00856, BspZ00056-00368
X003V-X259P    AprE-00370, Chemgen-00650, CP474-00593, Bpan01744-00544,
               DSM14391-00063, BspAI02518-00506, Bba02069-00477, BspZ00056-00384
X009E-X040E    AprE-01082, Bpan01744-02141, DSM14391-00863, BspAI02518-00637,
               Bba02069-00755, BspZ00056-01485
X009E-X069S    AprE-01083, Chemgen-00788, DSM14391-00864, BspAI02518-01198,
               Bba02069-00756
X009E-X076D    AprE-01025, Chemgen-00712, Bpan01744-01257, DSM14391-00839,
               BspAI02518-01178, Bba02069-00757, BspZ00056-01485
X009E-X078N    AprE-01850, Chemgen-00714, Bpan01744-02075, DSM14391-00962,
               BspAI02518-01186, Bba02069-00654
X009E-X166Q    AprE-01959, Chemgen-00797, Bpan01744-01122, BspAI02518-01991,
               Bba02069-00871, BspZ00056-01478
X009E-X185Q    AprE-01081, Chemgen-00798, Bpan01744-01123, DSM14391-01005,
               BspAI02518-01187, Bba02069-00866
X009E-X218S    AprE-01111, Bpan01744-00795, DSM14391-00971, BspAI02518-01811,
               Bba02069-00865
X009E-X259P    AprE-01096, Chemgen-00741, Bpan01744-01124, DSM14391-00867,
               BspAI02518-02004, Bba02069-00758, BspZ00056-00875
X040E-X069S    AprE-01951, CP474-00603, ZP-00454-00052, DSM14391-00968,
               Bba02069-00863, BspZ00056-00863
X040E-X076D    AprE-01108, CP474-00581, ZP-00454-00062, Bpan01744-01310,
               DSM14391-00969, BspAI02518-00689, Bba02069-00824
X040E-X078N    AprE-01844, Chemgen-00715, CP474-00613, ZP-00454-00072,
               Bpan01744-00574, DSM14391-00217, BspAI02518-00585, Bba02069-00653
X040E-X166Q    AprE-01107, Chemgen-01467, CP474-00564, ZP-00454-00043,
               BspAI02518-01177, Bba02069-00499, BspZ00056-00864
X040E-X185Q    AprE-01080, Chemgen-00716, CP474-00574, ZP-00454-00053,
               Bpan01744-00717, DSM14391-00985, BspAI02518-00764, Bba02069-
               00377, BspZ00056-00663
X040E-X218S    AprE-01054, Chemgen-01219, CP474-00584, ZP-00454-00063,
               Bpan01744-00892, DSM14391-00977, BspAI02518-00819, Bba02069-
               01952, BspZ00056-00248
X040E-X259P    AprE-01105, CP474-00594, ZP-00454-00073, Bpan01744-01737,
               DSM14391-00849, BspAI02518-00838, Bba02069-00845, BspZ00056-00819
X069S-X076D    AprE-00498, Chemgen-00527, CP474-00591, ZP-00454-00004,
               Bpan01744-00088, DSM14391-00494, BspAI02518-00412, Bba02069-
               00518, BspZ00056-00863
X069S-X078N    AprE-00788, Chemgen-00436, CP474-00604, ZP-00454-00014,
               Bpan01744-00423, DSM14391-00005, BspAI02518-00590, Bba02069-
               00648, BspZ00056-00490
X069S-X166Q    AprE-00666, Chemgen-00394, CP474-00634, Bpan01744-00153,
               BspAI02518-01025, Bba02069-00505, BspZ00056-00389
X069S-X185Q    Chemgen-00617, CP474-00565, ZP-00454-00074, Bpan01744-00414,
               DSM14391-00136, BspAI02518-00646, Bba02069-00844, BspZ00056-00042
X069S-X218S    AprE-00594, Chemgen-00569, ZP-00454-00005, Bpan01744-00488,
               DSM14391-00432, BspAI02518-00405, Bba02069-00736, BspZ00056-00873
X069S-X259P    AprE-00758, Chemgen-00444, DSM14391-00096, BspAI02518-01920,
               Bba02069-00114, BspZ00056-00151
X076D-X078N    AprE-00774, Chemgen-00459, CP474-00601, ZP-00454-00025,
               Bpan01744-00337, DSM14391-00007, BspAI02518-00814, Bba02069-00644
X076D-X166Q    AprE-00920, Chemgen-00132, CP474-00631, ZP-00454-00075,
               Bpan01744-01035, BspAI02518-01184, Bba02069-00247, BspZ00056-00864
X076D-X185Q    AprE-00795, Chemgen-00804, CP474-00562, ZP-00454-00006,
               Bpan01744-01133, DSM14391-00974, BspAI02518-00802, Bba02069-
               00822, BspZ00056-00663
X076D-X218S    AprE-00912, Chemgen-00367, ZP-00454-00016, Bpan01744-01135,
               DSM14391-00168, BspAI02518-00535, Bba02069-00815, BspZ00056-00248
X076D-X259P    AprE-01836, Chemgen-00546, ZP-00454-00026, Bpan01744-01148,
               DSM14391-00976, BspAI02518-00695, Bba02069-00423, BspZ00056-00819
X078N-X166Q    AprE-00974, Chemgen-00560, CP474-00615, ZP-00454-00076,
               Bpan01744-00158, BspAI02518-01190, Bba02069-00111, BspZ00056-00297
X078N-X185Q    AprE-00488, Chemgen-00224, CP474-00625, ZP-00454-00007,
               Bpan01744-01629, DSM14391-00302, BspAI02518-01032
X078N-X218S    AprE-00904, Chemgen-00313, CP474-00635, Bpan01744-00991,
               DSM14391-00252, BspAI02518-00800, Bba02069-00655
X078N-X259P    AprE-00891, Chemgen-00350, CP474-00566, ZP-00454-00027,
               Bpan01744-01151, DSM14391-00113, BspAI02518-00937, Bba02069-
               00217, BspZ00056-00088
X166Q-X185Q    AprE-00944, Chemgen-00122, Bpan01744-00369, DSM14391-00171,
               BspAI02518-02195, Bba02069-00190, BspZ00056-00257
X166Q-X218S    AprE-00698, Chemgen-00335, CP474-00597, Bpan01744-00172,
               DSM14391-00828, BspAI02518-01002, Bba02069-00817, BspZ00056-00837
X166Q-X259P    AprE-00694, Chemgen-00010, Bpan01744-00048, DSM14391-00430,
               BspAI02518-01006, Bba02069-00043, BspZ00056-00133
X185Q-X218S    AprE-00924, Chemgen-00198, CP474-00617, Bpan01744-00094,
               DSM14391-00161, BspAI02518-01094, Bba02069-00493, BspZ00056-01030
X185Q-X259P    AprE-00447, Chemgen-00359, CP474-00579, Bpan01744-00339,
               DSM14391-00203, BspAI02518-00439, Bba02069-00005, BspZ00056-00377
X218S-X259P    AprE-00646, Chemgen-00309, CP474-00627, Bpan01744-00126,
               DSM14391-00844, BspAI02518-00548, Bba02069-00051, BspZ00056-00491

TABLE 41
Variants of AprE, Chemgen, DSM14391, BspZ00056, Bba02069,
BspAI02518, and Bpan01744 subtilisins with shared features
Shared feature in
BPN′ numbering    Variants with shared feature
X003V-X009E-X040E AprE-01102, Chemgen-00801, Bpan01744-00830, DSM14391-
                  00824, BspAI02518-01175, Bba02069-00812
X003V-X009E-X069S AprE-01101, Chemgen-01562, Bpan01744-01709, DSM14391-
                  00996, BspAI02518-01092, Bba02069-00811
X003V-X009E-X076D AprE-01051, Bpan01744-01195, DSM14391-00995, BspAI02518-
                  01095, Bba02069-00809
X003V-X009E-X078N AprE-01099, Bpan01744-01267, DSM14391-00823, BspAI02518-
                  01270, Bba02069-00854
X003V-X009E-X166Q AprE-01045, BspAI02518-01096, Bba02069-00744, BspZ00056-00909
X003V-X009E-X185Q AprE-01089, Chemgen-00792, Bpan01744-01140, DSM14391-
                  00868, BspAI02518-01276, Bba02069-00808
X003V-X009E-X218S AprE-01077, Bpan01744-01141, DSM14391-00822, BspAI02518-
                  01097, Bba02069-00829
X003V-X009E-X259P AprE-01047, Bpan01744-01315, DSM14391-00817, BspAI02518-
                  01282, Bba02069-00843
X003V-X040E-X069S AprE-01009, Bpan01744-00167, DSM14391-00836, BspAI02518-
                  00599, Bba02069-00747, BspZ00056-00094
X003V-X040E-X076D AprE-01048, Chemgen-01640, Bpan01744-00166, DSM14391-
                  00819, BspAI02518-00897, Bba02069-00833
X003V-X040E-X078N Bpan01744-00304, DSM14391-00837, BspAI02518-01179,
                  Bba02069-00855
X003V-X040E-X166Q Chemgen-00793, Bpan01744-01145, BspAI02518-00959,
                  Bba02069-00013, BspZ00056-00221
X003V-X040E-X185Q AprE-00380, Chemgen-01651, Bpan01744-00152, DSM14391-
                  01004, BspAI02518-00624, Bba02069-00664, BspZ00056-00116
X003V-X040E-X218S AprE-01915, Bpan01744-00417, DSM14391-00421, BspAI02518-
                  00564, Bba02069-00781, BspZ00056-00368
X003V-X040E-X259P AprE-01039, Chemgen-00724, Bpan01744-00544, DSM14391-
                  00829, BspAI02518-00506, Bba02069-00834, BspZ00056-00384
X003V-X069S-X076D AprE-01864, Chemgen-00816, Bpan01744-00169, DSM14391-
                  00831, BspAI02518-01085, Bba02069-00779, BspZ00056-00094
X003V-X069S-X078N AprE-01056, Chemgen-00808, Bpan01744-01142, BspAI02518-
                  01086, Bba02069-00030
X003V-X069S-X166Q AprE-00991, Chemgen-00807, Bba02069-00778, BspZ00056-00859
X003V-X069S-X185Q AprE-01078, Chemgen-01642, Bpan01744-00150, DSM14391-
                  00858, BspAI02518-00534
X003V-X069S-X218S AprE-01060, Chemgen-00727, Bpan01744-00677, DSM14391-
                  00992, BspAI02518-01087, Bba02069-00679
X003V-X069S-X259P AprE-01064, Chemgen-01658, Bba02069-00776, BspZ00056-00848
X003V-X076D-X078N AprE-00685, Chemgen-00729, Bpan01744-01265, DSM14391-
                  00833, BspAI02518-01088, Bba02069-00564
X003V-X076D-X166Q AprE-01340, Chemgen-00694, Bpan01744-01585, BspAI02518-
                  01250, Bba02069-00836, BspZ00056-00221
X003V-X076D-X185Q AprE-00729, Chemgen-00776, Bpan01744-00170, DSM14391-
                  01080, BspAI02518-01084, Bba02069-00827, BspZ00056-00116
X003V-X076D-X218S AprE-01867, Chemgen-00775, Bpan01744-01252, DSM14391-
                  01084, BspAI02518-01220, Bba02069-00825, BspZ00056-00368
X003V-X076D-X259P AprE-01062, Chemgen-00832, DSM14391-01058, BspAI02518-
                  01230, Bba02069-00842, BspZ00056-00384
X003V-X078N-X166Q Chemgen-00774, Bpan01744-00059, BspAI02518-01222,
                  Bba02069-00347
X003V-X078N-X185Q AprE-00515, Chemgen-00696, Bpan01744-01253, DSM14391-
                  00922, BspAI02518-01895, Bba02069-00121
X003V-X078N-X218S AprE-00353, Bpan01744-01254, DSM14391-01083, BspAI02518-
                  01231, Bba02069-00856
X003V-X078N-X259P AprE-01068, Chemgen-00834, Bpan01744-01260, DSM14391-
                  01054, BspAI02518-01992, Bba02069-00477
X003V-X166Q-X185Q AprE-00841, Chemgen-00738, BspAI02518-01110, Bba02069-
                  00760, BspZ00056-00808
X003V-X166Q-X218S AprE-00773, Bpan01744-00547, DSM14391-00214, Bba02069-
                  00840, BspZ00056-00785
X003V-X166Q-X259P AprE-01855, Chemgen-00739, DSM14391-00359, BspAI02518-
                  02118, Bba02069-00471, BspZ00056-00786
X003V-X185Q-X218S AprE-01842, Chemgen-00785, Bpan01744-01274, DSM14391-
                  00929, BspAI02518-01099, BspZ00056-00223
X003V-X185Q-X259P AprE-00370, Chemgen-00698, Bpan01744-00685, DSM14391-
                  01078, BspAI02518-00539, Bba02069-00838, BspZ00056-00891
X003V-X218S-X259P AprE-00753, Chemgen-00780, Bpan01744-01275, DSM14391-
                  00919, BspAI02518-01960, Bba02069-00764, BspZ00056-00787
X009E-X040E-X069S AprE-01674, Chemgen-01553, BspAI02518-01198, Bba02069-00846
X009E-X040E-X076D AprE-01069, Chemgen-00703, Bpan01744-01257, DSM14391-
                  00931, BspAI02518-01178, Bba02069-00886
X009E-X040E-X078N AprE-01997, Chemgen-00683, Bpan01744-02075, DSM14391-
                  00928, BspAI02518-01186, Bba02069-00755
X009E-X040E-X166Q AprE-01984, Chemgen-00702, Bpan01744-01122, BspAI02518-
                  01991, Bba02069-00847, BspZ00056-01478
X009E-X040E-X185Q AprE-01082, Bpan01744-01123, DSM14391-01025, BspAI02518-
                  01187, Bba02069-01933
X009E-X040E-X218S AprE-01529, Chemgen-00822, Bpan01744-00795, DSM14391-
                  01034, BspAI02518-01811, Bba02069-01674
X009E-X040E-X259P AprE-01125, Chemgen-01604, Bpan01744-01124, DSM14391-
                  00899, BspAI02518-02004, Bba02069-00763, BspZ00056-00875
X009E-X069S-X076D AprE-01071, Chemgen-00684, Bpan01744-00693, BspAI02518-
                  01238, Bba02069-00848
X009E-X069S-X078N AprE-01091, Chemgen-00700, Bpan01744-00777, DSM14391-
                  01035, Bba02069-00756
X009E-X069S-X166Q AprE-01871, Chemgen-00823, Bpan01744-00898, Bba02069-00849,
                  BspZ00056-00810
X009E-X069S-X185Q AprE-01083, BspAI02518-01102, Bba02069-00791
X009E-X069S-X218S AprE-01087, Chemgen-00681, DSM14391-01030, BspAI02518-
                  01165, Bba02069-00722
X009E-X069S-X259P AprE-01857, Chemgen-00829, Bpan01744-01231, DSM14391-
                  01042, Bba02069-00795, BspZ00056-01468
X009E-X076D-X078N AprE-01892, Chemgen-00825, Bpan01744-01236, DSM14391-
                  01040, BspAI02518-01105, Bba02069-00757
X009E-X076D-X166Q AprE-01093, Chemgen-01315, Bpan01744-01237, BspAI02518-
                  01235, Bba02069-00796, BspZ00056-01478
X009E-X076D-X185Q AprE-01025, Chemgen-00737, Bpan01744-01238, DSM14391-
                  00908, BspAI02518-01153, Bba02069-00725
X009E-X076D-X218S AprE-01052, Chemgen-00690, Bpan01744-01196, DSM14391-
                  01038, BspAI02518-02121, Bba02069-01905
X009E-X076D-X259P AprE-01860, Chemgen-01420, Bpan01744-01307, DSM14391-
                  01049, BspAI02518-01107, Bba02069-00793, BspZ00056-00875
X009E-X078N-X166Q AprE-01095, Chemgen-00826, BspAI02518-02083, Bba02069-00871
X009E-X078N-X185Q AprE-01850, Chemgen-01162, Bpan01744-01248, DSM14391-
                  00915, BspAI02518-01268, Bba02069-00866
X009E-X078N-X218S AprE-01019, Chemgen-00688, Bpan01744-01241, DSM14391-
                  01047, BspAI02518-01140, Bba02069-00865
X009E-X078N-X259P AprE-01014, Chemgen-00818, Bpan01744-01242, DSM14391-
                  01053, BspAI02518-01142, Bba02069-00758
X009E-X166Q-X185Q AprE-01959, Bpan01744-01165, BspAI02518-01167, Bba02069-
                  00794, BspZ00056-00783
X009E-X166Q-X218S AprE-01029, Chemgen-00687, Bpan01744-01244, DSM14391-
                  01043, BspAI02518-01138, Bba02069-00797, BspZ00056-00913
X009E-X166Q-X259P AprE-01028, Chemgen-00686, BspAI02518-01226, Bba02069-
                  00715, BspZ00056-01238
X009E-X185Q-X218S AprE-01111, Chemgen-00705, Bpan01744-00976, DSM14391-
                  00911, BspAI02518-01227, Bba02069-00799
X009E-X185Q-X259P AprE-01096, Chemgen-00685, Bpan01744-01351, DSM14391-
                  00912, BspAI02518-01148, Bba02069-00718
X009E-X218S-X259P AprE-01026, Bpan01744-01157, DSM14391-01045, BspAI02518-
                  01251, Bba02069-00801, BspZ00056-00884
X040E-X069S-X076D Chemgen-00878, Bpan01744-00088, DSM14391-01074,
                  BspAI02518-00412, Bba02069-00720
X040E-X069S-X078N AprE-01024, Chemgen-00876, Bpan01744-00423, BspAI02518-
                  00590, Bba02069-00863, BspZ00056-00490
X040E-X069S-X166Q AprE-01865, Chemgen-00868, Bpan01744-00153, BspAI02518-
                  01025, Bba02069-00802, BspZ00056-00389
X040E-X069S-X185Q AprE-01951, Bpan01744-00414, DSM14391-00689, BspAI02518-
                  00646, Bba02069-01826, BspZ00056-00042
X040E-X069S-X218S AprE-01823, Bpan01744-00488, DSM14391-01032, BspAI02518-
                  00405, Bba02069-00882, BspZ00056-00873
X040E-X069S-X259P AprE-01036, Chemgen-01310, DSM14391-01033, BspAI02518-
                  01920, Bba02069-00730, BspZ00056-00151
X040E-X076D-X078N AprE-00540, Chemgen-00761, Bpan01744-00337, DSM14391-
                  01352, BspAI02518-00814, Bba02069-00824
X040E-X076D-X166Q AprE-01032, Chemgen-00642, Bpan01744-01035, DSM14391-
                  00272, BspAI02518-01184, Bba02069-00693
X040E-X076D-X185Q AprE-01108, Chemgen-00660, Bpan01744-01133, DSM14391-
                  00469, BspAI02518-00802, Bba02069-00729
X040E-X076D-X218S AprE-01824, Chemgen-00641, Bpan01744-01135, DSM14391-
                  00953, BspAI02518-00535, Bba02069-00695
X040E-X076D-X259P AprE-01031, Bpan01744-01148, DSM14391-00686, BspAI02518-
                  00695, Bba02069-00696
X040E-X078N-X166Q AprE-01825, Bpan01744-00158, BspAI02518-01190, Bba02069-
                  00499, BspZ00056-00297
X040E-X078N-X185Q AprE-01844, Chemgen-01465, Bpan01744-01629, DSM14391-
                  01066, BspAI02518-01032, Bba02069-00377
X040E-X078N-X218S AprE-01030, Bpan01744-00991, DSM14391-01065, BspAI02518-
                  00800, Bba02069-01952
X040E-X078N-X259P AprE-01035, Chemgen-00758, Bpan01744-01151, DSM14391-
                  00659, BspAI02518-00937, Bba02069-00845, BspZ00056-00088
X040E-X166Q-X185Q AprE-01107, Chemgen-00872, Bpan01744-00369, BspAI02518-
                  02195, Bba02069-00692, BspZ00056-00257
X040E-X166Q-X218S AprE-01820, Chemgen-00873, Bpan01744-00172, DSM14391-
                  00947, BspAI02518-01002, Bba02069-00691, BspZ00056-00837
X040E-X166Q-X259P AprE-01831, Chemgen-00752, Bpan01744-00048, BspAI02518-
                  01006, Bba02069-00705, BspZ00056-00133
X040E-X185Q-X218S AprE-01054, Chemgen-00750, Bpan01744-00094, DSM14391-
                  00281, BspAI02518-01094, Bba02069-00706, BspZ00056-01030
X040E-X185Q-X259P AprE-01105, Chemgen-00851, Bpan01744-00339, DSM14391-
                  00293, BspAI02518-00439, Bba02069-00686, BspZ00056-00377
X040E-X218S-X259P AprE-01826, Chemgen-00845, Bpan01744-00126, DSM14391-
                  00960, BspAI02518-00548, Bba02069-00685, BspZ00056-00491
X069S-X076D-X078N AprE-01810, Chemgen-00844, Bpan01744-00810, DSM14391-
                  00945, BspAI02518-01243, Bba02069-00518, BspZ00056-00490
X069S-X076D-X166Q AprE-01812, Chemgen-00746, Bpan01744-01159, BspAI02518-
                  01245, Bba02069-00708, BspZ00056-00389
X069S-X076D-X185Q AprE-00498, Chemgen-00748, Bpan01744-00291, DSM14391-
                  01016, BspAI02518-01139, Bba02069-00883, BspZ00056-00042
X069S-X076D-X218S AprE-01403, Chemgen-01054, Bpan01744-00736, DSM14391-
                  01013, BspAI02518-00870, Bba02069-00684, BspZ00056-00873
X069S-X076D-X259P AprE-01829, Chemgen-00744, Bpan01744-00888, DSM14391-
                  01012, BspAI02518-01219, Bba02069-00683, BspZ00056-00151
X069S-X078N-X166Q AprE-01830, Chemgen-00861, BspAI02518-01205, Bba02069-00505
X069S-X078N-X185Q AprE-00788, Chemgen-00860, Bpan01744-01154, DSM14391-
                  00883, Bba02069-00844
X069S-X078N-X218S AprE-01693, Chemgen-00859, Bpan01744-01162, DSM14391-
                  01014, BspAI02518-01207, Bba02069-00736
X069S-X078N-X259P AprE-01873, Chemgen-00756, Bpan01744-01779, DSM14391-
                  00474, Bba02069-00114
X069S-X166Q-X185Q AprE-00666, Bpan01744-00174, BspAI02518-01166, Bba02069-
                  00703, BspZ00056-00110
X069S-X166Q-X218S AprE-00996, Chemgen-00862, Bpan01744-01885, DSM14391-
                  01007, BspAI02518-01201, Bba02069-01832, BspZ00056-00500
X069S-X166Q-X259P AprE-01117, Chemgen-00753, BspAI02518-01151, Bba02069-
                  00704, BspZ00056-00704
X069S-X185Q-X218S AprE-00594, Chemgen-00659, Bpan01744-00963, DSM14391-
                  01105, BspAI02518-01215, Bba02069-01679
X069S-X185Q-X259P AprE-00758, Chemgen-00863, DSM14391-00882, Bba02069-00930,
                  BspZ00056-00693
X069S-X218S-X259P AprE-01807, Chemgen-00864, Bpan01744-01174, DSM14391-
                  00264, BspAI02518-01202, Bba02069-00700, BspZ00056-00705
X076D-X078N-X166Q AprE-01805, Chemgen-00645, Bpan01744-00228, BspAI02518-
                  01199, Bba02069-00247, BspZ00056-00297
X076D-X078N-X185Q AprE-00774, Bpan01744-01182, DSM14391-01018, BspAI02518-
                  00999, Bba02069-00822
X076D-X078N-X218S AprE-01407, Chemgen-00865, Bpan01744-01173, DSM14391-
                  01021, BspAI02518-00507, Bba02069-00815
X076D-X078N-X259P AprE-01118, Chemgen-00866, Bpan01744-01957, DSM14391-
                  01019, BspAI02518-01218, Bba02069-00423, BspZ00056-00088
X076D-X166Q-X185Q AprE-00920, BspAI02518-01210, Bba02069-00699, BspZ00056-00257
X076D-X166Q-X218S AprE-01802, Chemgen-00770, Bpan01744-01170, DSM14391-
                  01100, BspAI02518-01211, Bba02069-00698, BspZ00056-00837
X076D-X166Q-X259P AprE-01819, Chemgen-01542, BspAI02518-01212, Bba02069-
                  01319, BspZ00056-00133
X076D-X185Q-X218S AprE-00912, Chemgen-00771, Bpan01744-01292, DSM14391-
                  00473, BspAI02518-01124, Bba02069-01333, BspZ00056-01030
X076D-X185Q-X259P AprE-01836, Bpan01744-01289, BspAI02518-01132, Bba02069-
                  01321, BspZ00056-00377
X076D-X218S-X259P AprE-01817, Chemgen-00846, DSM14391-00162, BspAI02518-
                  01122, Bba02069-01323, BspZ00056-00491
X078N-X166Q-X185Q AprE-00974, Chemgen-00847, BspAI02518-02142, Bba02069-00190
X078N-X166Q-X218S AprE-01722, Chemgen-00763, Bpan01744-01294, DSM14391-
                  01090, BspAI02518-01128, Bba02069-00817
X078N-X166Q-X259P AprE-01816, BspAI02518-01119, Bba02069-00043
X078N-X185Q-X218S AprE-00904, Bpan01744-01717, DSM14391-00170, BspAI02518-
                  01114, Bba02069-00493
X078N-X185Q-X259P AprE-00891, Chemgen-00849, Bpan01744-00034, DSM14391-
                  01086, BspAI02518-01263, Bba02069-00005
X078N-X218S-X259P AprE-01112, Chemgen-00768, Bpan01744-01952, DSM14391-
                  00263, BspAI02518-01115, Bba02069-00051
X166Q-X185Q-X218S AprE-00698, Chemgen-00765, Bpan01744-00899, DSM14391-
                  00893, BspAI02518-01265, Bba02069-01324
X166Q-X185Q-X259P AprE-00694, Chemgen-00870, BspAI02518-01117, Bba02069-
                  01335, BspZ00056-00710
X166Q-X218S-X259P AprE-01815, Chemgen-00880, DSM14391-01445, BspAI02518-
                  01116, Bba02069-01328, BspZ00056-00238
X185Q-X218S-X259P AprE-00646, Chemgen-00767, Bpan01744-01867, DSM14391-
                  00872, Bba02069-01329, BspZ00056-01490

Although the disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A subtilisin variant having at least 70% amino acid sequence identity to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, wherein the variant has at least one, two, three, four, or more features selected from the group consisting of: X003T, X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128R, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X211P, X217L, X218S, X248D, and X259P, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

2. The subtilisin variant of claim 1, wherein the variant has at least one, two or more features selected from the group consisting of X003V, X009E, X024Q, X040E, X069S, X076D, X078N, X079I, X087D, X118R, X124I, X128S, X129P, X130S, X145R, X166Q, X182E, X185Q, X210I, X217L, X218S, X248D, and X259P, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

3. The subtilisin variant of claim 1, wherein the variant has at least one, two or more features selected from the group consisting of X003V, X009E, X040E, X069S, X076D, X078N, X166Q, X185Q, X218S, and X259P, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

4. The subtilisin variant of claim 1, wherein the variant has at least one feature selected from the group consisting of, X003V-X009E, X003V-X024Q, X003V-X040E, X003V-X069S, X003V-X076D, X003V-X078N, X003V-X079I, X003V-X087D, X003V-X118R, X003V-X124I, X003V-X128S, X003V-X129P, X003V-X130S, X003V-X145R, X003V-X166Q, X003V-X185Q, X003V-X210I, X003V-X217L, X003V-X218S, X003V-X248D, X003V-X259P, X009E-X024Q, X009E-X040E, X009E-X069S, X009E-X076D, X009E-X078N, X009E-X079I, X009E-X087D, X009E-X118R, X009E-X124I, X009E-X128S, X009E-X129P, X009E-X130S, X009E-X145R, X009E-X166Q, X009E-X185Q, X009E-X210I, X009E-X217L, X009E-X218S, X009E-X248D, X009E-X259P, X024Q-X040E, X024Q-X069S, X024Q-X076D, X024Q-X078N, X024Q-X079I, X024Q-X087D, X024Q-X118R, X024Q-X124I, X024Q-X128S, X024Q-X129P, X024Q-X130S, X024Q-X145R, X024Q-X166Q, X024Q-X185Q, X024Q-X210I, X024Q-X217L, X024Q-X218S, X024Q-X248D, X024Q-X259P, X040E-X069S, X040E-X076D, X040E-X078N, X040E-X079I, X040E-X087D, X040E-X118R, X040E-X124I, X040E-X128S, X040E-X129P, X040E-X130S, X040E-X145R, X040E-X166Q, X040E-X185Q, X040E-X210I, X040E-X217L, X040E-X218S, X040E-X248D, X040E-X259P, X069S-X076D, X069S-X078N, X069S-X079I, X069S-X087D, X069S-X118R, X069S-X124I, X069S-X128S, X069S-X129P, X069S-X130S, X069S-X145R, X069S-X166Q, X069S-X185Q, X069S-X210I, X069S-X217L, X069S-X218S, X069S-X248D, X069S-X259P, X076D-X078N, X076D-X079I, X076D-X087D, X076D-X118R, X076D-X124I, X076D-X128S, X076D-X129P, X076D-X130S, X076D-X145R, X076D-X166Q, X076D-X185Q, X076D-X210I, X076D-X217L, X076D-X218S, X076D-X248D, X076D-X259P, X078N-X079I, X078N-X087D, X078N-X118R, X078N-X124I, X078N-X128S, X078N-X129P, X078N-X130S, X078N-X145R, X078N-X166Q, X078N-X185Q, X078N-X210I, X078N-X217L, X078N-X218S, X078N-X248D, X078N-X259P, X079I-X087D, X079I-X118R, X079I-X124I, X079I-X128S, X079I-X129P, X079I-X130S, X079I-X145R, X079I-X166Q, X079I-X185Q, X079I-X210I, X079I-X217L, X079I-X218S, X079I-X248D, X079I-X259P, X087D-X118R, X087D-X124I, X087D-X128S, X087D-X129P, X087D-X130S, X087D-X145R, X087D-X166Q, X087D-X185Q, X087D-X210I, X087D-X217L, X087D-X218S, X087D-X248D, X087D-X259P, X118R-X124I, X118R-X128S, X118R-X129P, X118R-X130S, X118R-X145R, X118R-X166Q, X118R-X185Q, X118R-X210I, X118R-X217L, X118R-X218S, X118R-X248D, X118R-X259P, X124I-X128S, X124I-X129P, X124I-X130S, X124I-X145R, X124I-X166Q, X124I-X185Q, X124I-X210I, X124I-X217L, X124I-X218S, X124I-X248D, X124I-X259P, X128S-X129P, X128S-X130S, X128S-X145R, X128S-X166Q, X128S-X185Q, X128S-X210I, X128S-X217L, X128S-X218S, X128S-X248D, X128S-X259P, X129P-X130S, X129P-X145R, X129P-X166Q, X129P-X185Q, X129P-X210I, X129P-X217L, X129P-X218S, X129P-X248D, X129P-X259P, X130S-X145R, X130S-X166Q, X130S-X185Q, X130S-X210I, X130S-X217L, X130S-X218S, X130S-X248D, X130S-X259P, X145R-X166Q, X145R-X185Q, X145R-X210I, X145R-X217L, X145R-X218S, X145R-X248D, X145R-X259P, X166Q-X185Q, X166Q-X210I, X166Q-X217L, X166Q-X218S, X166Q-X248D, X166Q-X259P, X185Q-X210I, X185Q-X217L, X185Q-X218S, X185Q-X248D, X185Q-X259P, X210I-X217L, X210I-X218S, X210I-X248D, X210I-X259P, X217L-X218S, X217L-X248D, X217L-X259P, X218S-X248D, X218S-X259P, X248D-X259P wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:1 (BPN′), wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

5. The subtilisin variant of any of the preceding claims, wherein said variant is derived from a parent or reference polypeptide having has 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22.

6. The subtilisin variant of any of the preceding claims, wherein said variant comprises an amino acid sequence having 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22.

7. The subtilisin variant of any preceding claim, wherein said variant has improved stability when compared to a reference subtilisin lacking the one, two, three, four or more features.

8. The subtilisin variant of claim 7, wherein the improved stability is measured as (i) a performance index (PI) of 1.1 or greater after 20 minutes at 30-70 degrees Celsius in a 10% detergent solution in comparison to the respective parent or (ii) an improved residual activity of at least 10% greater in comparison to the respective parent under the same assay conditions.

9. The subtilisin variant of claim 8, wherein the improved stability is measured as (i) a PI of 1.1 or greater after 20 minutes at 30-70 degrees Celsius in 10% detergent solution without protease-stabilizers in comparison to the respective parent or (ii) an improved residual activity of at least 10% greater in comparison to the respective parent under the same assay conditions in a 10% detergent solution without protease-stabilizers.

10. The subtilisin variant of any preceding claim, wherein the subtilisin variant has protease activity.

11. The subtilisin variant of claim 10, wherein the subtilisin variant has a cleaning performance index (PI) value of 1.0 or greater in comparison to a reference subtilisin.

12. A polynucleotide comprising a nucleotide sequence that encodes the subtilisin variant of any one of claims 1-11, wherein said polynucleotide is optionally isolated.

13. An expression vector or cassette comprising the polynucleotide of claim 12.

14. The expression vector or cassette of claim 13, wherein the polynucleotide is operably linked to a promoter.

15. A recombinant host cell comprising the vector or cassette of claim 13 or 14.

16. A composition comprising one or more subtilisin variant according to any preceding claim.

17. The composition according to claim 16, wherein said composition is selected from an enzyme composition and a detergent composition.

18. The composition according to claim 17, wherein said detergent composition is selected from a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning product.

19. The composition of any one of claims 16-18, wherein said composition further comprises one or more ions selected from calcium and/or zinc; one or more enzyme stabilizer; from about 0.001% to about 1.0 weight % of said subtilisin variant; one or more bleaching agent; one or more adjunct material; and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, DNase or nuclease, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, lysozymes, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, nucleases, additional serine proteases, and combinations thereof.

20. The composition of any one of claims 16-19, wherein said composition contains phosphate or is phosphate-free and/or contains boron or is boron-free.

21. The composition of any one of claims 16-19, wherein said composition does not contain a protease stabilizer.

22. The composition of any one of claims 16-21, wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, paste or unit dose composition.

23. A method of cleaning, comprising contacting a surface or an item in need of cleaning with the subtilisin variant of any one of claims 1-11 or the composition of any one of claims 16-21; and optionally further comprising the step of rinsing said surface or item after contacting said surface or item with said variant or composition, wherein, optionally, said item is dishware or fabric.

24. A composition comprising the subtilisin variant of any one of claims 1-11, wherein said composition is a disinfectant, industrial or institutional cleaning, medical instrument cleaning, contact lens cleaning, wound cleaning, or textile processing composition.

25. The variant of any one of claims 1-11, wherein the variant does not have an amino acid sequence identical to a naturally occurring molecule.

26. The composition according to claim 17, wherein said enzyme composition is an enzyme granule.