Subtilisin variants multiply-substituted subtilising variants having a substitution at position 76

FIELD OF THE INVENTION

The present invention relates to novel carbonyl hydrolase variants having an amino acid sequence wherein a plurality of amino acid residues of a precursor carbonyl hydrolase, specifically those at positions corresponding or equivalent to residue +76 in combination with one or more of the residues selected from the group consisting of +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 in

Bacillus amyloliquefaciens

subtilisin, have been substituted with a different amino acid. Such mutant/variant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding a naturally-occurring or recombinant carbonyl hydrolase to encode the substitution of a plurality of these amino acid residues in a precursor amino acid sequence alone or in combination with other substitution, insertion or deletion in the precursor amino acid sequence.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolase. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R.

Sci. Amer.,

131:74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: the subtilisins and the mammalian chymotrypsin related and homologous bacterial serine proteases (e.g., trypsin and

S. gresius

trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977),

Ann. Rev. Biochem.,

46:331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisin is a serine endoprotease (MW 27,500) which is secreted in large amounts from a wide variety of Bacillus species and other microorganisms. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1983),

Hoppe

-

Sevler's Z. Physiol. Chem.,

364:1537-1540. The three-dimensional crystallographic structure of

Bacillus amyloliquefaciens

subtilisin to 2.5 Å resolution has also been reported. Wright, C. S., et al. (1969),

Nature,

221:235-242; Drenth, J., et al. (1972),

Eur. J. Biochem.,

26:177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972),

Biochemistry,

11:2439-2449) or product complexes (Robertus, J. D., et al. (1976),

J. Biol. Chem.,

251:1097-1103) have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983),

Mol. Cell. Biochem.,

51:5-32; Svendsen, B. (1976),

Carlsberg Res. Comm.,

41:237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965),

J. Biol. Chem.,

244:5333-5338) and the side chain of serine at residue 221 converted to cysteine by chemical modification (Polgar, et al. (1981),

Biochimica et Biophysica Acta,

667:351-354.)

U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606) discloses the modification of subtilisin amino acid residues corresponding to positions in

Bacillus amyloliquefaciens

subtilisin tyrosine −1, aspartate +32, asparagine +155, tyrosine +104, methionine +222, glycine +166, histidine +64, glycine +169, phenylalanine +189, serine +33, serine +221, tyrosine +217, glutamate +156 and alanine +152. U.S. Pat. No. 5,182,204 discloses the modification of the amino acid +224 residue in

Bacillus amyloliquefaciens

subtilisin and equivalent positions in other subtilisins which may be modified by way of substitution, insertion or deletion and which may be combined with modifications to the residues identified in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606) to form useful subtilisin mutants or variants. U.S. Pat. No. 5,155,033 discloses similar mutant subtilisins having a modification at an equivalent position to +225 of

B. amyloliquefaciens

subtilisin. U.S. Pat. Nos. 5,185,258 and 5,204,015 disclose mutant subtilisins having a modification at positions +123 and/or +274. The disclosure of these patents is incorporated herein by reference, as is the disclosure of U.S. patent application Ser. No. 07/898,382, which discloses the modification of many amino acid residues within subtilisin, including specifically +99, +101, +103, +107, +126, +128, +135, +197 and +204. All of these patents/applications are commonly owned. U.S. Pat. No. 4,914,031 discloses certain subtilisin analogs, including a subtilisin modified at position +76. The disclosure of this patent is also incorporated herein by reference. The particular residues identified herein and/or the specific combinations claimed herein, however, are not identified in these references.

Accordingly, it is an object herein to provide carbonyl hydrolase (preferably subtilisin) variants containing the substitution of a plurality of amino acid residues in the DNA encoding a precursor carbonyl hydrolase corresponding to positions +76 in combination with one or more positions selected from the group +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 in

Bacillus amyloliquefaciens

subtilisin. Such variants generally have at least one property which is different from the same property of the carbonyl hydrolase precursor from which the amino acid sequence of said variant is derived.

It is a further object to provide DNA sequences encoding such carbonyl hydrolase variants, as well as expression vectors containing such variant DNA sequences.

Still further, another object of the invention is to provide host cells transformed with such vectors, as well as host cells which are capable of expressing such DNA to produce carbonyl hydrolase variants either intracellularly or extracellularly.

The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of a prior invention or priority based on earlier filed applications.

SUMMARY OF THE INVENTION

The invention includes non-naturally-occurring carbonyl hydrolase variants having a different proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor carbonyl hydrolase from which the amino acid sequence of the variant is derived. The precursor carbonyl hydrolase may be a naturally-occurring carbonyl hydrolase or recombinant hydrolase. Specifically, such carbonyl hydrolase variants have an amino acid sequence not found in nature, which is derived by replacement of a plurality of amino acid residues of a precursor carbonyl hydrolase with different amino acids. The plurality of amino acid residues of the precursor enzyme correspond to position +76 in combination with one or more of the following residues +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274, where the numbered position corresponds to naturally-occurring subtilisin from

Bacillus amyloliquefaciens

or to equivalent amino acid residues in other carbonyl hydrolases or subtilisins, such as

Bacillus lentus

subtilisin. The carbonyl hydrolase variants of the present invention comprise replacement of amino acid residue +76 in combination with one or more additional modifications. Preferably the variant enzymes of the present invention comprise the substitution, deletion or insertion of amino acid residues in the following combinations: 76/99; 76/101; 76/103; 76/104; 76/107; 76/123; 76/99/101; 76/99/103; 76/99/104; 76/101/103; 76/101/104; 76/103/104; 76/104/107; 76/104/123; 76/107/123; 76/99/101/103; 76/99/101/104; 76/99/103/104; 76/101/103/104; 76/103/104/123; 76/104/107/123; 76/99/101/103/104; 76/99/103/104/123; 76/99/101/103/104/123; 76/103/104/128; 76/103/104/260; 76/103/104/265; 76/103/104/197; 76/103/104/105; 76/103/104/135; 76/103/104/126; 76/103/104/107; 76/103/104/210; 76/103/104/126/265 and/or 76/103/104/222. Most preferably the variant enzymes of the present invention comprise the substitution, deletion or insertion of an amino acid residue in the following combination of residues: 76/99; 76/104; 76/99/104; 76/103/104; 76/104/107; 76/101/103/104; 76/99/101/103/104 and 76/101/104 of

B. amyloliquefaciens

subtilisin.

The invention also includes variant DNA sequences encoding such carbonyl hydrolase or subtilisin variants. These variant DNA sequences are derived from a precursor DNA sequence which encodes a naturally-occurring or recombinant precursor enzyme. The variant DNA sequences are derived by modifying the precursor DNA sequence to encode the substitution of one or more specific amino acid residues encoded by the precursor DNA sequence corresponding to positions 76, 99, 101, 103, 104, 107, 123, 27, 105, 109, 126, 128, 135, 156, 166, 195, 197, 204, 206, 210, 216, 217, 218, 222, 260, 265 and/or 274 in

Bacillus amyloliquefaciens

or any combination thereof. Although the amino acid residues identified for modification herein are identified according to the numbering applicable to

B. amyloliquefaciens

(which has become the conventional method for identifying residue positions in all subtilisins), the preferred precursor DNA sequence useful in the present invention is the DNA sequence of

Bacillus lentus

as shown in

FIG. 6

(Seq ID No.11).

The variant DNA sequences of the present invention encode the insertion or substitution of the amino acid residue 76 in combination with one or more additional modification. Preferably the variant DNA sequences encode the substitution or insertion of amino acid residues in the following combinations: 76/99; 76/101; 76/103; 76/104; 76/107; 76/123; 76/99/101; 76/99/103; 76/99/104; 76/101/103; 76/101/104; 76/103/104; 76/104/107; 76/104/123; 76/107/123; 76/99/101/103; 76/99/101/104; 76/99/103/104; 76/101/103/104; 76/103/104/123; 76/104/107/123; 76/99/101/103/104; 76/99/103/104/123; 76/99/101/103/104/123; 76/103/104/128; 76/103/104/260; 76/103/104/265; 76/103/104/197; 76/103/104/105; 76/103/104/135; 76/103/104/126; 76/103/104/107; 76/103/104/210; 76/103/104/126/265 and/or 76/103/104/222. Most preferably the variant DNA sequences encode for the modification of the following combinations of residues: 76/99; 76/104; 76/99/104; 76/103/104; 76/104/107; 76/101/103/104; 76/99/101/103/104 and 76/101/104. These recombinant DNA sequences encode carbonyl hydrolase variants having a novel amino acid sequence and, in general, at least one property which is substantially different from the same property of the enzyme encoded by the precursor carbonyl hydrolase DNA sequence. Such properties include proteolytic activity, substrate specificity, stability, altered pH profile and/or enhanced performance characteristics.

The present invention encompasses the substitution of any of the nineteen naturally occurring L-amino acids at the designated amino acid residue positions. Such substitutions can be made in any precursor subtilisin (procaryotic, eucaryotic, mammalian, etc.). Preferably, the substitution to be made at each of the identified amino acid residue positions include but are not limited to: substitutions at position 76 including D, H, E, G, F, K, P and N; substitutions at position 99 including D, T, N, Q, G and S; substitutions at position 101 including G, D, K, L, A, E, S and R; substitutions at position 103 including Q, T, D, E, Y, K, G, R, S and A; substitutions at position 104 including all nineteen naturally-occurring amino acids; substitutions at position 107 including V, L, M, Y, G, E, F, T, S, A, N and I; substitutions at position 123 including N, T, I, G, A, C and S; substitutions at position 27 including K, N, C, V and T; substitutions at position 105 including A, D, G, R and N; substitutions at position 107 including A, L, V, Y, G, F, T, S and A; substitutions at position 109 including S, K, R, A, N and D; substitutions at position 126 including A, F, I, V and G; substitutions at position 128 including G, L and A; substitutions at position 135 including A, F, I, S and V; substitutions at position 156 including D, E, A, G, Q and K; substitutions at position 166 including all nineteen naturally-occurring amino acids; substitutions at position 195 including E; substitutions at position 197 including E; substitutions at position 204 including A, G, C, S and D; substitutions at position 206 including L, Y, N, D and E; substitutions at position 210 including L, I, S, C and F; substitutions at position 216 including V, E, T and K; substitutions at position 217 including all nineteen naturally-occurring amino acids; substitutions at position 218 including S, A, G, T and V; substitutions at position 222 including all nineteen naturally-occurring amino acids; substitutions at position 260 including P, N, G, A, S, C, K and D; substitutions at position 265 including N, G, A, S, C, K, Y and H; and substitutions at position 274 including A and S. The specifically preferred amino acid(s) to be substituted at each such position are designated below in Table I. Although specific amino acids are shown in Table I, it should be understood that any amino acid may be substituted at the identified residues.

TABLE I

Amino Acid

Preferred Amino Acid to

Residue

be Substituted/Inserted

+76

D,H

+99

D,T,N,G

+101

R,G,D,K,L,A,E

+103

A,Q,T,D,E,Y,K,G,R

+104

I,Y,S,L,A,T,G,F,M,W,D,V,N

+107

V,L,Y,G,F,T,S,A,N

+123

S,T,I

+27

K

+105

A,D,

+109

S,K,R

+126

A,I,V,F

+128

G,L

+135

I,A,S

+156

E,D,Q

+166

D,G,E,K,N,A,F,I,V,L

+195

E

+197

E

+204

A,G,C

+206

L

+210

I,S,C

+216

V

+217

H,I,Y,C,A,G,F,S,N,E,K

+218

S

+222

A,Q,S,C,I,K

+260

P,A,S,N,G

+265

N,A,G,S

+274

A,S

Further, the invention includes expression vectors containing such variant carbonyl hydrolase DNA sequences, as well as host cells transformed with such vectors which are capable of producing such variants. The invention also relates to detergent compositions comprising the carbonyl hydrolase variants of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C

depict the DNA and amino acid sequence for

Bacillus amyloliquefaciens

subtilisin and a partial restriction map of this gene (Seq. ID No.6).

FIG. 2

depicts the conserved amino acid residues among subtilisins from

Bacillus amyloliquefaciens

(BPN)′ and

Bacillus lentus

(wild-type).

FIGS. 3A and 3B

depict the amino acid sequence of four subtilisins. The top line represents the amino acid sequence of subtilisin from

Bacillus amyloliquefaciens

subtilisin (also sometimes referred to as subtilisin BPN′) (Seq. ID No.7). The second line depicts the amino acid sequence of subtilisin from

Bacillus subtilis

(Seq. ID No.8). The third line depicts the amino acid sequence of subtilisin from

B. licheniformis

(Seq. ID No.9). The fourth line depicts the amino acid sequence of subtilisin from

Bacillus lentus

(also referred to as subtilisin 309 in PCT WO89/06279.) (Seq. ID No.10). The symbol * denotes the absence of specific amino acid residues as compared to subtilisin BPN′.

FIG. 4

depicts the construction of plasmid GGA274.

FIG. 5

depicts the construction of GGT274 which is an intermediate to certain expression plasmids used in this application.

FIGS. 6A and 6B

depict the DNA and amino acid sequence of subtilisin from

Bacillus lentus

(Seq. ID No.11). The mature subtilisin protein is coded by the codons beginning at the codon GCG (334-336) corresponding to Ala.

FIGS. 7A and 7B

depict the DNA and amino acid sequence of a preferred embodiment of the invention (N76D/S103A/V104I) (Seq. ID No.12). The DNA in this figure has been modified by the methods described to encode aspartate at position 76, alanine at position 103 and isoleucine at position 104. The mature subtilisin variant protein is coded by the codons beginning at the codon GCG (334-336) corresponding to Ala.

FIG. 8

depicts the construction of vector pBCDAICAT.

FIG. 9

depicts the construction of vector pUCCATFNA.

FIG. 10

shows the stability of a preferred mutant enzyme compared to wild-type, in a liquid detergent formulation.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that in vitro mutations in

B. lentus

subtilisin at an amino acid residue equivalent to +76 in

Bacillus amyloliquefaciens

subtilisin produces subtilisin variants exhibiting altered stability (e.g., modified autoproteolytic stability) over precursor subtilisins. (See Tables IV and VI.)

It has also been discovered that in vitro mutation at residues equivalent to +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 in

Bacillus amyloliquefaciens

subtilisin, alone or in combination with each other and in any combination with +76 mutations, produce subtilisin variants exhibiting altered proteolytic activity, altered thermal stability, altered pH profile, altered substrate specificity and/or altered performance characteristics.

Carbonyl hydrolases are enzymes which hydrolyze compounds containing

bonds in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally-occurring carbonyl hydrolases principally include hydrolases, e.g., peptide hydrolases such as subtilisins or metalloproteases. Peptide hydrolases include α-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid proteases are included, as well as endo and exo-proteases.

“Recombinant carbonyl hydrolase” refers to a carbonyl hydrolase in which the DNA sequence encoding the naturally-occurring carbonyl hydrolase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the carbonyl hydrolase amino acid sequence. Suitable modification methods are disclosed herein, and in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606), U.S. Pat. Nos. 5,204,015 and 5,185,258, the disclosure of which are incorporated herein by reference.

Subtilisins are bacterial or fungal carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “subtilisin” means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this. series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases. Examples include but are not limited to the subtilisins identified in

FIG. 3

herein.

“Recombinant subtilisin” refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a variant (or mutant) DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally-occurring subtilisin amino acid sequence. Suitable methods to produce such modification, and which may be combined with those disclosed herein, include those disclosed in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606), U.S. Pat. Nos. 5,204,015 and 5,185,258.

“Non-human carbonyl hydrolases” and the DNA encoding them may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as

E. coli

or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic organisms from which carbonyl hydrolase and their genes may be obtained include yeast such as

Saccharomyces cerevisiae,

fungi such as Aspergillus sp. and non-human mammalian sources such as, for example, bovine sp. from which the gene encoding the carbonyl hydrolase chymosin can be obtained. As with subtilisins, a series of carbonyl hydrolases can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human carbonyl hydrolase as used herein has a functional definition which refers to carbonyl hydrolases which are associated, directly or indirectly, with procaryotic and eucaryotic sources.

A “carbonyl hydrolase variant” has an amino acid sequence which is derived from the amino acid sequence of a “precursor carbonyl hydrolase.” The precursor carbonyl hydrolases (such as a subtilisin) include naturally-occurring carbonyl hydrolases (subtilisin) and recombinant carbonyl hydrolases (subtilisin). The amino acid sequence of the carbonyl hydrolase variant is “derived” from the precursor hydrolase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor carbonyl hydrolase (subtilisin) rather than manipulation of the precursor carbonyl hydrolase (subtilisin) enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods known to those skilled in the art (see, for example, EP 0 328299, WO89/06279 and the U.S. patents and applications already referenced herein).

Specific residues corresponding to position +76 in combination with one or more of the following positions +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 of

Bacillus amyloliquefaciens

subtilisin are identified herein for mutation. Preferably the modified residues are selected from the following combinations: 76/99; 76/101; 76/103; 76/104; 76/107; 76/123; 76/99/101; 76/99/103; 76/99/104; 76/101/103; 76/101/104; 76/103/104; 76/104/107; 76/104/123; 76/107/123; 76/99/101/103; 76/99/101/104; 76/99/103/104; 76/101/103/104; 76/103/104/123; 76/104/107/123; 76/99/101/103/104; 76/99/103/104/123; 76/99/101/103/104/123; 76/103/104/128; 76/103/104/260; 76/103/104/265; 76/103/104/197; 76/103/104/105; 76/103/104/135; 76/103/104/126; 76/103/104/107; 76/103/104/210; 76/103/104/126/265 and/or 76/103/104/222; and most preferably are 76/99; 76/104; 76/99/104; 76/103/104; 76/104/107; 76/101/103/104; 76/99/101/103/104 and 76/101/104. These amino acid position numbers refer to those assigned to the mature

Bacillus amyloliquefaciens

subtilisin sequence presented in FIG.

1

. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor carbonyl hydrolases containing amino acid residues at positions which are “equivalent” to the particular identified residues in

Bacillus amyloliquefaciens

subtilisin. In a preferred embodiment of the present invention, the precursor subtilisin is

Bacillus lentus

subtilisin and the substitutions, deletions or insertions are made at the equivalent amino acid residue in

B. lentus

corresponding to those listed above.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of

Bacillus amyloliquefaciens

subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in

Bacillus amyloliquefaciens

subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor carbonyl hydrolase is directly compared to the

Bacillus amyloliquefaciens

subtilisin primary sequence and particularly to a set of residues known to be invariant in subtilisins for which sequence is known.

FIG. 2

herein shows the conserved residues as between

B. amyloliquefaciens

subtilisin and

B. lentus

subtilisin. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of

Bacillus amyloliquefaciens

subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, in

FIG. 3

the amino acid sequence of subtilisin from

Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis

(

carlsbergensis

) and

Bacillus lentus

are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These conserved residues (as between BPN′ and

B. lentus

) are identified in FIG.

2

.

These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of

Bacillus amyloliquefaciens

subtilisin in other carbonyl hydrolases such as subtilisin from

Bacillus lentus

(PCT Publication No. WO89/06279 published Jul. 13, 1989), the preferred subtilisin precursor enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which is highly homologous to the preferred

Bacillus lentus

subtilisin. The amino acid sequences of certain of these subtilisins are aligned in

FIGS. 3A and 3B

with the sequence of

Bacillus amyloliquefaciens

subtilisin to produce the maximum homology of conserved residues. As can be seen, there are a number of deletions in the sequence of

Bacillus lentus

as compared to

Bacillus amyloliquefaciens

subtilisin. Thus, for example, the equivalent amino acid for Val165 in

Bacillus amyloliquefaciens

subtilisin in the other subtilisins is isoleucine for

B. lentus

and

B. licheniformis.

Thus, for example, the amino acid at position +76 is asparagine (N) in both

B. amyloliquefaciens

and

B. lentus

subtilisins. In the preferred subtilisin variant of the invention, however, the amino acid equivalent to +76 in

Bacillus amyloliquefaciens

subtilisin is substituted with aspartate (D). A comparison of certain of the amino acid residues identified herein for substitution versus the most preferred substitution for each such position is provided in Table II for illustrative purposes.

TABLE II

+76

+99

+101

+103

+104

+107

+123

B. amyloliquefaciens

N

D

S

Q

Y

I

N

(wild-type)

B. lentus

(wild-type)

N

S

S

S

V

I

N

Most Preferred

D

D

R

A

I/Y

V

S

Substitution

Equivalent residues may also be defined by determining homology at the level of tertiary structure for a precursor carbonyl hydrolase whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the precursor carbonyl hydrolase and

Bacillus amyloliquefaciens

subtilisin (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the carbonyl hydrolase in question to the

Bacillus amyloliquefaciens

subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

R factor = \frac{Σ_{h} &LeftBracketingBar; Fo (h) &RightBracketingBar; - &LeftBracketingBar; Fc (h) &RightBracketingBar;}{Σ_{h} &LeftBracketingBar; Fo (h) &RightBracketingBar;}

Equivalent residues which are functionally analogous to a specific residue of

Bacillus amyloliquefaciens

subtilisin are defined as those amino acids of the precursor carbonyl hydrolases which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the

Bacillus amyloliquefaciens

subtilisin. Further, they are those residues of the precursor carbonyl hydrolase (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of

Bacillus amyloliquefaciens

subtilisin. The coordinates of the three dimensional structure of

Bacillus amyloliquefaciens

subtilisin are set forth in EPO Publication No. 0 251 446 (equivalent to U.S. patent application Ser. No. 08/212,291, the disclosure of which is incorporated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a variant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally-occurring sequence. The carbonyl hydrolase variants of the present invention include the mature forms of carbonyl hydrolase variants, as well as the pro- and prepro-forms of such hydrolase variants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the carbonyl hydrolase variants.

“Prosequence” refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a carbonyl hydrolase which when removed results in the appearance of the “mature” form of the carbonyl hydrolase. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. A preferred prosequence for producing carbonyl hydrolase variants, specifically subtilisin variants, is the putative prosequence of

Bacillus amyloliquefaciens

subtilisin, although other subtilisin prosequences may be used. In Examples 1 and 2 the putative prosequence from the subtilisin from

Bacillus lentus

(ATCC 21536) was used.

A “signal sequence” or “presequence” refers to any sequence of amino acids bound to the N-terminal portion of a carbonyl hydrolase or to the N-terminal portion of a prohydrolase which may participate in the secretion of the mature or pro forms of the hydrolase. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the subtilisin gene or other secretable carbonyl hydrolases which participate in the effectuation of the secretion of subtilisin or other carbonyl hydrolases under native conditions. The present invention utilizes such sequences to effect the secretion of the carbonyl hydrolase variants as defined herein. A preferred signal sequence used in the Examples comprises the first seven amino acid residues of the signal sequence from

Bacillus subtilis

subtilisin fused to the remainder of the signal sequence of the subtilisin from

Bacillus lentus

(ATCC 21536).

A “prepro” form of a carbonyl hydrolase variant consists of the mature form of the hydrolase having a prosequence operably linked to the amino terminus of the hydrolase and a “pre” or “signal” sequence operably linked to the amino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606) to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing subtilisin is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in U.S. Pat. No. 5,264,366. Other host cells for expressing subtilisin include

Bacillus subtilis

I168 (also described in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606) and U.S. Pat. No. 5,264,366, the disclosure of which are incorporated herein by reference), as well as any suitable Bacillus strain such as

B. licheniformis, B. lentus,

etc.

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the carbonyl hydrolase variants or expressing the desired carbonyl hydrolase variant. In the case of vectors which encode the pre- or prepro-form of the carbonyl hydrolase variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

“Operably linked,” when describing the relationship between two DNA regions, simply means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The genes encoding the naturally-occurring precursor carbonyl hydrolase may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the hydrolase of interest, preparing genomic libraries from organisms expressing the hydrolase, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced. The

B. lentus

gene used in the Examples was cloned as described in Example 1 of U.S. Pat. No. 5,185,258, the disclosure of which is incorporated herein. The BPN′ gene used in Example 5 was cloned as described in Example 1 in U.S. Pat. RE No. 34,606, the disclosure of which is incorporated herein.

The cloned carbonyl hydrolase is then used to transform a host cell in order to express the hydrolase. The hydrolase gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promotor if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the hydrolase gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the hydrolase gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosomal limitations. However, it is within the scope herein to integrate multiple copies of the hydrolase gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination.

The genes used in the present examples are a natural

B. lentus

gene and a natural

B. amyloliquefaciens

gene. Alternatively, a synthetic gene encoding a naturally-occurring or mutant precursor carbonyl hydrolase (subtilisin) may be produced. In such an approach, the DNA and/or amino acid sequence of the precursor hydrolase (subtilisin) is determined. Multiple, overlapping synthetic single-stranded DNA fragments are thereafter synthesized, which upon hybridization and ligation produce a synthetic DNA encoding the precursor hydrolase. An example of synthetic gene construction is set forth in Example 3 of U.S. Pat. No. 5,204,015, the disclosure of which is incorporated herein by reference.

Once the naturally-occurring or synthetic precursor carbonyl hydrolase gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor carbonyl hydrolase. Such modifications include the production of recombinant carbonyl hydrolases as disclosed in U.S. Pat. No. 4,760,025 (U.S. Pat. RE No. 34,606) and EPO Publication No. 0 251 446 and the production of carbonyl hydrolase variants described herein.

The following cassette mutagenesis method may be used to facilitate the construction and identification of the carbonyl hydrolase variants of the present invention, although other methods including site-directed mutagenesis may be used. First, the naturally-occurring gene encoding the hydrolase is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the hydrolase gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the hydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

As used herein, proteolytic activity is defined as the rate of hydrolysis of peptide bonds per milligram of active enzyme. Many well known procedures exist for measuring proteolytic activity (K. M. Kalisz, “Microbial Proteinases,”

Advances in Biochemical Engineering/Biotechnology,

A. Fiechter ed., 1988). In addition to or as an alternative to modified proteolytic activity, the variant enzymes of the present invention may have other modified properties such as K

m

, k

cat

, k

cat

/K

m

ratio and/or modified substrate specificity and/or modified pH activity profile. These enzymes can be tailored for the particular substrate which is anticipated to be present, for example, in the preparation of peptides or for hydrolytic processes such as laundry uses.

In one aspect of the invention the objective is to secure a variant carbonyl hydrolase having altered proteolytic activity as compared to the precursor carbonyl hydrolase, since increasing such activity (numerically larger) enables the use of the enzyme to more efficiently act on a target substrate. Also of interest are variant enzymes having altered thermal stability and/or altered substrate specificity as compared to the precursor. Preferably the carbonyl hydrolase to be mutated is a subtilisin. Specific amino acids useful to obtain such results in subtilisin-type carbonyl hydrolases at residues equivalent to +76, +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 or any combination thereof in

Bacillus amyloliquefaciens

subtilisin are presented in the Examples. In some instances, lower proteolytic activity may be desirable, for example a decrease in proteolytic activity would be useful where the synthetic activity of the carbonyl hydrolases is desired (as for synthesizing peptides). One may wish to decrease this proteolytic activity, which is capable of destroying the product of such synthesis. Conversely, in some instances it may be desirable to increase the proteolytic activity of the variant enzyme versus its precursor. Additionally, increases or decreases (alteration) of the stability of the variant, whether alkaline or thermal stability, may be desirable. Increases or decreases in k

cat

, K

m

or k

cat

/K

m

are specific to the substrate used to determine these kinetic parameters.

In another aspect of the invention, it has been determined that residues equivalent to +76 in combination with a number of other modifications in subtilisin are important in modulating overall stability and/or proteolytic activity of the enzyme. Thus, as set forth in the Examples, the Asparagine (N) in

Bacillus lentus

subtilisin at equivalent position +76 can be substituted with Aspartate (D) in the preferred embodiment in combination with modification of one or more of the following amino acid residues +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274, to produce enhanced stability and/or enhanced activity of the resulting mutant enzyme.

The most preferred embodiments of the invention are set forth in the Examples. These include the following specific combinations of substituted residues: N76D/S99D; N76D/V104I; N76D/S99D/V104I; N76D/S103A/V104I; N76D/V104I/I107V; N76D/V104Y/I107V and N76D/S101R/S103A/V104I. Also described in the Examples are all mutant combinations claimed in the present invention. These substitutions are preferably made in

Bacillus lentus

(recombinant or native-type) subtilisin, although the substitutions may be made in any Bacillus subtilisin.

Based on the results obtained with this and other variant subtilisins, it is apparent that residues in carbonyl hydrolases (preferably subtilisin) equivalent to positions +76, +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 in

Bacillus amyloliquefaciens

are important to the proteolytic activity, performance and/or stability of these enzymes and the cleaning or wash performance of such variant enzymes.

Many of the carbonyl hydrolase variants of the invention, especially subtilisin, are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the carbonyl hydrolase mutants of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S. Pat. No. 4,261,868 to Jiri Flora, et al. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 (previously incorporated by reference). The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the subtilisin variants of the present invention may be used for any purpose that native or wild-type subtilisins are used. Thus, these variants can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, etc. The variants of the present invention may comprise enhanced performance in a detergent composition (as compared to the precursor). As used herein, enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.

Subtilisins of the invention can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.

The addition of subtilisins of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range, and the temperature is below the described subtilisin's denaturing temperature. In addition, subtilisins of the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLE 1

Construction for the Expression of GG36 Gene in

B. subtilis

The cloning and the construction for expression of the subtilisin gene from

B. lentus

was performed essentially the same as that described in U.S. Pat. No. 5,185,258. The plasmid GGA274 (described in

FIG. 4

herein) was further modified in the following manner, as shown in FIG.

5

. The PstI site that was introduced during the construction of the GGA274 plasmid was removed by the oligonucleotide directed mutagenesis described below, with an oligonucleotide having the following sequence: 5′ GAAGCTGCA

A

CTCGTTAAA 3′ (Seq. ID No.1). The underlined “A” residue eliminated the recognition sequence of restriction enzyme PstI and changed the corresponding amino acid residue from alanine to threonine at position 274. Threonine at position 274 is the wild-type residue originally found in the cloned

B. lentus

subtilisin gene sequences. The DNA segment encoding subtilisin was excised from the plasmid GGA274 or its derivatives (GGT274 shown in

FIG. 5

) by EcoRI and BamHI digest. The DNA fragment was subcloned back into Bacteriophage M13-based vectors, such as MP19, for mutagenesis. After mutagenesis, the EcoRI and HindIII digest, followed by cloning, were performed to move the mutated subtilisin gene back into an expression plasmid like GGA274 for the expression and the recovery of mutated subtilisin proteins.

EXAMPLE 2

Oligonucleotide-Directed Mutagenesis

Oligonucleotide-directed mutagenesis was performed as described in Zoller, M., et al. (1983),

Methods Enzymol.,

100:468-500. As an example, a synthetic oligonucleotide of the sequence 5′ GCTGCT

C

TA

G

ACAATTCG 3′ (Seq. ID No.2) was used to change the amino acid residue at position 76 from asparagine (N) to aspartic acid (D), or N76D. The underlined “G” and “C” residues denote changes from the wild-type gene sequence. The

CA

keeps the leucine at position +75 and changes the amino acid sequence to introduce an XbaI recognition site of the XbaI restriction enzyme (TCTAGA), while the change at GAC changes asparagine at +76 to aspartate.

For mutagenesis at positions 99, 101, 103 and 104, different oligonucleotides can be used depending on the combination of mutations desired. For example, an oligonucleotide of the sequence 5′ GTATTAGGGGCG

GA

CGGT

CG

AGG

CG

C

CA

TCAGCTCGATT 3′ (Seq. ID No.3) was used to simultaneously make the following changes: S99D; S101R; S103A and V104I in a single subtilisin molecule. Similarly, oligonucleotides of the sequence 5′ TCAGGTTCGGTC

TCGAG

C

G

TTGCCCAAGGATTG 3′ (Seq. ID No.4) and 5′ CACGTTGCTA

GC

TTGAGTTTAG 3′ (Seq. ID No.5) were utilized to generate I107V and N123S, respectively. Again, the underlined residues denote changes from wild-type sequences which produced desired changes either in amino acid sequences or restriction enzyme recognition sequences.

EXAMPLE 3

Proteolytic Activity of Subtilisin Variants

Following the methods of Example 2, the variants listed in Table III were made. Proteolytic activity of each of these subtilisin variants is shown in Table III. The kinetic parameters k

cat

, K

M

, and k

cat

/K

M

were measured for hydrolysis of the synthetic peptide substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide using the method described in P. Bonneau, et al. (1991)

J. Am. Chem. Soc.,

Vol. 113, No. 3, p. 1030. Briefly, a small aliquot of subtilisin variant stock solution was added to a 1 cm cuvette containing substrate dissolved in 0.1M Tris-HCL buffer, pH 8.6, and thermostated at 25° C. The reaction progress was followed spectrophotometrically by monitoring the absorbance of the reaction product p-nitroaniline at 410 nm. Kinetic parameters were obtained by using a non-linear regression algorithm to fit the reaction velocity and product concentration for each reaction to the Michaelis-Menten equation.

TABLE III

Kinetic Parameters k

cat

, K

M

and k

cat

/K

M

Measured for

Bacillus lentus

Subtilisin and Variants

k

cat

/

Enzyme

k

cat

(s

−1

)

K

M

(M)

K

M

(s

−1

M

−1

)

B. lentus

Subtilisin

170

0.00078

2.18 × 10

5

N76D

219

0.0008

2.74 × 10

5

N76D/S99D

88

0.00061

1.44 × 10

5

N76D/S103A

400

0.0014

2.86 × 10

5

N76D/V104I

459

0.0011

4.17 × 10

5

N76D/I107V

219

0.0011

1.99 × 10

5

N76D/N123S

115

0.0018

6.40 × 10

4

N76D/S99D/S101R

146

0.00038

3.84 × 10

5

N76D/S99D/S103A

157

0.0012

1.31 × 10

5

N76D/S99D/V104I

247

0.00097

2.55 × 10

5

N76D/S101R/S103A

405

0.00069

5.90 × 10

5

N76D/S101R/V104I

540

0.00049

1.10 × 10

6

N76D/S103A/V104I

832

0.0016

5.20 × 10

5

N76D/V104I/I107V

497

0.00045

1.10 × 10

6

N76D/V104Y/I107V

330

0.00017

1.90 × 10

6

N76D/V104I/N123S

251

0.0026

9.65 × 10

4

N76D/I107V/N123S

147

0.0035

4.20 × 10

4

N76D/S99D/S101R/S103A

242

0.00074

3.27 × 10

5

N76D/S99D/S101R/V104I

403

0.00072

5.60 × 10

5

N76D/S99D/S103A/V104I

420

0.0016

2.62 × 10

5

N76D/S101R/S103A/V104I

731

0.00065

1.12 × 10

6

N76D/S103A/V104I/N1235

321

0.0026

1.23 × 10

5

N76D/V104I/I107V/N1235

231

0.003

7.70 × 10

4

N76D/S99D/S101R/S103A/V104I

624

0.00098

6.37 × 10

5

N76D/S99D/S103A/V104I/N123S

194

0.0043

4.51 × 10

4

N76D/S99D/S101R/S103A/V104I/

311

0.0023

1.35 × 10

5

N123S

The results listed in Table III indicate that all of the subtilisin variants tested retain proteolytic activity. Further, detailed analysis of the data reveal that proteolytic activity was significantly altered for

Bacillus lentus

subtilisin by the various combinations of substitutions at amino acid residues equivalent to positions 76, 99, 101, 103, 104, 107 and 123 in

Bacillus amyloliquefaciens.

EXAMPLE 4

Thermal Stability of Subtilisin Variants

A comparison of thermal stability observed for

Bacillus lentus

subtilisin and the variants of the present invention made by the process of Example 2 is shown in Table IV. Purified enzyme, 15 ug/ml in 0.1 M glycine 0.01% Tween-80 pH 10.0, with or without 50 mM CaCl

2

, was aliquotted into small tubes and incubated at 10° C. for 5 minutes, 10° C. to 60° C. over 1 minute, and 60° C. for 20 minutes. Tubes were then placed on ice for 10 minutes. Aliquots from the tubes were assayed for enzyme activity by addition to 1 cm cuvettes containing 1.2 mM of the synthetic peptide substrate succinyl-L-ala-L-Ala-L-Pro-L-Phe-p-nitroanilide dissolved in 0.1 M tris-HCL buffer, pH 8.6, thermostatted at 25° C. The initial linear reaction velocity was followed spectrophotometrically by monitoring the absorbance of the reaction product p-nitroaniline at 410 nm as a function of time. Data are presented as percent activity prior to heating. The results listed in Table IV indicate that a vast majority of variants exhibit thermal stability comparable to

Bacillus lentus

subtilisin (24 out of 26) in the test condition with 50 mM CaCl

2

added. In the test condition without 50 mM CaCl

2

added, a vast majority of variants (19 out of 26) are significantly more stable than

Bacillus lentus

subtilisin. Further, the variants N76D/S99D, N76D/V104I, N76D/S99D/V104I, N76D/S103A/V104I, N76D/V104I/I107V, N76D/V104Y/I107V and N76D/S101R/S103A/V104I are significantly more stable than the single substitution variant N76D in the test condition without 50 mM CaCl

2

added.

TABLE IV

Thermal Stability Measured for

Bacillus lentus

Subtilisin and Variants

At pH 10, 60° C., +/− 50 mM CaCl

2

Added

% Initial

Activity Remaining

Enzyme

−CaCl

2

+CaCl

2

B. lentus

Subtilisin

2

96

N76D

34

97

N76D/S99D

49

98

N76D/S103A

26

92

N76D/V104I

58

98

N76D/I107V

32

96

N76D/N123S

0

97

N76D/S99D/S101R

30

100

N76D/S99D/S103A

36

100

N76D/S99D/V104I

48

97

N76D/S101R/S103A

26

100

N76D/S101R/V104I

38

100

N76D/S103A/V104I

58

100

N76D/V104I/I107V

60

97

N76D/V104Y/I107V

48

74

N76D/V104I/N123S

0

98

N76D/I107V/N123S

16

100

N76D/S99D/S101R/S103A

38

100

N76D/S99D/S101R/V104I

33

100

N76D/S99D/S103A/V104I

38

98

N76D/S101R/S103A/V104I

40

99

N76D/S103A/V104I/N123S

1

98

N76D/V1041/X107V/N123S

3

99

N76D/S99D/S101R/S103A/V104I

36

99

N76D/S99D/S103A/V104I/N123S

2

95

N76D/S99D/S101R/S103A/V104I/N123S

0

100

EXAMPLE 5

Oligonucleotide-Directed Mutagenesis with Single-Stranded DNA Template Generated from Phagemid

A. Construction of

B. lentus

Variants

The mutagenesis protocol was essentially the same as described above in Example 2. The single-stranded DNA template was generated by phagemid method. To construct the phagemid vector for generating the single-stranded DNA template we first constructed the vector pBCDAICAT. The flow chart of vector construction is outlined in FIG.

8

. First, the ClaI to ClaI fragment encoding the CAT gene from pC194 plasmid (Horinouchi, S. and Weisblum, B.,

J. Bacteriol.,

150:8-15, 1982) was cloned into the AccI site of polylinker region of pUC19 (New England BioLabs, Beverly, Mass.) to make plasmid PUCCHL and the EcoRI-DraI 0.6 KB fragment from the 5′ end of the GG36DAI encoding DNA was cloned into the EcoRI and EcoRV sites of pBSKS plasmid (Stratagene, Inc., San Diego, Calif.) to make pBC2SK5. The single EcoRI site of the plasmid pBC2SK5 was eliminated by EcoRI digestion, followed by filling in catalyzed-by-T4 DNA polymerase, and religation to generate the plasmid pBC2SK-5R which does not have the EcoRI site. The EcoRI-DraI fragment which was cloned into pBCSK-5R was isolated as a PstI-HindIII fragment and cloned into the PstI-HindIII site of the pUCCHL (part of the polylinker of pUC19) to generate plasmid pUCCHL5R. The encoding sequence of GG36DAI gene was excised as an EcoRI-BamHI fragment and cloned into the EcoRI-BamHI sites of pUCCHL5R to make PUCCAT. The large EcoRI-HindIII fragment of pUCCAT was then cloned into the EcoRI and HindIII sites of BS2KS+ to generate the plasmid pBCDAICAT.

To generate single-stranded DNA,

E. coli

-containing pBCDAICAT were infected with phage R408 (obtained from Stratagene, San Diego, Calif.) following the protocol described in Russel, M., Kidd, S. and Kelley, M. R., GENE 45:333-338, 1986. Once the single-stranded DNA template was available, standard mutagenesis methods as described above in Example 2 were carried out. The construction of certain mutants is detailed below for illustrative purposes.

For the construction of

B. lentus

(GG36) N76D/S103A/V104I/L217H, an EcoRI-BamHI DNA fragment encoding GG36 N76D/S103A/V104I was used in the construction of pUCCAT (see

FIG. 8

) to generate the plasmid pBCDAICAT. After the single-stranded DNA template was made following the protocol described above, a mutagenesis primer with the following sequence

* *** ** x ClaI

5′ TAT GCC AGC

CAC

AAC GGT AC

T

TCG ATG GCT 3′ (Seq. ID No.13) was used to make the L217H. As before, the underlined residues denote the nucleotide changes that were made and the x ClaI indicates that the existing ClaI site was eliminated after the mutagenesis. The mutagenesis protocol was as described in Example 2. After the mutagenesis, plasmid DNA was first screened for the elimination of the ClaI site and those clones missing the ClaI site were subjected to DNA sequence analysis to verify the DNA sequence which made the L to H change at the 217th amino acid residue.

B. Construction of BPN′ Variants and their Expression in

B. subtilis

The construction of

B. amyloliquefaciens

(BPN′) N76D/Q103A/Y104I/Y217L was done in a similar fashion except in two consecutive steps. N76D was first introduced into BPN′ Y217L to make BPN′ N76D/Y217L and a second mutagenesis was done to convert BPN′ N76D/Y217L to BPN′ N76D/Q103A/Y104I/Y217L. To generate the single-stranded DNA template for the first mutagenesis, an EcoRI-BamHI fragment encoding BPN′ Y217L subtilisin (derived from the Y217L plasmid described in Wells, J., et al.,

PNAS,

84, 5167, 1087) was used to construct a plasmid pUCCATFNA (see FIG.

9

). The pUCCATFNA plasmid containing BPN′ Y217L was used to construct the pBCFNACAT plasmid (FIG.

9

). Single-stranded DNA was generated as described above. To generate BPN′ N76D/Y217L, an oligonucleotide primer with the sequence

* *** ** x XbaI

5′ C ACA GTT GCG GCT CT

A

G

AT AAC TCA ATC GGT G 3′ (Seq. ID No.14) was used to generate the change N76D. Single-stranded DNA was then prepared from the pBCFNACAT plasmid containing the BPN′ N76D/Y217L (the pBCFNACAT plasmid after N76D mutagenesis) and mutagenized with another oligonucleotide with the sequence

* *** ** x PvuII

5′ GCT GAC GGT TCC GGC

GCT

ATT

AG

T

TGG ATC ATT 3′ (Seq. ID No.15) to obtain BPN′ N76D/Q103A/Y104I/Y217L. All steps involved in the cloning, the single-stranded DNA preparation, the mutagenesis, and the screening for mutants were carried out as described above.

Expression of the BPN′ gene and its variants were achieved by integrating plasmid DNA (PBCFNACAT containing the different variants' BPN′ gene) directly into a protease-deficient strain of

Bacillus subtilis

as described in U.S. Pat. RE No. 34,606.

Numerous variants were made as per the teachings of Examples 2 and 5. Kinetics data and stability data were generated for such variants. The kinetics data were generated using the methods described in Example 3 and are provided in Table V. The stability data were generated as detailed herein. Results are shown in Table VI.

Thermal Stability Assay Procedure

Purified enzyme was buffer-exchanged into 0.1 M glycine pH 10.0, 0.01% Tween-80 by applying the enzyme to a column consisting of Sephadex G-25 equilibrated with this buffer and eluting the enzyme from the column using the same buffer.

To a tube containing 0.1 M glycine, 0.01% Tween-80 pH 10.0 thermostatted at 60° C., the buffer-exchanged enzyme was added to give a final enzyme concentration of 15 ug/ml.

Aliquots were removed from the 60° C. incubation at various times and immediately assayed for enzyme activity by addition to a 1 cm cuvette containing 1.2 mM of the synthetic peptide substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide dissolved in 0.1 M tris-HCL buffer, pH 8.6, thermostatted at 25° C. The initial linear reaction velocity was followed spectrophotometrically by monitoring the absorbance of the reaction product p-nitroaniline at 410 nm as a function of time.

Half-life, which is the length of time required for 50% enzyme inactivation, was determined from the first-order plot of reaction velocity as a function of the time of incubation at 60° C.

The data are presented in Table VI as percent of the half-life determined for

Bacillus lentus

subtilisin (GG36) under identical conditions.

TABLE V

kcat

KW

kcat/KM

Enzyme

(s

−1

)

(mM)

(s

−1

M

−1

)

B. lentus

subtilisin

170

0.78

2.20E + 05

N76D/S103G/V104I*

380

1.4

2.70E + 05

N76D/S103A/V104F

730

0.33

2.20E + 06

N76D/S103A/V104N

790

2.8

2.80E + 05

N76D/S103A/V104S

170

0.83

2.00E + 05

N76D/S103A/V104T

370

1.9

2.00E + 05

N76D/S103A/V104W

880

0.31

2.80E + 06

N76D/S103A/V104Y

690

0.5

1.40E + 06

K27R/N76D/V104Y/N123S

500

1.2

4.20E + 05

N76D/S101G/S103A/V104I*

620

1.3

4.80E + 05

N76D/S103A/V104I/S105A*

550

1.3

4.20E + 05

N76D/S103A/V104I/S105D*

440

1.7

2.60E + 05

N76D/S103A/V104T/I107A*

120

5.7

2.10E + 04

N76D/S103A/V104T/I107L*

310

3.2

9.70E + 04

N76D/S103A/V104I/L126A

90

2.2

4.10E + 04

N76D/S103A/V104I/L126F

180

1.9

9.50E + 04

N76D/S103A/V104I/L126I

100

2.4

4.20E + 04

N76D/S103A/V104I/L126V

64

3.2

2.00E + 04

N76D/S103A/V104I/S128G*

560

1.7

3.30E + 05

N76D/S103A/V104I/S128L*

430

3.8

1.10E + 05

N76D/S103A/V104I/L135A

140

0.76

1.80E + 05

N76D/S103A/V104I/L135F

390

0.69

5.70E + 05

N76D/S103A/V104I/L135I

110

0.73

1.50E + 05

N76D/S103A/V104I/L135V

140

0.86

1.60E + 05

N76D/S103A/V104I/5156E*

170

2.6

6.50E + 04

N76D/S103A/V104I/5166D*

160

3.5

4.60E + 04

N76D/S103A/V104I/D197E

510

1.4

3.60E + 05

N76D/S103A/V104I/N204A*

530

1.1

4.80E + 05

N76D/S103A/V104I/N204G*

580

1.4

4.10E + 05

N76D/S103A/V104I/N204C*

370

1.3

2.90E + 05

N76/S103A/V104I/P210I*

500

1.2

4.20E + 05

N76D/S103A/V104I/L217H*

80

0.63

1.30E + 05

N76D/S103A/V104I/M222A

70

3.1

2.30E + 04

N76D/S103A/V104I/M222S

80

3.1

2.60E + 04

N76D/S103A/V104I/T260P

660

1.5

4.40E + 05

N76D/S103A/V104I/S265N

590

1.3

4.50E + 05

K27R/N76D/V104Y/I107V/N123S

220

1.4

1.60E + 05

K27R/N76D/V104Y/N123S/D197E

430

1.1

3.90E + 05

K27R/N76D/V104Y/N123S/N204C

400

1.1

3.60E + 05

K27R/N76D/V104Y/N123S/Q206L

440

1.2

3.70E + 05

K27R/N76D/V104Y/N123S/S216V

440

1.2

3.70E + 05

K27R/N76D/V104Y/N123S/N218S

760

0.98

7.80E + 05

K27R/N76D/V104Y/N123S/T260P

410

1.2

3.40E + 05

K27R/N76D/V104Y/N123S/T274A

390

1

3.90E + 05

N76D/S103A/V104I/L126F/S265N

170

2.1

8.10E + 04

N76D/S103A/V104I/S156E/S166D*

40

6.3

6.40E + 03

K27R/N76D/V104Y/N123S/G19SE/G197E

410

0.98

4.20E + 05

K27R/N76D/V104Y/N123S/G195E/N218S

540

0.66

8.20E + 05

K27R/N76D/V104Y/N123S/D197E/N218S

770

0.79

9.80E + 05

K27R/N76D/V104Y/N123S/N204C/N218S

610

0.99

6.20E + 05

K27R/N76D/V104Y/N123S/Q206L/N218S

580

0.78

7.40E + 05

K27R/N76D/V104Y/N123S/N218S/T260P

660

1

6.60E + 05

K27R/N76D/V104Y/N123S/N218S/T274A

590

0.89

6.60E + 05

K27R/N76D/V104Y/Q109S/N123S/N218S/

520

1

5.20E + 05

T274A

K27R/N76D/V104Y/N123S/G195E/D197E/

460

0.65

7.10E + 05

N218S

B. amyloliquefaciens subtilisin

(BPN′)

50

0.14

3.60E + 05

BPN′-N76D/Y217L*

380

0.46

8.30E + 05

*These mutants made as per Example 5, all others made as per Example 2

TABLE VI

Thermal Stability

(% half-life of

Enzyme

native enzyme)

B. lentus subtilisin

100

N76D

590

N76D/S99D

840

N76D/S103A

390

N76D/V104I

660

N76D/I107V

710

N76D/N123S

70

N76D/S99D/S101R

610

N76D/S99D/S103A

590

N76D/S99D/V104I

910

N76D/S101R/S103A

930

N76D/S101R/V104I

500

N76D/S103A/V104I

460

N76D/S103G/V104I*

370

N76D/S103A/V104F

480

N76D/S103A/V104N

230

N76D/S103A/V104S

230

N76D/S103A/V104T

370

N76D/S103A/V104W

280

N76D/S103A/V104Y

400

N76D/V104I/I107V

940

N76D/V104Y/I107V

820

N76D/V104I/N123S

80

N76D/I107V/N123S

150

K27R/N76D/V104Y/N123S

100

N76D/S99D/S101R/S103A

570

N76D/S99D/S101R/V104I

1000

N76D/S99D/S103A/V104I

680

N76D/S101G/S103A/V104I*

390

N76D/S101R/S103A/V104I

470

N76D/S103A/V104I/S105A*

360

N76D/S103A/V104I/S105D*

370

N76D/S103A/V104T/I107A*

270

N76D/S103A/V104T/I107L*

230

N76D/S103A/V104I/N123S

110

N76D/V104I/I107V/N123S

220

N76D/S103A/V104I/L126A

270

N76D/S103A/V104I/L126F

950

N76D/S103A/V104I/L126I

410

N76D/S103A/V104I/L126V

320

N76D/S103A/V104I/S128G*

640

N76D/S103A/V104I/S128L*

760

N76D/S103A/V104I/L135A

230

N76D/S103A/V104I/L135F

200

N76D/S103A/V104I/L135I

510

N76D/S103A/V104I/L135V

500

N76D/S103A/V104I/S156E*

120

N76D/S103A/V104I/S166D*

590

N76D/S103A/V104I/D197E

460

N76D/S103A/V104I/N204A*

230

N76D/S103A/V104I/N204G*

240

N76D/S103A/V104I/N204C*

500

N76D/S103A/V104I/P210I*

1370

N76D/S103A/V104I/L217H*

60

N76D/S103A/V104I/M222A

520

N76D/S103A/V104I/M222S

490

N76D/S103A/V104I/T260P

490

N76D/S103A/V104I/S265N

360

K27R/N76D/V104Y/I107V/N123S

210

K27R/N76D/V104Y/N123S/D197E

120

K27R/N76D/V104Y/N123S/N204C

110

K27R/N76D/V104Y/N123S/Q206L

380

K27R/N76D/V104Y/N123S/S216V

140

K27R/N76D/V104Y/N123S/N218S

270

K27R/N76D/V104Y/N123S/T260P

40

K27R/N76D/V104Y/N123S/T274A

60

N76D/S99D/S101R/S103A/V104I

590

N76D/S99D/S103A/V104I/N123S

110

N76D/S103A/V104I/L126F/S265N

810

N76D/S103A/V104I/S156E/S166D*

220

K27R/N76D/V104Y/N123S/G195E/G197E

90

K27R/N76D/V104Y/N123S/G195E/N218S

250

K27R/N76D/V104Y/N123S/D197E/N218S

270

K27R/N76D/V104Y/N123S/N204C/N218S

460

K27R/N76D/V104Y/N123S/Q206L/N218S

1400

K27R/N76D/V104Y/N123S/N218S/T260P

310

K27R/N76D/V104Y/N123S/N218S/T274A

180

N76D/S99D/S101R/S103A/V104I/N123S

90

K27R/N76D/V104Y/Q109S/N123S/N218S/T274A

230

K27R/N76D/V104Y/N123S/G195E/D197E/N218S

240

B. amyloliquefaciens subtilisin

(BPN′)

100

BPN′-N76D/Y217L*

420

*These mutants made as per Example 5, all others made as per Example 2

EXAMPLE 6

Wash Performance Test

The wash performance of the variants described in the previous examples was evaluated by measuring the removal of stain from EMPA 116 (blood/milk/carbon black on cotton) cloth swatches (Testfabrics, Inc., Middlesex, N.J. 07030).

Six EMPA 116 swatches, cut to

3×4-½

inches with pinked edges, were placed in each pot of a Model 7243S Terg-O-Tometer (United States Testing Co., Inc., Hoboken, N.J.) containing 1000 ml of water, 15 gpg hardness (Ca

++

:Mg

++

::3:1::w:w), 7 g of detergent, and enzyme as appropriate. The detergent base was WFKl detergent from wfk-Testgewebe GmbH, Adlerstrasse 42, Postfach 13 07 62, D-47759 Krefeld, Germany:

Component

% of Final Formulation

Zeoiite A

25%

Sodium sulfate

25%

Soda Ash

10%

Linear alkylbenzenesulfonate

8.8%

Alcohol ethoxylate (7-8 EO)

4.5%

Sodium soap

3%

Sodium silicate

3%

(SiO

2

:Na

2

O::3.3:1)

To this base detergent, the following additions were made:

Component

% of Final Formulation

Sodium perborate monohydrate

13%

Copolymer (Sokalan CP5)

4%

TAED (Mykon ATC Green)

3%

Enzyme

0.5%

Brightener (Tinopal AMS-GX)

0.2%

Sodium perborate monohydrate was obtained from Degussa Corporation, Ridgefield-Park, N.J. 07660. Sokalan CP5 was obtained from BASF Corporation, Parsippany, N.J. 07054. Mykon ATC Green (TAED, tetraacetylethylenediamine) was obtained from Warwick International, Limited, Mostyn, Holywell, Clwyd CH8 9HE, England. Tinopal AMS GX was obtained from Ciba-Geigy Corporation, Greensboro, N.C. 27419.

Six EMPA 116 swatches were washed in detergent with enzyme for 30 minutes at 60° C. and were subsequently rinsed twice for 5 minutes each time in 1000 ml water. Enzymes were added at final concentrations of 0.05 to 1 ppm for standard curves, and 0.25 ppm for routine analyses. Swatches were dried and pressed, and the reflectance from the swatches was measured using the L value on the L*a*b* scale of a Minolta Chroma Meter, Model CR-200 (Minolta Corporation, Ramsey, N.J. 07446). Performance is reported as a percentage of the performance of

B. lentus

(GG36) protease and was calculated by dividing the amount of

B. lentus

(GG36) protease by the amount of variant protease that was needed to provide the same stain removal performance X 100. The data are shown in Table VII.

TABLE VII

Enzyme

Wash Performance

B. lentus

subtilisin

100

N76D

310

N76D/S103A

230

N76D/V104I

130

N76D/I107V

160

N76D/S99D/S101R

370

N76D/S99D/S103A

290

N76D/S101R/S103A

130

N76D/S101R/V104I

300

N76D/S103A/V104I

320

N76D/S103G/V104I

160

N76D/S103A/V104F

210

N76D/S103A/V104N

110

N76D/S103A/V104T

170

N76D/V104I/I107V

210

N76D/S99D/S101R/S103A

220

N76D/S99D/S101R/V104I

140

N76D/S101G/S103A/V104I

170

N76D/S101R/S103A/V104I

150

N76D/S103A/V104I/S105A

170

N76D/S103A/V104T/I107A

120

N76D/S103A/V104T/I107L

110

N76D/S103A/V104I/L126F

110

N76D/S103A/V104I/S128G

280

N76D/S103A/V104I/L135I

160

N76D/S103A/V104I/L135V

160

N76D/S103A/V104I/D197E

170

N76D/S103A/V104I/N204A

160

N76D/S103A/V104I/N204G

150

N76D/S103A/V104I/P210I

470

N76D/S103A/V104I/M222A

100

N76D/S103A/V104I/T260P

280

N76D/S103A/V104I/S265N

190

EXAMPLE 7

Protease Stability in a Liquid Detergent Formulation

A comparison of protease stability toward inactivation in a liquid detergent formulation was made for

Bacillus lentus

subtilisin and it's variant enzyme N76D/S103A/V104I according to the procedure outlined herein. The detergent formulation used for the study was a commercially purchased bottle of Tide Ultra liquid lanudry detergent made in the USA by Procter & Gamble Company. Heat treatment of the detergent formulation was necessary to inactivate in-situ protease. This was accomplished by incubating the detergent at 96° C. for a period of 4.5 hours. Concentrated preparations of the

B. lentus

subtilisin and N76D/S103A/V104I variant, in the range of 20 grams/liter enzyme, were then added to the heat-treated Tide Ultra at room-temperature to a final concentratrion of 0.3 grams/liter enzyme in the detergent formulation. The heat-treated detergent with protease added was then incubated in a water bath thermostatted at 50° C. Aliquots were removed from the incubation tubes at 0, 24, 46, 76, and 112 hour time intervals and assayed for enzyme activity by addition to a 1 cm cuvette containing 1.2 mM of the synthetic peptide substrate suc-Ala-Ala-Pro-phe-p-nitroanilide dissolved in 0.1M tris-HCL buffer, pH 8.6, and thermostatted at 25° C. The initial linear reaction velocity was followed spectrophotometrically by monitoring the absorbance of the reaction product p-nitroaniline at 410 nm as a function of time. As shown in

FIG. 10

, the N76D/S103A/V104I variant was observed to have significantly greater stability towards inactivation than the native

B. lentus

enzyme. Estimated half-lives for inactivation in the Tide Ultra detergent formulation for the two enzymes, under the specified test conditions, are 45 hours for

B. lentus

subtilisin and 125 hours for the N76D/S103A/V104I variant.

Throughout this application reference is made to various amino acids by way of common one- and three-letter codes. Such codes are identified in Dale, J. W. (1989),

Molecular Genetics of Bacteria,

John Wiley & Sons, Ltd., Appendix B.

Although the preferred embodiments of the invention have been described above, it will be obvious to those skilled in the art to which the invention pertains, that, after understanding the invention as a whole, various changes and equivalent modifications may be made without departing from the scope of the invention as defined by the appended claims.

19 base pairs

nucleic acid

single

linear

DNA (genomic)

1
GAAGCTGCAA CTCGTTAAA 19

18 base pairs

nucleic acid

single

linear

DNA (genomic)

2
GCTGCTCTAG ACAATTCG 18

39 base pairs

nucleic acid

single

linear

DNA (genomic)

3
GTATTAGGGG CGGACGGTCG AGGCGCCATC AGCTCGATT 39

33 base pairs

nucleic acid

single

linear

DNA (genomic)

4
TCAGGTTCGG TCTCGAGCGT TGCCCAAGGA TTG 33

22 base pairs

nucleic acid

single

linear

DNA (genomic)

5
CACGTTGCTA GCTTGAGTTT AG 22

1497 base pairs

nucleic acid

single

linear

DNA (genomic)

6
GGTCTACTAA AATATTATTC CATACTATAC AATTAATACA CAGAATAATC TGTCTATTGG 60
TTATTCTGCA AATGAAAAAA AGGAGAGGAT AAAGAGTGAG AGGCAAAAAA GTATGGATCA 120
GTTTGCTGTT TGCTTTAGCG TTAATCTTTA CGATGGCGTT CGGCAGCACA TCCTCTGCCC 180
AGGCGGCAGG GAAATCAAAC GGGGAAAAGA AATATATTGT CGGGTTTAAA CAGACAATGA 240
GCACGATGAG CGCCGCTAAG AAGAAAGATG TCATTTCTGA AAAAGGCGGG AAAGTGCAAA 300
AGCAATTCAA ATATGTAGAC GCAGCTTCAG TCACATTAAA CGAAAAAGCT GTAAAAGAAT 360
TGAAAAAAGA CCCGAGCGTC GCTTACGTTG AAGAAGATCA CGTAGCACAT GCGTACGCGC 420
AGTCCGTGCC TTACGGCGTA TCACAAATTA AAGCCCCTGC TCTGCACTCT CAAGGCTACA 480
CTGGATCAAA TGTTAAAGTA GCGGTTATCG ACAGCGGTAT CGATTCTTCT CATCCTGATT 540
TAAAGGTAGC AAGCGGAGCC AGCATGGTTC CTTCTGAAAC AAATCCTTTC CAAGACAACA 600
ACTCTCACGG AACTCACGTT GCCGGCACAG TTGCGGCTCT TAATAACTCA ATCGGTGTAT 660
TAGGCGTTGC GCCAAGCGCA TCACTTTACG CTGTAAAAGT TCTCGGTGCT GACGGTTCCG 720
GCCAATACAG CTGGATCATT AACGGAATCG AGTGGGCGAT CGCAAACAAT ATGGACGTTA 780
TTAACATGAG CCTCGGCGGA CCTTCTGGTT CTGCTGCTTT AAAAGCGGCA GTTGATAAAG 840
CCGTTGCATC CGGCGTCGTA GTCGTTGCGG CAGCCGGTAA CGAAGGCACT TCCGGCAGCT 900
CAAGCACAGT GGGCTACCCT GGTAAATACC CTTCTGTCAT TGCAGTAGGC GCTGTTGACA 960
GCAGCAACCA AAGAGCATCT TTCTCAAGCG TAGGACCTGA GCTTGATGTC ATGGCACCTG 1020
GCGTATCTAT CCAAAGCACG CTTCCTGGAA ACAAATACGG GGCGTACAAC GGTACGTCAA 1080
TGGCATCTCC GCACGTTGCC GGAGCGGCTG CTTTGATTCT TTCTAAGCAC CCGAACTGGA 1140
CAAACACTCA AGTCCGCAGC AGTTTAGAAA ACACCACTAC AAAACTTGGT GATTCTTTGT 1200
ACTATGGAAA AGGGCTGATC AACGTACAAG CGGCAGCTCA GTAAAACATA AAAAACCGGC 1260
CTTGGCCCCG CCGGTTTTTT ATTATTTTTC TTCCTCCGCA TGTTCAATCC GCTCCATAAT 1320
CGACGGATGG CTCCCTCTGA AAATTTTAAC GAGAAACGGC GGGTTGACCC GGCTCAGTCC 1380
CGTAACGGCC AACTCCTGAA ACGTCTCAAT CGCCGCTTCC CGGTTTCCGG TCAGCTCAAT 1440
GCCATAACGG TCGGCGGCGT TTTCCTGATA CCGGGAGACG GCATTCGTAA TCGGATC 1497

275 amino acids

amino acid

single

linear

protein

7
Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu
1 5 10 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
35 40 45
Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His
50 55 60
Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95
Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu
100 105 110
Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala
130 135 140
Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly
145 150 155 160
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala
165 170 175
Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val
180 185 190
Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205
Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn
225 230 235 240
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys
245 250 255
Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270
Ala Ala Gln
275

275 amino acids

amino acid

single

linear

protein

8
Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu
1 5 10 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala
35 40 45
Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His
50 55 60
Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80
Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95
Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu
100 105 110
Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser
130 135 140
Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly
145 150 155 160
Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala
165 170 175
Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala
180 185 190
Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205
Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr
225 230 235 240
Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr
245 250 255
Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270
Ala Ala Gln
275

274 amino acids

amino acid

single

linear

protein

9
Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val
1 5 10 15
Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala
35 40 45
Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly
50 55 60
Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val
65 70 75 80
Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn
85 90 95
Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp
100 105 110
Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala
115 120 125
Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg
130 135 140
Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Asn Ser Gly Ser
145 150 155 160
Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val
165 170 175
Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly
180 185 190
Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr
195 200 205
Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro
210 215 220
His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu
225 230 235 240
Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu
245 250 255
Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala
260 265 270
Ala Gln

269 amino acids

amino acid

single

linear

protein

10
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala
1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
115 120 125
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile
225 230 235 240
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265

1140 base pairs

nucleic acid

single

linear

DNA (genomic)

11
ATGAAGAAAC CGTTGGGGAA AATTGTCGCA AGCACCGCAC TACTCATTTC TGTTGCTTTT 60
AGTTCATCGA TCGCATCGGC TGCTGAAGAA GCAAAAGAAA AATATTTAAT TGGCTTTAAT 120
GAGCAGGAAG CTGTCAGTGA GTTTGTAGAA CAAGTAGAGG CAAATGACGA GGTCGCCATT 180
CTCTCTGAGG AAGAGGAAGT CGAAATTGAA TTGCTTCATG AATTTGAAAC GATTCCTGTT 240
TTATCCGTTG AGTTAAGCCC AGAAGATGTG GACGCGCTTG AACTCGATCC AGCGATTTCT 300
TATATTGAAG AGGATGCAGA AGTAACGACA ATGGCGCAAT CAGTGCCATG GGGAATTAGC 360
CGTGTGCAAG CCCCAGCTGC CCATAACCGT GGATTGACAG GTTCTGGTGT AAAAGTTGCT 420
GTCCTCGATA CAGGTATTTC CACTCATCCA GACTTAAATA TTCGTGGTGG CGCTAGCTTT 480
GTACCAGGGG AACCATCCAC TCAAGATGGG AATGGGCATG GCACGCATGT GGCCGGGACG 540
ATTGCTGCTT TAAACAATTC GATTGGCGTT CTTGGCGTAG CGCCGAGCGC GGAACTATAC 600
GCTGTTAAAG TATTAGGGGC GAGCGGTTCA GGTTCGGTCA GCTCGATTGC CCAAGGATTG 660
GAATGGGCAG GGAACAATGG CATGCACGTT GCTAATTTGA GTTTAGGAAG CCCTTCGCCA 720
AGTGCCACAC TTGAGCAAGC TGTTAATAGC GCGACTTCTA GAGGCGTTCT TGTTGTAGCG 780
GCATCTGGGA ATTCAGGTGC AGGCTCAATC AGCTATCCGG CCCGTTATGC GAACGCAATG 840
GCAGTCGGAG CTACTGACCA AAACAACAAC CGCGCCAGCT TTTCACAGTA TGGCGCAGGG 900
CTTGACATTG TCGCACCAGG TGTAAACGTG CAGAGCACAT ACCCAGGTTC AACGTATGCC 960
AGCTTAAACG GTACATCGAT GGCTACTCCT CATGTTGCAG GTGCAGCAGC CCTTGTTAAA 1020
CAAAAGAACC CATCTTGGTC CAATGTACAA ATCCGCAATC ATCTAAAGAA TACGGCAACG 1080
AGCTTAGGAA GCACGAACTT GTATGGAAGC GGACTTGTCA ATGCAGAAGC GGCAACACGC 1140

1140 base pairs

nucleic acid

single

linear

DNA (genomic)

12
ATGAAGAAAC CGTTGGGGAA AATTGTCGCA AGCACCGCAC TACTCATTTC TGTTGCTTTT 60
AGTTCATCGA TCGCATCGGC TGCTGAAGAA GCAAAAGAAA AATATTTAAT TGGCTTTAAT 120
GAGCAGGAAG CTGTCAGTGA GTTTGTAGAA CAAGTAGAGG CAAATGACGA GGTCGCCATT 180
CTCTCTGAGG AAGAGGAAGT CGAAATTGAA TTGCTTCATG AATTTGAAAC GATTCCTGTT 240
TTATCCGTTG AGTTAAGCCC AGAAGATGTG GACGCGCTTG AACTCGATCC AGCGATTTCT 300
TATATTGAAG AGGATGCAGA AGTAACGACA ATGGCGCAAT CAGTGCCATG GGGAATTAGC 360
CGTGTGCAAG CCCCAGCTGC CCATAACCGT GGATTGACAG GTTCTGGTGT AAAAGTTGCT 420
GTCCTCGATA CAGGTATTTC CACTCATCCA GACTTAAATA TTCGTGGTGG CGCTAGCTTT 480
GTACCAGGGG AACCATCCAC TCAAGATGGG AATGGGCATG GCACGCATGT GGCCGGGACG 540
ATTGCTGCTT TAGACAACTC GATTGGCGTT CTTGGCGTAG CGCCGAGCGC GGAACTATAC 600
GCTGTTAAAG TATTAGGGGC GAGCGGTTCA GGCGCCATCA GCTCGATTGC CCAAGGATTG 660
GAATGGGCAG GGAACAATGG CATGCACGTT GCTAATTTGA GTTTAGGAAG CCCTTCGCCA 720
AGTGCCACAC TTGAGCAAGC TGTTAATAGC GCGACTTCTA GAGGCGTTCT TGTTGTAGCG 780
GCATCTGGGA ATTCAGGTGC AGGCTCAATC AGCTATCCGG CCCGTTATGC GAACGCAATG 840
GCAGTCGGAG CTACTGACCA AAACAACAAC CGCGCCAGCT TTTCACAGTA TGGCGCAGGG 900
CTTGACATTG TCGCACCAGG TGTAAACGTG CAGAGCACAT ACCCAGGTTC AACGTATGCC 960
AGCTTAAACG GTACATCGAT GGCTACTCCT CATGTTGCAG GTGCAGCAGC CCTTGTTAAA 1020
CAAAAGAACC CATCTTGGTC CAATGTACAA ATCCGCAATC ATCTAAAGAA TACGGCAACG 1080
AGCTTAGGAA GCACGAACTT GTATGGAAGC GGACTTGTCA ATGCAGAAGC GGCAACACGC 1140

30 base pairs

nucleic acid

single

linear

DNA (genomic)

13
TATGCCAGCC ACAACGGTAC TTCGATGGCT 30

31 base pairs

nucleic acid

single

linear

DNA (genomic)

14
CACAGTTGCG GCTCTAGATA ACTCAATCGG T 31

33 base pairs

nucleic acid

single

linear

DNA (genomic)

15
GCTGACGGTT CCGGCGCTAT TAGTTGGATC ATT 33

Number	Name	Date	Kind
4914031	Zukowski et al.	Apr 1990	A
5013657	Bryan et al.	May 1991	A
5155033	Estell et al.	Oct 1992	A
5182204	Estell et al.	Jan 1993	A
5185258	Caldwell et al.	Feb 1993	A
5204015	Caldwell et al.	Apr 1993	A
RE34606	Estell et al.	May 1994	E
5316935	Arnold et al.	May 1994	A
5324653	van Eekelen et al.	Jun 1994	A
5336611	van Eekelen et al.	Aug 1994	A
5340735	Christianson et al.	Aug 1994	A
5397705	Zukowski et al.	Mar 1995	A
5665587	Aaslyng et al.	Sep 1997	A
5677272	Ghosh et al.	Oct 1997	A
5679630	Baeck et al.	Oct 1997	A
5700676	Bott et al.	Dec 1997	A
5741694	Hastrup et al.	Apr 1998	A

Number	Date	Country
0 251 446	Jan 1988	EP
0 328 229	Aug 1989	EP
0 398 539	Nov 1990	EP
0 405 901	Jan 1991	EP
0 405 902	Jan 1991	EP
WO8808028	Oct 1988	WO
WO8906279	Jul 1989	WO
WO8909819	Oct 1989	WO
WO9100345	Jan 1991	WO
WO9106637	May 1991	WO
WO9208778	May 1992	WO
WO9211357	Jul 1992	WO
WO9221760	Dec 1992	WO
WO9402618	Feb 1994	WO
WO9423053	Oct 1994	WO

	Number	Date	Country
Parent	08/137240	Oct 1993	US
Child	08/322678		US

Subtilisin variants multiply-substituted subtilising variants having a substitution at position 76

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Disclaimer

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

US Referenced Citations (17)

Foreign Referenced Citations (15)

Non-Patent Literature Citations (16)

Continuation in Parts (1)

Entry
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Graycar, T.P. et al. (1992) Altering the Proteolytic Activity of Subtillsin Through Protein Engineering in Annals of the New York Academy of Sciences:Enzyme Engineering XI, vol. 672, 71-79, Clark D.S. and Estell, D.A., eds, New York Academy of Sciences, New York.
Bott, R.R. et al. (1992) Using Structural Comparison as a Guide in Protein Engineering in Annals of the New York Academy of Sciences:Enzyme Engineering XIa, vol. 672, 10-19, Clark D.S. and Estell, D.A., eds, New York Academy of Sciences, New York.
Wells, J.A. et al. (1987) Proc Natl Acad Sci USA 84:1219-1223.
Siezen, R.J. et al. (1991) Protein Engineering 4(7):719-737.