Patent Application
20220127592
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
The present invention relates to a method for producing a protein hydrolysate using a polypeptide having endopeptidase activity and a polypeptide having carboxypeptidase activity and the use of these enzymes for hydrolysing a protein substrate. In addition the present invention relates to polypeptides having carboxypeptidase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
Protein hydrolysates are used as an additive or ingredient in various food products. The advantages of using protein hydrolysates in food products are reduced allergenicity, easier digestion and absorbtion and thereby also faster absorption of the nutrients. Such products can be used in medical nutrition, infant nutrition, health foods, sports nutritions or can be used for enhancing the protein content of the food product. Also protein hydrolysates are used for enhancing the flavour of the food product, e.g., by adding umami taste to the product.
Conventionally, protein hydrolysates are produced chemically by hydrolysing protein (e.g., de-fatted soy flour or wheat gluten) with hydrochloric acid. The hydrolysates are cheap to produce, but the chemical hydrolysis results in byproducts, which are undesirable in food products.
An alternative method for hydrolysing protein is enzymatic hydrolysis, where protein substrate is subjected to peptidases, e.g., as Flavourzyme® (Novozymes) or Alcalase® (Novozymes). The peptidases used for hydrolysing can be either endopeptidases or exopeptidases, where the exopeptidases are categorised in aminopeptidases and carboxypeptidases. Endoproteases attack proteins and peptides within the molecule. Exoproteases attack from the terminal of the molecule, where aminopeptidases and carboxypeptidases cleaves off amino acids or peptides from the protein substrate from the amino-terminal end or the carboxy-terminal end, respectively.
WO 2016/210395 concerns use of aminopeptidases for producing protein hydrolysate. EP 0946106 describes a method for producing a protein hydrolysate with a proteolytic enzyme mixture comprising only one exopeptidase.
The invention provides a method for producing a protein hydrolysate which method comprises:
a. providing an aqueous solution or suspension of a protein substrate; and
b. exposing the aqueous solution or suspension of the protein substrate to a polypeptide having endopeptidase activity and to a polypeptide having carboxypeptidase activity, to obtain the protein hydrolysate;
wherein the polypeptide having carboxypeptidase activity is characterised by having a Pro/ACHA*100 ratio of at least 30.
The present invention provides polypeptides having carboxypeptidase activity and polynucleotides encoding the polypeptides.
Accordingly, the present invention relates to polypeptides having carboxypeptidase activity selected from the group consisting of:
a. a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10;
b. a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or the cDNA sequence thereof;
c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more positions; and
d. a fragment of the polypeptide of (a), (b) or (c) that has carboxypeptidase activity.
The invention further concerns a liquid or granulate composition comprising the polypeptide of the invention. And the invention concerns a whole broth formulation or cell culture composition comprising the polypeptide of the invention.
The invention also concerns a polynucleotide encoding the inventive polypeptide, a nucleoic acid construct or expression vector comprising the polynucleotide.
Based on the activities measured in Assay II, the ACHA can be calculated as the average of the specific activity of the carboxypeptidase on the following substrates: Z-Ala-Ala-OH, Z-Ala-Val-OH, Z-Ala-Ile-OH, Z-Ala-Leu-OH, Z-Ala-Met-OH, Z-Ala-Phe-OH and Z-Ala-Trp-OH. Z-Ala-Pro-OH is not included in this calculation.
Based on the activities measured in Assay II, the ACLA can be calculated as the average of the specific activity of the carboxypeptidase on the following two substrates: Z-Ala-Lys-OH, Z-Ala-Arg-OH.
Based on the activities measured in Assay I and Assay II and the calculation of ACHA, the Pro/ACHA*100 ratio can be calculated as the activity on Pro as measured in Assay I divided by ACHA (the average of the activity on the hydrophobic amino acids Ala, Val, Ile, Met, Phe, Leu and Trp measured in Assay II) and multiplied with 100.
Carboxypeptidase or polypeptide having carboxypeptidase activity: The term “carboxypeptidase” means a protein or a polypeptide having carboxypeptidase activity (3.4.16.X, 3.4.17.X, 3.4.18.X) that catalyzes the cleavage of the peptide bond at the carboxy-terminal end of the protein or peptide. For purposes of the present invention, carboxypeptidase activity is determined according to the procedure described in the Assay II, where a polypeptide having carboxypeptidase activity is capable of cleaving at least one of the substrates in the assay (e.g., Z-Ala-Val-OH). In one aspect, the polypeptides of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8 or 10. The terms “polypeptide having carboxypeptidase activity” and “carboxypeptidase” are used interchangeably.
Endopeptidase, endoprotease or polypeptide having endopeptidase activity: The term “endopeptidase” means a protein or a polypeptide having endopeptidase activity (3.4.19.X, 3.4.21.X, 3.4.22.X, 3.4.23.X, 3.4.24.X, 3.4.25.X) that catalyzes the cleavage of the peptide bonds within the protein or peptide molecule. For purposes of the present invention, endopeptidase activity is determined according to the procedure described in Assay III. The terms “polypeptide having endopeptidase activity”, “endoprotease” and “endopeptidase” are used interchangeably.
Aminopeptidase or polypeptide having aminopeptidase activity: The term “aminopeptidase” means a protein or a polypeptide having aminopeptidase activity (3.4.11.X) that catalyzes the cleavage of the peptide bond at the amino-terminal end of the protein or peptide. For purposes of the present invention, aminopeptidase activity is determined according to the procedure described in the Assay IV. The terms “polypeptide having aminopeptidase activity” and “aminopeptidase” are used interchangeably.
Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Expression: The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
Fragment: The term “fragment” means a polypeptide or a catalytic or carboxypeptidase binding domain having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has carboxypeptidase activity.
Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample; e.g., a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.
Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1-426 of SEQ ID NO: 2, amino acids 1-443 of SEQ ID NO: 4, amino acids 1-444 of SEQ ID NO: 6, amino acids 1-477 SEQ ID NO: 8 or amino acids 1-553 of SEQ ID NO: 10.
It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having carboxypeptidase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 358 to 1687 of SEQ ID NO: 1 and nucleotides 1 to 45 of SEQ ID NO: 1 encode a signal peptide.
Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)
Variant: The term “variant” means a polypeptide having carboxypeptidase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
The present invention concerns a method for producing a protein hydrolysate comprising:
a. providing an aqueous solution or suspension of a protein substrate; and
b. exposing the aqueous solution or suspension of the protein substrate to a polypeptide having endopeptidase activity and polypeptide having carboxypeptidase activity to obtain the protein hydrolysate;
wherein the polypeptide having carboxypeptidase activity is characterised by having a Pro/ACHA*100 ratio of at least 30.
By the use of two different polypeptides, a polypeptide having endopeptidase activity and a polypeptide having carboxypeptidase activity, the protein substrate is attacked in two different ways. The polypeptide having endopeptidase activity attacks the protein substrate by cleaving the peptide bonds within the protein molecule, whereby the protein substrate is hydrolysed to smaller peptides, which can be attacked by the polypeptide having carboxypeptidase activity. The method thereby provides protein hydrolysates with a high degree of hydrolysis, which are suitable for use in various products, e.g., food products, cosmetic products, medical products.
Any type of protein substrate can be used for protein hydrolysis. Usually the applications of the protein hydrolysate determines the type of protein substrate used. The protein substrate can be from animal or vegetable source. In one embodiment the animal protein is selected from milk protein such as casein or whey proteins.
In one embodiment the protein substrate is a vegetable protein selected from the group consisting of cereal, legumes and oilseed. The protein substrate can be from a cereal such as wheat, barley, oat, rye, triticale, maize, rice, sorghum, buckwheat, quinoa, chia, millet or fonio. In a preferred embodiment, the protein is gluten. In a particularly preferred embodiment, the protein is wheat gluten. Another source of vegetable protein is legumes, where the protein can be from peas, beans, lentils or chick peas. Protein from oilseeds can be used in the inventive method e.g soy bean, cotton seed, peanut, rape seed, sunflower seed, palm kernel, coconut, corn, safflower seed, sunflower seed and lin seed. In a preferred embodiment the protein substrate is soy protein. The soy protein can be defatted soy (e.g., flakes or flour), soy protein concentrates and soy isolates. The protein content in defatted soy products accounts for 50% of the dry matter, whereas soy protein concentrates and soy isolates may contain 70% protein and 90% protein, respectively.
The concentration of the protein substrate in the aqueous solution or suspension should be in the range of 5-35% depending on which protein substrate that is used.
In one embodiment of the invention the concentration of the protein substrate is in the range of 5-30%, in the range of 5-25%, in the range of 5-20%, in the range of 5-15% or in the range of 10-15%.
The concentration of the polypeptides used in the method depends on the amount of protein substrate used. In one embodiment of the invention the concentration of the polypeptide having carboxypeptidase activity is in the range of 0.01-4 mg enzyme protein/gram protein substrate. In one embodiment of the invention the concentration of polypeptide is in the range of 0.01-3.9 mg enzyme protein/gram protein substrate, such as in the range of 0.1-3.8 mg enzyme protein/gram protein substrate, in the range of 0.1-3.6, in the range of 0.2-3.4, in the range of 0.2-3.2, in the range of 0.3-3.0, in the range of 0.3-2.8, in the range of 0.4-2.6, in the range of 0.4-2.4, in the range of 0.5-2.2, in the range of 0.5-2.0.
In one embodiment of the invention, the concentration of the polypeptide having endopeptidase activity is in the range of 0.1-3.0% w/w on protein, such as is in the range of 0.1-2.8% w/w on protein, such as in the range of 0.1-2.6% w/w on protein, in the range of 0.1-2.4% w/w on protein, in the range of 0.1-2.2% w/w on protein, in the range of 0.1-2.0% w/w on protein, in the range of 0.1-1.8% w/w on protein, in the range of 0.1-1.6% w/w on protein, in the range of 0.1-1.4% w/w on protein, in the range of 0.1-1.2% w/w on protein, in the range of 0.1-1.0% w/w on protein, in the range of 0.2-1.0% w/w on protein, in the range of 0.3-1.0% w/w on protein, in the range of 0.4-1.0% w/w on protein or in the range of 0.5-1.0% w/w on pro.
The polypeptide having carboxypeptidase activity used in the inventive method can be any polypeptide having carboxypeptidase activity and which has a Pro/ACHA*100 ratio of at least 30. The Pro/ACHA*100 ratio is at least 35, at least 40, such as at least 41, at least 42, at least 45, at least 50, at least 51, at least 50, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60 or at least 61.
Some polypeptide having carboxypeptide activity may have an activity on Proline when measured with Assay I of at least 0.15. In one embodiment of the invention the activity on Proline when measured with Assay I is at least 0.16, at least 0.17, at least 0.18, at least 0.19, at least 0.2, at least 0.21, at least 0.22, at least 0.23, at least 0.24, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6 or at least 0.625.
The polypeptides having carboxypeptidase activity tend to have a higher activity on hydrophobic amino acids. In one embodiment the polypeptide having carboxypeptidase activity has an average hydrophobic activity of at least 0.51 when measured in Assay II.
In one embodiment, the polypeptide having carboxypeptidase activity has an ACHA value of at least 0.52 when measured in Assay II, such as at least 0.53, at least 0.54, at least 0.56, at least 0.57, at least 0.58 or at least 0.59.
Some polypeptides having carboxypeptidase activity may have an an ACLA value in the range of 0.16-0.25, such as in the range of 0.17-0.25, in the range of 0.18-0.25, in the range of 0.19-0.25.
Specific polypeptides having carboxypeptidase activity which may be used in the inventive method are described in the below.
When producing a protein hydrolysate according to the invention, the temperature of the aqueous solution/suspension comprising the protein substrate should be within the temperature range where the enymes work, so the temperature should be above 5° C. or above 10° C. In one embodiment, the temperature is in the range of 10-90° C., such as in the range of 20-80° C., in the range of 30-70° C., in the range of 40-60° C., in the range of 45-55° C. or in the range of 50-60° C.
In the inventive method, the protein substrate is exposed to the polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity for at least 4 hours. In one embodiment the protein substrate is exposed to the polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity for 3-48 hours, such as 4-48 hours, 4-36 hours, 6-32 hours, 8-28 hours, 10-26 hours, 12-24 hours, 12-22 hours, 12-20 hours, 12-18 hours, 12-16 hours or for 12-14 hours.
The pH of the aqueous solution/suspension is in the range of 5-8, such as in the range of 5.5-6.5 or in the range of 6.0-7.0.
When producing the protein hydrolysate, the aqueous solution/suspension comprising the protein substrate should be exposed to the polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity either at the same time, or the protein substrate should be exposed to the polypeptide having endopeptidase activity first. The advantage of adding the polypeptide having endopeptidase activity to the aqueous solution/suspension comprising the protein substrate first, is that the polypeptide having endopeptidase activity can hydrolyse the protein substrate and cut into peptides, which are available for the polypeptide having carboxypeptidase activity to hydrolyse.
The hydrolysis can be stopped when the hydrolysed proteins have the desired degree of hydrolysis, however by the inventive method protein hydrolysates with a high degree of hydrolysis can be produced. In one embodiment, the method is carried out until the obtained protein hydrolysate has a degree of hydrolysis (% DH) in the range of 40-70%, such as in the range of 45-70%, in the range of 50-70%, in the range of 55-70%, in the range of 60-70%, in the range of 65-70%.
The polypeptide having endopeptidase activity may be obtained from a strain of Bacillus, preferably Bacillus licheniformis or Bacillus subtilis, a strain of Staphylococcus, preferably Staphylococcus aureus. Or the polypeptide having endopeptidase activity may be obtained from fungal source, e.g., a strain of Streptomyces, preferably Streptomyces thermovularis or Streptomyces griseus, a strain of Actinomyces species, a strain of Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae, or a strain of Trichoderma, preferably Trichoderma reesei, or Fusarium, preferably Fusarium venenatum. The polypeptide having endopeptidase activity may be a substilisin. In some embodiments, the endopeptidase is comprised in the products Alcalase® (Novozymes A/S), Flavourzyme® (Novozymes A/S), Savinase® (Novozymes A/S), Esperase® (Novozymes A/S), Alphalase® (Dupont).
The protein substrate can be hydrolysed to an even higher degree of hydrolysis by exposing the protein substrate to a polypeptide having aminopeptidase activity or a fermentation broth supernatant of an Aspergillus strain having aminopeptidase activity. Amino peptidase 2 described in WO 2016/210395 (Dupont Nutrition Biosciences APS) can be used in the present invention, especially aminopeptidase 2 defined in SEQ ID NOs: 1-8 or WO 2016/210395 is preferred. Other suitable aminopeptidases are described in WO 97/29179 (Gist Brocades BV) and WO 2003/102195 (DSM IP Assets BV). The protein substrate may be exposed to the polypeptide having aminopeptidase activity at the same time as being exposed to the polypeptide having endopeptidase activity. In one embodiment of the invention, the protein substrate is exposed to the polypeptide having endopeptidase activity and then subsequently to the polypeptide having carboxypeptidase activity and the polypeptide having aminopeptidase activity. In one embodiment, the protein substrate is exposed to the polypeptide having aminopeptidase activity before, simultanously or after the protein substrate is exposed to the polypeptide having carboxypeptidase activity.
The concentration of the polypeptide having aminopeptidase activity should be in the range of 0.05-4 mg enzyme protein per gram protein substrate. In one embodiment, the concentration of the polypeptide having aminopeptidase activity may be 0.05-3.5 mg enzyme protein per gram protein substrate, such as 0.05-3.0 mg enzyme protein per gram protein substrate, 0.05-2.5 mg enzyme protein per gram protein substrate, 0.05-2.0 mg enzyme protein per gram protein substrate, 0.06-2.0 mg enzyme protein per gram protein substrate, 0.07-2.0 mg enzyme protein per gram protein substrate, 0.08-2.0 mg enzyme protein per gram protein substrate, 0.09-2.0 mg enzyme protein per gram protein substrate or 0.1-2.0 mg enzyme protein per gram protein substrate.
In one embodiment, the invention concerns an isolated polypeptide having carboxypeptidase activity, selected from the group consisting of:
a. a polypeptide having [at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10;
b. a polypeptide encoded by a polynucleotide having at least 60% sequence identty to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;
c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more positions; and
d. a fragment of the polypeptide of (a), (b) or, (c), or (d) that has carboxypeptidase activity.
In one embodiment, the invention concerns a protein hydrolysate produced by the inventive method.
In one embodiment, the invention concerns a protein hydrolysate comprising free amino acids, wherein the amount of free amino acids is at least 20 g per 100 gram protein, the total amount of Ala, Val, Ile, Leu, Met, Phe and Trp is at least 7 g per 100 g protein and at least 1.0 g Pro per 100 g protein.
In one embodiment, the amount of free amino acids is at least 25 g per 100 g protein hydrolysate, such as at least 30 g per 100 g hydrolysate, at least 30 g per 100 g hydrolysate, at least 35 g per 100 g hydrolysate or at least 40 g per 100 g hydrolysate.
In one embodiment, the amount of Ala, Val, Ile, Leu, Met, Phe and Trp is at least 13 g per 100 g protein substrate and at least 1.8 g per 100 g protein hydrolysate.
In one embodiment, the hydrolysate further has a degree of hydrolysation (% dH) in the range of 30-70%, such as in the range of 45-70%, in the range of 50-70%, in the range of 55-70%, in the range of 60-70%, in the range of 65-70%.
SEQ ID NO: 1 is the sequence of a polynucleotide derived from Penicillium emersonii.
SEQ ID NO: 2 is the amino acid sequence of the polypeptide encoded by SEQ ID NO: 1.
SEQ ID NO: 3 is the sequence of a polynucleotide derived from Myceliophthora heterothallica.
SEQ ID NO: 4 is the amino acid sequence of the polypeptide encoded by SEQ ID NO: 3.
SEQ ID NO: 5 is the sequence of a polynucleotide derived from Chaetomium strumarium.
SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded by SEQ ID NO: 5.
SEQ ID NO: 7 is the sequence of a polynucleotide derived from Lasiodiplodia theobromae.
SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded by SEQ ID NO: 7.
SEQ ID NO: 9 is the sequence of a polynucleotide derived from Thermoascus aurantiacus.
SEQ ID NO: 10 is the amino acid sequence of the polypeptide encoded by SEQ ID NO: 9.
SEQ ID NO: 11 is the amino acid sequence of a substilisin protease.
SEQ ID NO: 12 is the amino acid sequence of a substilisin protease.
SEQ ID NO: 13 is the amino acid sequence of a substilisin protease.
SEQ ID NO: 14 is the amino acid sequence of carboxypeptidase CPY.
SEQ ID NO: 15 is the amino acid sequence of carboxypeptidase CP1.
In an embodiment, the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have carboxypeptidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 70% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 80% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 85% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 90% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 95% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 2.
In an embodiment, the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have carboxypeptidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 70% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 80% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 85% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 90% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 95% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 4.
In an embodiment, the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have carboxypeptidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 70% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 80% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 85% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 90% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 95% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 6.
In an embodiment, the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have carboxypeptidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 70% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 80% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 85% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 90% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 95% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 8.
In an embodiment, the present invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have carboxypeptidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 70% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 75% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 80% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 85% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 90% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 95% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In a particular embodiment, the invention relates to polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 10 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least 100% of the carboxypeptidase activity of the mature polypeptide of SEQ ID NO: 10.
In an embodiment, the polypeptide has been isolated. A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2, 4 or 6, 8, 10 or an allelic variant thereof; or is a fragment thereof having carboxypeptidase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2, 4, 6. 8 or 10. In another aspect, the polypeptide comprises or consists of amino acids −119-426 of SEQ ID NO: 2. In another aspect, the polypeptide comprises or consists of amino acids −111-443 of SEQ ID NO: 4. In another aspect, the polypeptide comprises or consists of amino acids −110-444 of SEQ ID NO: 6. In another aspect, the polypeptide comprises or consists of amino acids 1-477 of SEQ ID NO: 8. In another aspect, the polypeptide comprises or consists of amino acids −7-553 of SEQ ID NO: 10.
The polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having carboxypeptidase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having carboxypeptidase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1, 3, 5, 7, 9 or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1, 3, 5, 7 or 9; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7 or 9; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In another embodiment, the present invention relates to a polypeptide having carboxypeptidase activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7 or 9 or the cDNA sequence thereof of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a further embodiment, the polypeptide has been isolated.
In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8 or 10 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 2, 4, 6, 8 or 10 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for carboxypeptidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
A polypeptide having carboxypeptidase activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.
In one embodiment, the polypeptide is a Penicillium polypeptide, e.g., a polypeptide obtained from Penicillium emersonii.
In one embodiment, the polypeptide is a Myceliophthora polypeptide, e.g., a polypeptide obtained from Myceliophthora heterothallica.
In one embodiment, the polypeptide is a Chaetomium polypeptide, e.g., a polypeptide obtained from Chaetomium strumarium.
In one embodiment, the polypeptide is a Lasiodiplodia polypeptide, e.g., a polypeptide obtained from Lasiodiplodia theobromae.
In one embodiment, the polypeptide is a Thermoascus polypeptide, e.g., a polypeptide obtained from Thermoascus aurantiacus.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and variant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3′-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).
The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5′-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCI B 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide. In one aspect, the cell is an Aspergillus cell. In another aspect, the cell is an Aspergillus oryzae cell.
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
The polypeptide may be detected using methods known in the art that are specific for the polypeptides having carboxypeptidase activity. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.
The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
The invention is further summarized in the following paragraphs:
1. A method for producing a protein hydrolysate comprising:
a. providing an aqueous solution or suspension of a protein substrate; and
b. exposing the aqueous solution or suspension of the protein substrate to a polypeptide having endopeptidase activity and to a polypeptide having carboxypeptidase activity, to obtain the protein hydrolysate;
wherein the polypeptide having carboxypeptidase activity is characterised by having a Pro/ACHA*100 ratio of at least 30.
2. The method of paragraph 1, wherein the Pro/ACHA*100 ratio is at least 35, at least 40, such as at least 41, at least 42, at least 45, at least 50, at least 51, at least 50, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60 or at least 61.
3. The method of paragraph 1 or 2, wherein the protein substrate is a vegetable protein or an animal protein.
4. The method of paragraph 3, wherein the vegetable protein substrate is selected from the group consisting of cereal, legumes and oilseed.
5. The method of paragraph 4, wherein the vegetable protein substrate from cereal is selected from the group comprising wheat, barley, oat, rye, triticale, maize, rice, sorghum, buckwheat, quinoa, chia, millet or fonio.
6. The method of paragraph 5, wherein the protein substrate is gluten, preferably wheat gluten.
7. The method of paragraph 4, wherein the vegetable protein substrate from legumes is selected from the group consisting of peas, beans, lentils and chick peas.
8. The method of paragraph 4, wherein the vegetable protein substrate from oilseed is selected from soy bean, cotton seed, peanut, rape seed, sunflower seed, palm kernel, coconut, corn, safflower seed, sunflower seed and lin seed.
9. The method of paragraph 8, wherein the protein substrate is soy protein.
10. The method of paragraph 3, wherein the animal protein is selected from milk proteins such as casein or whey proteins.
11. The method of any of paragraphs 1-10, wherein the concentration of the protein substrate in the aqueous solution or suspension is in the range of 5-35%.
12. The method of paragraph 11, wherein the concentration of the protein substrate is in the range of 5-30%, in the range of 5-25%, in the range of 5-20%, in the range of 5-15% or in the range of 10-15%.
13. The method of any of paragraphs 1-12, wherein the polypeptide having carboxypeptidase activity has an activity of at least 0.15 on Proline when measured in Assay I, such as at least 0.16, at least 0.17, at least 0.18, at least 0.19, at least 0.2, at least 0.21, at least 0.22, at least 0.23, at least 0.24, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6 or at least 0.625.
14. The method of any of paragraphs 1-13, wherein the polypeptide having carboxypeptidase activity has an average hydrophobic activity of at least 0.51 when measured in Assay II.
15. The method of any of paragraphs 1-14, wherein the polypeptide having carboxypeptidase activity has an ACHA value of at least 0.52, such as at least 0.53, at least 0.54, at least 0.56, at least 0.57, at least 0.58 or at least 0.59.
16. The method of any of paragraphs 1-15, wherein the polypeptide having carboxypeptidase activity has an ACLA value in the range of 0.15-0.25.
17. The method of paragraph 16, wherein the polypeptide having carboxypeptidase activity has an ACLA value in the range of 0.16-0.25, such as in the range of 0.17-0.25, in the range of 0.18-0.25, in the range of 0.19-0.25.
18. The method of any of paragraphs 1-17, wherein the concentration of the polypeptide having endopeptidase activity is in the range of 0.1-3.0% w/w on protein.
19. The method of paragraph 18, wherein the concentration is in the range of 0.1-2.8%, such as in the range of 0.1-2.6%, in the range of 0.1-2.4%, in the range of 0.1-2.2%, in the range of 0.1-2.0%, in the range of 0.1-1.8%, in the range of 0.1-1.6%, in the range of 0.1-1.4%, in the range of 0.1-1.2%, in the range of 0.1-1.0%, in the range of 0.2-1.0%, in the range of 0.3-1.0%, in the range of 0.4-1.0% or in the range of 0.5-1.0%.
20. The method of any of paragraphs 1-19, wherein the concentration of the polypeptide having carboxypeptidase activity is in the range of 0.01-4 mg enzyme protein/gram protein substrate.
21. The method of paragraph 20, wherein the concentration is in the range of 0.01-3.9 mg enzyme protein/gram protein substrate, such as in the range of 0.1-3.8 mg enzyme protein/gram protein substrate, in the range of 0.1-3.6, in the range of 0.2-3.4, in the range of 0.2-3.2, in the range of 0.3-3.0, in the range of 0.3-2.8, in the range of 0.4-2.6, in the range of 0.4-2.4, in the range of 0.5-2.2, in the range of 0.5-2.0.
22. The method of any of paragraphs 1-21, wherein the protein substrate is exposed to the polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity for at least 3 hours.
23. The method of paragraph 22, wherein the protein substrate is exposed to the polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity for 3-48 hours, such as 4-48 hours, such as 4-36 hours, 6-32 hours, 8-28 hours, 10-26 hours, 12-24 hours, 12-22 hours, 12-20 hours, 12-18 hours, 12-16 hours or for 12-14 hours.
24. The method of any of paragraphs 1-23, wherein method is carried out at a temperature above 10° C.
25. The method of paragraph 24, wherein the temperature is in the range of 10-90° C., such as in the range of 20-80° C., in the range of 30-70° C., in the range of 40-60° C., in the range of 45-55° C. or in the range of 50-60° C.
26. The method of any of paragraphs 1-25, wherein the pH of the aqueous solution or the aqueous suspension is in the range of 5-8, such as in the range of 5.5-6.5 or in the range of 6.0-7.0.
27. The method of any of paragraphs 1-26, wherein the protein substrate is exposed to polypeptide having endopeptidase activity and the polypeptide having carboxypeptidase activity at the same time.
28. The method of any of paragraphs 1-27, wherein the protein substrate is exposed to the polypeptide having endopeptidase activity before being exposed to the polypeptide having carboxypeptidase activity.
29. The method of any of paragraphs 1-28, wherein the protein substrate is further exposed to a polypeptide having aminopeptidase activity such as aminopeptidase 2.
30. The method of paragraph 29, wherein the aminopeptidase is the polypeptide of SEQ ID NO: 16.
31. The method of paragraph 29, wherein the protein substrate is exposed to the polypeptide having aminopeptidase activity at the same time as being exposed to the polypeptide having endopeptidase activity.
32. The method of paragraph 29, wherein the protein substrate is exposed to the polypeptide having aminopeptidase activity before, simultanously or after the protein substrate is exposed to the polypeptide having carboxypeptidase activity.
33. The method of paragraph 29, wherein the concentration of the polypeptide having aminopeptidase activity is in the range of 0.05-4 mg enzyme protein per gram protein substrate, such as 0.05-3.5 mg enzyme protein per gram protein substrate, 0.05-3.0 mg enzyme protein per gram protein substrate, 0.05-2.5 mg enzyme protein per gram protein substrate, 0.05-2.0 mg enzyme protein per gram protein substrate, 0.06-2.0 mg enzyme protein per gram protein substrate, 0.07-2.0 mg enzyme protein per gram protein substrate, 0.08-2.0 mg enzyme protein per gram protein substrate, 0.09-2.0 mg enzyme protein per gram protein substrate or 0.1-2.0 mg enzyme protein per gram protein substrate.
34. The method of any of paragraphs 1-33, wherein the method is carried out until the obtained protein hydrolysate has a degree of hydrolysis (% DH) in the range of 30-70%, such as in the range of 45-70%, in the range of 50-70%, in the range of 55-70%, in the range of 60-70%, in the range of 65-70%.
35. The method of any of paragraphs 1-34, wherein the polypeptide having endopeptidase activity is a subtilisin.
36. The method of paragraph 35, wherein the substilisin is selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13.
37. The method of any of paragraphs 1-36, wherein the polypeptide having carboxypeptidase activity is obtained from fungal source.
38. The method of paragraph 37, wherein the polypeptide having carboxypeptidase activity is defined in paragraphs 39-45.
39. An isolated polypeptide having carboxypeptidase activity, selected from the group consisting of:
a. a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10;
b. a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or the cDNA sequence thereof;
c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more positions; and
d. a fragment of the polypeptide of (a), (b) or (c) that has carboxypeptidase activity.
40. The polypeptide of paragraph 39, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.
41. The polypeptide of paragraph 39 or 40, which is encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 or the cDNA sequence thereof.
42. The polypeptide of any of paragraphs 39-41, comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10; or the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.
43. The polypeptide of paragraph 42, wherein the mature polypeptide is amino acids 1-426 of SEQ ID NO: 2, amino acids 1-443 of SEQ ID NO: 4, amino acids 1-444 of SEQ ID NO: 6, amino acids 1-477 of SEQ ID NO: 8 or amino acids 1-553 of SEQ ID NO: 10.
44. The polypeptide of any of paragraphs 39-43, which is a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a substitution, deletion, and/or insertion at one or more positions.
45. The polypeptide of paragraph 39, which is a fragment of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, wherein the fragment has carboxypeptidase activity.
46. A liquid or granulate composition comprising the polypeptide of any of paragraphs 39-45.
47. A whole broth formulation or cell culture composition comprising the polypeptide of any of paragraphs 39-45.
48. A polynucleotide encoding the polypeptide of any of paragraphs 39-45.
49. A nucleic acid construct or expression vector comprising the polynucleotide of paragraph 48 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
50. A recombinant host cell comprising the polynucleotide of paragraph 49 operably linked to one or more control sequences that direct the production of the polypeptide.
51. A method of producing the polypeptide of any of paragraphs 39-45, comprising cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide.
52. The method of paragraph 51, further comprising recovering the polypeptide.
53. A method for producing a polypeptide having carboxypeptidase activity, comprising cultivating the host cell of paragraph 50 under conditions conducive for production of the polypeptide.
54. The method of paragraph 53, further comprising recovering the polypeptide.
55. Use of a polypeptide having endopeptidase activity and a polypeptide having carboxypeptidase activity for hydrolysing a protein substrate to obtain a protein hydrolysate, wherein the polypeptide having carboxypeptidase activity is characterised by having a Pro/ACHA*100 ratio of at least 30.
56. Use of paragraph 55, wherein the endopeptidase and a polypeptide having carboxypeptidase activity is used in a method according to any of paragraphs 1-38.
57. A protein hydrolysate produced by the method of any of paragraphs 1-38.
58. A protein hydrolysate comprising free amino acids, wherein the amount of free amino acids is at least 20 g per 100 gram protein, the total amount of Ala, Val, Ile, Leu, Met, Phe and Trp is at least 7 g per 100 g protein and at least 1.0 g Pro per 100 g protein.
59. The protein hydrolysate of paragraph 58, wherein the amount of free amino acids is at least 25 g per 100 g protein hydrolysate, such as at least 30 g per 100 g hydrolysate, at least 30 g per 100 g hydrolysate, at least 35 g per 100 g hydrolysate or at least 40 g per 100 g hydrolysate.
60. The protein hydrolysate of paragraph 58 or 59, wherein the amount of Ala, Val, Ile, Leu, Met, Phe and Trp is at least 13 g per 100 g protein substrate and at least 1.8 g per 100 g protein hydrolysate.
61. The protein hydrolysate of any of paragraphs 58-60, wherein the hydrolysate further has a degree of hydrolysation (% dH) in the range of 30-70%, such as in the range of 45-70%, in the range of 50-70%, in the range of 55-70%, in the range of 60-70%, in the range of 65-70%.
Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mM CaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0
Substrate: Z-L-alanyl-L-Pro-OH substrate (Z-Ala-Pro-OH, Bachem, C-1185) was dissolved in DMSO to a concentration of 40 mM.
25 μL of assay buffer, 25 μL deionized water, 5 μL of the Z-Ala-Pro-OH substrate and 10 μL of enzyme diluted to a concentration of 5 μg/mL were added to the wells of a 96 well microtiter plate (MTP). The MTP was incubated at 55° C. for 30 minutes with shaking at 1000 rpm in an Eppendorf Thermomixer comfort. Then the MTP was transferred to a fridge kept at 4° C. for 10 minutes. 100 μL of 2.0 M HEPES pH 10.0 and then 100 μL of a 6% solution of ninhydrin (Sigma) dissolved in 100% ethanol were added to the wells containing the enzyme and substrate solution. The MTP was incubated at 80° C. for 5 minutes with moderate shaking (350 rpm). The MTP was then kept at room temperature for 10 minutes and the absorbance of the sample at 450 nm was then measured.
The activity of the enzyme towards (or on) the Z-Ala-Pro-OH substrate was calculated as the absorption at 450 nm minus the background absorption of a blank (blank sample with 10 μL assay buffer added instead of enzyme solution). Results was rounded off to the second decimal digit.
Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mM CaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0
Stop reagent: 17.9 g trichloro acetic acid (Sigma), 29.9 g sodium acetate trihydrate (Sigma) and 19.0 mL concentrated acetic acid (Sigma) were mixed and deionized water was added to a final volume of 500 mL
OPA reagent: 47.6 g disodium tetraborate decahydrate (Sigma) and 12.5 g sodium dodecyl sulfate (Sigma) were dissolved in 1 L deionized water
Substrate working solution: The Z-L-alanyl-L-XXX-OH substrate listed in table 1 were dissolved in DMSO or deionized water to a concentration of 40 mM (Z-Ala-XXX-OH, where XXX is one of the 17 amino acid listed below).
Carboxypeptidase activity can be determined using the substrates listed in Table 1 as follows.
50 μL of assay buffer, 10 μL of enzyme diluted to a concentration of 5 μg/m L, 5 μL of the Z-Ala-XXX-OH substrate were added to the wells of a 96 well microtiter plate (MTP). The MTP was incubated at 55° C. for 30 minutes with shaking at 1000 rpm in an Eppendorf Thermomixer comfort. Then the MTP was transferred to a fridge kept at 4° C. for 10 minutes. 100 μL of stop reagent was added to the wells containing the enzyme and substrate solution and the MTP was shaken for 10 seconds at 1000 rpm to ensure complete mixing of the two solutions. 80 mg ortho-phtaldialdehyde (OPA, Sigma) were dissolved in 2 mL ethanol (Sigma) and 88 mg DL-Dithiothreitol (Sigma) were dissolved in 2 mL deionized water. The two solutions were added to 80 mL of OPA reagent and the solutions were stirred at room temperature. After 5 minutes mixing the solution volume was adjusted to 100 mL with deionized water (OPA solution). 225 μL of the freshly-prepared OPA solution was added to the wells of a new MTP. 30 μL supernatant from the MTP containing enzymes and substrate was added to the well of the second MTP. The solution was mixed for 10 seconds at room temperature and absorbance of the sample at 340 nm was measured after 2 minutes of addition of the OPA solution.
The activity of the enzyme for a substrate was calculated as the absorption at 340 nm minus the background absorption of a blank (blank sample with 10 μL assay buffer added instead of enzyme solution). Results were rounded off to the second decimal digit.
Substrate: Protazyme OL (Megazyme T-PROL 1000).
Temperature: 30° C.
Assay buffer: 100 mM HEPES (Sigma), 50 mM KCl (Sigma), 1 mM CaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0
A Protazyme OL tablet (from Megazyme) was suspended in 2.0 ml 0.01% Triton X-100 by gentle stirring. 500 μL of this suspension and 500 μL assay buffer were mixed in an Eppendorf tube and placed on ice. 20 μL Alcalase sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm). The reaction was stopped by transferring the tube back to the ice bath. Then the tube was centrifuged in an ice cold centrifuge for a few minutes and 200 μL supernatant was transferred to a microtiter plate. The absorbance at 650 nm (OD650) was read as a measure of protease activity. A blank sample (20 μL 0.01% Triton X-100 instead of enzyme) was prepared and was included in the assay. The enzymatic activity was calculated as OD 650(sample)−OD650(blank).
Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mM CaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0
Substrate listed in table 1B were dissolved in DMSO to a concentration of 40 mM (H-XXX-pNA, where XXX is one of the 18 amino acid listed below).
100 μL of assay buffer, 5 μL of the H-XXX-pNA substrate and 10 μL of enzyme diluted to a concentration of 5 μg/mL were added to the wells of a 96 well microtiter plate (MTP). The MTP was incubated at 55° C. for 60 minutes with shaking at 1000 rpm in an Eppendorf Thermomixer comfort. Then the absorbance of the sample at 405 nm was measured.
The activity of the enzyme towards (or on) the H-XXX-pNA substrate was calculated as the absorption at 405 nm minus the background absorption of a blank (blank sample with 10 μL assay buffer added instead of enzyme solution). Results was rounded off to the second decimal digit.
20 μl of a diluted sample with a protein concentration of 0.06-0.1% was added to a microtiterplate followed by 200 μl OPA solution. The plate was shaken and absorbance read immediately at 340 nm. OPA solution was in 100 ml MQ water: 0.504 g sodium bicarbonate, 0.429 g sodium carbonate decahydrate, 88 mg dithiothreitol, 1 ml 10% SDS, 80 mg o-phthaldialdehyde (OPA) in 2 ml 96% ethanol. A standard curve using 125 mg L-serine in 250 ml MQ water and diluted 2, 4, 8, 16, 32, 64 fold was included, and the response of the samples calculated by comparing to this.
Amino acid content was analysed using a ThermoFischer WPS3000 high pressure liquid chromatography system comprising a quaternary pump, an autosampler, a column oven, and a tuneable fluorescence detector. Prior to the analysis, samples were filtered using 0.22 μm PVDF filters and norvaline added as an internal standard. Samples were analysed after automated pre-column derivatization. 28 μL milli-Q water, 10 μL of 0.4 M borate buffer pH 10.2, 2 μL sample, 2 μL ortho-phthaldialdehyde 2.5 g/L in 0.1 M borate buffer pH 10.2 and 2 μL fluorenylmethyl chloroformate 0.6 g/L in acetonitrile were collected and mixed by pipetting up and down in a mixing vial. 100 μL milli-Q water was added followed by mixing, and 10 μL was finally injected for chromatographic analysis on a Kinetex 5 μm EVO C18 100 Å LC column (150 mm×4.6 mm) with a corresponding SecurityGuard ULTRA cartridge guard column. Solvents were: A: 20 mM potassium phosphate buffer pH 7.2 and B: 50% methanol, 50% acetonitrile. The pump was set to a constant flow rate of 1 ml/minute, and a linear gradient implemented from 0 to 26 min using 3% solvent B up to 60% solvent B. After 26 min, the solvent composition was changed to 3% solvent B and the column equilibrated until 35 min. Column temperature was 40° C. Primary amino acids were excited at 340 nm and emission wavelength was 460 nm while for the secondary amino acids the excitation wavelength was 288 nm and the emission wavelength 308 nm. Samples were analysed by comparison to an amino acids standard mix in a concentration range up to 0.3125 mM. All 20 amino acids were analysed except cysteine. Histidine and glycine coeluted.
A fungal strain was isolated from a compost sample collected from Yunnan province, China by the dilution plate method with PDA medium at 45° C. It was then purified by transferring a single conidium onto a YG agar plate. The strain was identified as Penicillium emersonii, based on both morphological characteristics and ITS rDNA sequence.
PDA medium was composed of 39 grams of potato dextrose agar and deionized water to 1 liter.
YG agar plates were composed of 5.0 g of yeast extract, 10.0 g of glucose, 20.0 g of agar, and deionized water to 1 liter.
YPG medium contained 0.4% of yeast extract, 0.1% of KH2PO4, 0.05% of MgSO4.7H2O, 1.5% glucose in deionized water.
YPM medium contained 1% yeast extract, 2% of peptone, and 2% of maltose in deionized water.
Genomic DNA Extraction of Penicillium emersonii
Penicillium emersonii strain was inoculated onto a PDA plate and incubated for 3 days at 45° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 45° C. with shaking at 160 rpm. The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, Calif., USA) and frozen in liquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to a fine powder, and genomic DNA was isolated using an Andybio Large-Scale Column Fungal DNAout Kit (Bioserver Inc., BeiJing, China).
The extracted genomic DNA samples were delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The raw reads were assembled at BGI using program SOAPdenovo (Li et al., 2010, Genome Research 20(2): 265-72). The assembled sequences were analyzed using standard bioinformatics methods for gene finding and functional prediction. GeneID (Parra et al., 2000, Genome Research 10(4): 511-515) was used for gene prediction. BlastaII version 2.2.10 (Altschul et al., 1990, J. Mol. Biol. 215(3): 403-410, National Center for Biotechnology Information (NCBI), Bethesda, Md., USA) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, Md., USA) were used to predict function based on structural homology. The carboxypeptidase gene, S10_Pe1, was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7: 263) and SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) were used to identify start codons. The SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet. 16(6): 276-277) was used to predict the isoelectric points and molecular weights of the deduced amino acid sequences.
Cloning of the Carboxypeptidase Gene of Penicillium emersonii from Genomic DNA
The expression vector pCaHj505 was used for gene cloning. It contained the TAKA-amylase promoter derived from Aspergillus oryzae and the Aspergillus niger glucoamylase terminator elements. Furthermore pCaHj505 had pUC18 derived sequences for selection and propagation in E. coli, and an amdS gene, which encoded an acetoamidase gene derived from Aspergillus nidulans for selection of an amds* Aspergillus transformant.
The carboxypeptidase gene, S10_Pe1, SEQ ID NO: 1 for the genomic DNA sequence and SEQ ID NO: 2 for the deduced amino acid sequence, was selected for expression cloning.
Based on the DNA information obtained from genome sequencing, oligonucleotide primers, shown below, were designed to amplify the coding sequence of the carboxypeptdase genes from the genomic DNA of Penicillium emersonii NN051602. The primers were synthesized by Invitrogen, Beijing, China.
Lowercase characters of the forward primer represent the coding region of the gene and lowercase characters of the reverse primer represent the flanking region of the gene, while captalized characters represent a region homologous to insertion sites of pCaHj505 (WO 2013/029496). The 4 underlined characters ahead of the coding sequence in the forward primer represent the Kozak sequence as the initiation of translation process.
Ten picomoles of the forward and reverse primers above, primer1 and primer2, were used in a PCR reaction for amplification of the Penicillium emersonii carboxypeptidase gene S10_Pe1. The PCR reaction was composed of 2 μl of genomic DNA of Penicillium emersonii NN051602, 10 μl of 5× Phusion® HF Buffer (Finnzymes Oy, Espoo, Finland), 1.5 μl of DMSO, 1.5 ul of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 μl. The amplification was performed using a Peltier Thermal Cycler (MJ Research Inc., South San Francisco, Calif., USA) programmed for denaturing at 98° C. for 1 minute; 7 cycles of denaturing each at 98° C. for 30 seconds, annealing at 65° C. for 30 seconds with 1° C. decrease per cycle, and elongation at 72° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes; and a final extension at 72° C. for 5 minutes. The heat block then went to a 4° C. soak cycle.
The PCR product was isolated by 1.0% agarose gel electrophoresis using 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product band of approximately 1.8 kb from the reaction was visualized under UV light. The PCR product was then purified from solution by using an Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.
Plasmid pCaHj505 was digested with Bam HI and Xho I, isolated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.
In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) was used to clone the PCR fragment directly into the expression vector pCaHj505, without the need for restriction digestion.
The purified PCR fragment and the digested vector were ligated together using the In-Fusion® HD Cloning Kit according to the manufacturer's instructions resulting in plasmid p505-S10_Pe1 (FIG. 1), in which the transcription of the carboxypeptidase polypeptide coding sequence was under the control of an Aspergillus oryzae alpha-amylase gene promoter. In brief, 0.8 ul of 30 ng/ul of pCaHj505, digested with Bam HI and Xho I, and 3.2 ul of the purified PCR fragment containing ˜60 ng of the Penicillium emersonii carboxypeptidase gene PCR fragment were added to 1 ul of 5× In-Fusion® HD Enzyme Premix. The reaction was incubated at 50° C. for 15 minutes. The ligation reaction was used to transform E. coli TOP10 competent cells (TIANGEN Biotech Co. Ltd., Beijing, China). E. coli transformants containing an expression construct were detected by colony PCR. Colony PCR is a method for quick screening of plasmid inserts directly from E. coli colonies. Briefly, a single colony was transferred to a premixed PCR solution in a PCR tube, including PCR buffer, MgCl2, dNTPs, Taq DNA polymerase and primer pairs from which the PCR fragment was generated. Several colonies were screened. After the PCR, reactions were analyzed by 1.0% agarose gel electrophoresis using TBE buffer. Plasmid DNA was prepared using a QIAPREP® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany) from the colony showing an insert with the expected size. The carboxypeptidase gene coding sequence inserted in p505-S10_Pe1 was confirmed by DNA sequencing using 3730XL DNA Analyzers (Applied Biosystems Inc, Foster City, Calif., USA).
Expression of the Penicillium emersonii Carboxypeptidase Gene in Aspergillus oryzae
Aspergillus oryzae strain MT3568 was used for heterologous expression of the gene encoding the Penicillium emersonii carboxypeptidase gene. A. oryzae MT3568 is an amdS (acetamidase) disrupted derivative of A. oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene.
Protoplasts were prepared according to the method described as “Transformation of Aspergillus Expression Host” in Example 2 of US 2014/0179588. Three μg of p505-S10_Pe1 were used to transform Aspergillus oryzae MT3568.
The transformation of Aspergillus oryzae MT3568 with p505-S10_Pe1 yielded about 10 transformants. Four transformants were isolated to plate for reisolation and were then inoculated separately into 3 ml of YPM medium in 24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation, 20 μl of supernatant from each culture were analyzed on NUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM MES (Invitrogen Corporation, Carlsbad, Calif., USA) according to the manufacturer's instructions. The resulting gel was stained with INSTANTBLUE™ (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures showed that the majority of the transformants had a smear band of approximately 60 kDa. The expression strain was designated as O82P5E.
Fermentation of Aspergillus oryzae Expression Strain O82P5E
Two slants of the expression strain O82P5E, was washed with 10 ml of YPM and inoculated into 18 2-liter flasks each containing 400 ml of YPM medium, shaking at 30° C., 80 rpm. The culture was harvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane (Millipore, Bedford, Mass., USA).
The culture broth harvested in example 1 was precipitated with ammonium sulfate (80% saturated). Precipitates were re-dissolved in 200 ml of 20 mM phosphate-buffered saline (PBS) pH 6.0, and ammonium sulfate was replenished to get final concentration 1.2 M. Crude protein solution was filtered through a 0.45 μm filter, and then applied to a 50 ml self-packed Phenyl Sepharose 6 Fast Flow (low sub) column (GE Healthcare, Buckinghamshire, UK) equilibrated with 20 mM PBS pH 6.0 and 1.2 M ammonium sulfate buffer. Proteins were eluted with a linear 1.2 M-0 M ammonium sulfate gradient. Fractions were analyzed by SDS-PAGE using a Mini-PROTEAN TGX Stain-Free 4-15% Precast Gel (Bio-Rad Laboratories, CA, United States). Carboxypeptidase activities of fractions were assessed by halo zone assay on skim-milk agarose plate at pH 5.0, 50° C. Fractions were pooled containing recombinant protein bands and showing positive activities. Then the pooled solution was concentrated by ultrafiltration.
Cloning and Expression of a S10 Peptidase from Myceliophthora heterothallica Gene
A fungal strain was isolated and based on both morphological and molecular characterization (ITS sequencing) classified as Myceliophthora heterothallica. The Myceliophthora heterothallica strain was annotated as Myceliophthora heterothallica CBS 202.75 strain and fully genome sequenced. The genomic DNA sequence of a S10 peptidase polypeptide encoding sequence was identified in the genome of Myceliophthora heterothallica CBS 202.75 strain and the genomic DNA sequence and deduced amino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The genomic DNA sequence of 1720 nucleotides contains 1 intron of 55 bp (nucleotides 503 to 557). The genomic DNA fragment encodes a polypeptide of 554 amino acids.
The Aspergillus expression vector pDau109 (WO 2005/042735) consists of an expression cassette based on the partly duplicated Aspergillus niger neutral amylase II (NA2) promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpl) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the vector is the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source and the amplicillin resistance gene (beta lactamase) allowing for facile selection for positive recombinant E. coli clones using commercially available and highly competent strains on commonly used LB ampicillin plates. pDau109 contains a multiple cloning site situated between the promoter region and terminator, allowing for insertion of the gene of interest in front of the promoter region.
The gene encoding the Myceliophthora heterothallica CBS 202.75 S10 peptidase (SEQ ID NO: 3) was PCR amplified from genomic DNA isolated from Myceliophthora heterothallica CBS 202.75 strain. The PCR product encoding the Myceliophthora heterothallica CBS 202.75 S10 peptidase (SEQ ID NO: 3) was cloned into the pDau109 Aspergillus expression vector using the unique restriction sites BamHI and HindII and transformed into E. coli (Top10, Invitrogen). Expression plasmids containing the insert were purified from the E. coli transformants, and sequenced with vector primers and gene specific primers in order to determine a representative plasmid expression clone that was free of PCR errors. The plasmid expression clone was transformed into A. oryzae and a recombinant A. oryzae clone containing the integrated expression construct were grown in liquid culture. Expression of the Myceliophthora heterothallica CBS 202.75 S10 peptidase was verified by SDS-page. The enzyme containing supernatant was sterile filtered before purification.
Substrate: Z-Ala-Phe-OH (Bachem C-1155).
Temperature: 37° C.
Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, pH 6.0.
100 μl Z-Ala-Phe-OH substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 25× in 0.01% Triton X-100) was mixed with 150 μl Assay buffer in an Eppendorf tube and placed on ice. 50 μl peptidase sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to 37° C. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm). The tube was then transferred back to the ice bath and when the tube had cooled, 500 μl Stop reagent (17.9 g TCA+29.9 g Na-acetate trihydrate+19.0 ml conc. CH3COOH and deionised water ad 500 ml) was added and the tube was vortexed and left for 15 min at room temperature (to ensure complete precipitation). The tube was centrifuged (15000×g, 3 min, room temp), 30 μl supernatant was transferred to a microtiter plate and 225 μl freshly prepared OPA-reagent (3.81 g disodium tetraborate and 1.00 g SDS were dissolved in approx. 80 ml deionised water—just before use 80 mg ortho-phtaldialdehyde dissolved in 2 ml ethanol was added and then 1.0 ml 10% (w/v) DTE and finally the volume was adjusted ad 100 ml with deionised water) was added. After 2 minutes, A340 was read in a MTP reader. The A340 measurement relative to an enzyme blind (50 μl 0.01% Triton X-100 instead of peptidase sample) was a measure of carboxypeptidase activity.
Purification of the S10 Carboxypeptidase from Myceliophthora heterothallica:
The S10 carboxypeptidase was expressed in A. oryzae.
The culture broth was centrifuged (20000×g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filtration unit in order to remove the rest of the Aspergillus host cells. The 0.2 μm filtrate was transferred to 20 mM MES/NaOH, 0.5 mM CaCl2, pH 6.0 on a G25 sephadex column (from GE Healthcare). The G25 sephadex transferred carboxypeptidase was applied to a Q-sepharose FF column (from GE Healthcare) equilibrated in 20 mM MES/NaOH, pH 6.0. After washing the column extensively with the equilibration buffer, the S10 carboxypeptidase was eluted with a linear gradient between the equilibration buffer and 20 mM MES/NaOH, 5 mM CaCl2, 500 mM NaCl, pH 6.0 over five column volumes. The eluted fractions were analysed for carboxypeptidase activity and active fractions were further analysed by SDS-PAGE. Fractions with one band at approx. 50 kDa were pooled as the purified product and was used for further characterization.
Characteristics for the Purified S10 Carboxypeptidase from Myceliophthora heterothallica:
The relative molecular weight as determined by SDS-PAGE was approx. Mr=50 kDa.
The major N-terminal sequence determined by EDMAN degradation was: TVDPSKL (60%). Two minor N-terminal sequences were also determined: KTVDPSK (20%) and VKTVDPS (20%) suggesting that that the N-termini were somewhat ragged.
The S10 carboxypeptidase from Myceliophthora heterothallica was glycosylated and therefore the purified carboxypeptidase was treated with Endo H before Intact MS analysis. The measured peak pattern could be assigned to the mature sequence.
Cloning and Expression of a S10 Peptidase from Chaetomium strumarium Gene
A fungal strain was isolated and based on both morphological and molecular characterization (ITS sequencing) classified as Chaetomium strumarium. The Chaetomium strumarium strain was annotated as Chaetomium strumarium strain and fully genome sequenced. The genomic DNA sequence of a S10 peptidase polypeptide encoding sequence was identified in the genome of Chaetomium strumarium strain and the genomic DNA sequence and deduced amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The genomic DNA sequence of 1722 nucleotides contains 1 intron of 57 bp (nucleotides 503 to 559). The genomic DNA fragment encodes a polypeptide of 554 amino acids.
The Aspergillus expression vector pDau109 (WO 2005/042735) consists of an expression cassette based on the partly duplicated Aspergillus niger neutral amylase II (NA2) promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpl) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the vector is the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source and the amplicillin resistance gene (beta lactamase) allowing for facile selection for positive recombinant E. coli clones using commercially available and highly competent strains on commonly used LB ampicillin plates. pDau109 contains a multiple cloning site situated between the promoter region and terminator, allowing for insertion of the gene of interest in front of the promoter region.
The gene encoding the Chaetomium strumarium S10 peptidase (SEQ ID NO: 5) was PCR amplified from genomic DNA isolated from Chaetomium strumarium strain. The PCR product encoding the Chaetomium strumarium S10 peptidase (SEQ ID NO: 5) was cloned into the pDau109 Aspergillus expression vector using the unique restriction sites BamHI and HindIII and transformed into E. coli (Top10, Invitrogen). Expression plasmids containing the insert were purified from the E. coli transformants, and sequenced with vector primers and gene specific primers in order to determine a representative plasmid expression clone that was free of PCR errors. The plasmid expression clone was transformed into A. oryzae and a recombinant A. oryzae clone containing the integrated expression construct were grown in liquid culture. Expression of the Chaetomium strumarium S10 peptidase was verified by SDS-page. The enzyme containing supernatant was sterile filtered before purification.
The strain Lasiodiplodia theobromae was isolated from soil, Yunnan, China in 1999.
Cloning of S10 Carboxypeptidase from Lasiodiplodia theobromae
The carboxypeptidase with nucleotide sequence SEQ ID NO: 7 was PCR amplified from genomic DNA isolated from Lasiodiplodia theobromae and cloned into the expression vector pCaHj505 (WO 2013/029496).
The final expression plasmid was transformed into the Aspergillus oryzae MT3568 expression host. A. oryzae MT3568 is a derivative of A. oryzae JaL355 (WO02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene. The carboxypeptidase gene was integrated by homologous recombination into the A. oryzae MT3568 host cell genome upon transformation.
The gene coding for amdS was used as marker. Transformants were selected on pyrG media agar supplemented with 10 mM acetamide. One recombinant Aspergillus oryzae clone containing the carboxypeptidase expression construct was selected and was cultivated on a rotary shaking table in 4 2-liter baffled Erlenmeyer flasks each containing 400 ml YPM (1% Yeast extract, 2% Peptone and 2% Maltose). After 3 days cultivation time at 30° C., enzyme containing supernatants were harvested by filtration using a 0.22 μm 1-liter bottle top vacuum filter (Corning Inc., Corning, N.Y., USA). The protein sequence is SEQ ID NO: 8.
The culture supernatant of O23GV9 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM Bis-Tris at pH7.5. The solution was filtered with 0.45 um filter and then loaded into Q Sepharose Fast Flow column (GE Healthcare) equilibrated with 20 mM Bis-Tris at pH7.5. A gradient of NaCl concentration from zero to 1 M was applied as elution buffer, and then elution fractions and flow-through fraction were collected separately for SDS-PAGE analysis. The fractions with target protein were pooled together for evaluation.
A fungal strain designated was isolated from a soil sample collected from Yunnan Province, China, by dilution on PDA plates at 45° C. and then purified by transferring a single conidium onto a YG agar plate. The strain was identified as Thermoascus aurantiacus, based on both morphological characteristics and ITS rDNA sequence.
PDA medium was composed of 39 grams of potato dextrose agar and deionized water to 1 liter.
YPG medium contained 0.4% of yeast extract, 0.1% of KH2PO4, 0.05% of MgSO4.7H2O, 1.5% glucose in deionized water.
YPM medium contained 1% yeast extract, 2% of peptone, and 2% of maltose in deionized water.
Genomic DNA Extraction of Thermoascus aurantiacus
Thermoascus aurantiacus strain was inoculated onto a PDA plate and incubated for 3 days at 45° C. in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 45° C. with shaking at 160 rpm. The mycelia were collected by filtration through MIRACLOTH® and frozen in liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using a DNeasy® Plant Maxi Kit following the manufacturer's instructions.
The extracted genomic DNA samples were delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The raw reads were assembled at BGI using program SOAPdenovo (Li et al., 2010, Genome Research 20(2): 265-72). The assembled sequences were analyzed using standard bioinformatics methods for gene finding and functional prediction. GeneID (Parra et al., 2000, Genome Research 10(4): 511-515) was used for gene prediction. BlastaII version 2.2.10 (Altschul et al., 1990, J. Mol. Biol. 215(3): 403-410, National Center for Biotechnology Information (NCBI), Bethesda, Md., USA) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, Md., USA) were used to predict function based on structural homology. The carboxypeptidase gene, S10_Ta, was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7: 263) and SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) were used to identify start codons. The SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet. 16(6): 276-277) was used to predict the isoelectric points and molecular weights of the deduced amino acid sequences.
Cloning of the Carboxypeptidase Genes of Thermoascus aurantiacus from Genomic DNA
The expression vector pCaHj505 was used for gene cloning. It contained the TAKA-amylase promoter derived from Aspergillus oryzae and the Aspergillus niger glucoamylase terminator elements. Furthermore pCaHj505 had pUC18 derived sequences for selection and propagation in E. coli, and an amdS gene, which encoded an acetoamidase gene derived from Aspergillus nidulans for selection of an amds+ Aspergillus transformant.
The carboxypeptidase gene, S10_Ta, SEQ ID NO: 9 for the genomic DNA sequence and SEQ ID NO: 10 for the deduced amino acid sequence, was selected for expression cloning.
Based on the DNA information obtained from genome sequencing, oligonucleotide primers, shown below, were designed to amplify the coding sequence of the carboxypeptdase genes from the genomic DNA of Thermoascus aurantiacus NN044936. The primers were synthesized by Invitrogen, Beijing, China.
Lowercase characters of the forward primer represent the coding region of the gene and lowercase characters of the reverse primer represent the flanking region of the gene, while captalized characters represent a region homologous to insertion sites of pCaHj505 (WO 2013/029496). The 4 underlined characters ahead of the coding sequence in the forward primer represent the Kozak sequence as the initiation of translation process.
Ten picomoles of the forward and reverse primers above, primer1 and primer2 were used in a PCR reaction for amplification of the Thermoascus aurantiacus carboxypeptidase gene S10_Ta. The PCR reaction was composed of 2 μl of genomic Thermoascus aurantiacus, 10 μl of 5× Phusion® HF Buffer (Finnzymes Oy, Espoo, Finland), 1.5 μl of DMSO, 1.5 ul of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 μl. The amplification was performed using a Peltier Thermal Cycler (MJ Research Inc., South San Francisco, Calif., USA) programmed for denaturing at 98° C. for 1 minute; 7 cycles of denaturing each at 98° C. for 30 seconds, annealing at 65° C. for 30 seconds with 1° C. decrease per cycle, and elongation at 72° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes; and a final extension at 72° C. for 5 minutes. The heat block then went to a 4° C. soak cycle.
The PCR product was isolated by 1.0% agarose gel electrophoresis using 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product band of approximately 2.3 kb from the reaction was visualized under UV light. The PCR product was then purified from solution by using an Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.
Plasmid pCaHj505 was digested with Bam HI and Xho I, isolated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.
In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) was used to clone the PCR fragment directly into the expression vector pCaHj505, without the need for restriction digestion.
The purified PCR fragment and the digested vector were ligated together using the In-Fusion® HD Cloning Kit according to the manufacturer's instructions resulting in plasmid p505-S10_Ta, in which the transcription of the carboxypeptidase polypeptide coding sequence was under the control of an Aspergillus oryzae alpha-amylase gene promoter. In brief, 0.8 ul of 30 ng/ul of pCaHj505, digested with Bam HI and Xho I, and 3.2 ul of the purified PCR fragment containing ˜60 ng of the Thermoascus aurantiacus carboxypeptidase gene PCR fragment were added to 1 ul of 5× In-Fusion® HD Enzyme Premix. The reaction was incubated at 50° C. for 15 minutes. The ligation reaction was used to transform E. coli TOP10 competent cells (TIANGEN Biotech Co. Ltd., Beijing, China). E. coli transformants containing an expression construct were detected by colony PCR. Colony PCR is a method for quick screening of plasmid inserts directly from E. coli colonies. Briefly, a single colony was transferred to a premixed PCR solution in a PCR tube, including PCR buffer, MgCl2, dNTPs, Taq DNA polymerase and primer pairs from which the PCR fragment was generated. Several colonies were screened. After the PCR, reactions were analyzed by 1.0% agarose gel electrophoresis using TBE buffer. Plasmid DNA was prepared using a QIAPREP® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany) from the colony showing an insert with the expected size. The carboxypeptidase gene coding sequence inserted in p505-S10_Ta was confirmed by DNA sequencing using 3730XL DNA Analyzers (Applied Biosystems Inc, Foster City, Calif., USA).
Expression of the Thermoascus aurantiacus Carboxypeptidase Gene in Aspergillus oryzae
Aspergillus oryzae strain MT3568 was used for heterologous expression of the gene encoding the Thermoascus aurantiacus carboxypeptidase gene. A. oryzae MT3568 is an amdS (acetamidase) disrupted derivative of A. oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy was restored by disrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene.
Protoplasts were prepared according to the method described as “Transformation of Aspergillus Expression Host” in Example 2 of US 2014/0179588. Three μg of p505-S10_Ta were used to transform Aspergillus oryzae MT3568.
The transformation of Aspergillus oryzae MT3568 with p505-S10_Ta yielded about 10 transformants. Four transformants were isolated to plate for reisolation and were then inoculated separately into 3 ml of YPM medium in 24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation, 20 μl of supernatant from each culture were analyzed on NUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM MES (Invitrogen Corporation, Carlsbad, Calif., USA) according to the manufacturer's instructions. The resulting gel was stained with INSTANTBLUE™ (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures showed that the majority of the transformants had a smear band of approximately 65 kDa. The expression strain was designated as O13U8S.
Fermentation of Aspergillus oryzae Expression Strain O13U8S.
A slant of the expression strain O13U8S, was washed with 10 ml of YPM and inoculated into 4 2-liter flasks each containing 400 ml of YPM medium, shaking at 30° C., 80 rpm. The culture was harvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane (Millipore, Bedford, Mass., USA).
The culture broth harvested in example 8 was precipitated with ammonium sulfate (80% saturated). Precipitates were re-dissolved in 80 ml of 20 mM PBS pH 6.0, and ammonium sulfate was replenished to get final concentration 1.2 M. Crude protein solution was filtered through a 0.45 μm filter, and then applied to a 50 ml self-packed Phenyl Sepharose 6 Fast Flow (low sub) column (GE Healthcare, Buckinghamshire, UK) equilibrated with 20 mM PBS pH 6.0 and 1.2 M ammonium sulfate buffer. Proteins were eluted with a linear 1.2 M-0 M ammonium sulfate gradient. Fractions were analyzed by SDS-PAGE using a Mini-PROTEAN TGX Stain-Free 4-15% Precast Gel (Bio-Rad Laboratories, CA, United States). Carboxypeptidase activities of fractions were assessed by halo zone assay on skim-milk agarose plate at pH 5.0, 50° C. Fractions were pooled containing recombinant protein bands and showing positive activities. Then the pooled solution was concentrated by ultrafiltration.
The activity of carboxypeptidases of SEQ ID NO: 2, 4, 6, 8 and 10 were tested in Assay I and II together with two benchmark carboxypeptidases, i.e., CP1 (SEQ ID NO: 15) and CPY (SEQ ID NO: 14) from A. oryzae. Results are given in the below table 2. Calculated values for ACHA, ACLA and Pro/ACHA*100 are given in table 3.
The result show that the carboxypeptidases of SEQ ID NO: 2, 4 and 6 all have an activity of at least 0.15 on Proline, whereas the prior art carboxypeptidase CPY has an activity of 0.13.
As seen in the table the new carboxypeptidases are all characterised by having a higher ratio of activity on Pro versus average activity on hydrolphobic amino acids (Pro/ACHA*100) compared to the benchmark carboxypeptidases (SEQ ID NO: 14 and 15).
The carboxy-peptidases (exo-peptidases) were screened on top of Alcalase (Alc) and Flavourzyme (FZ using a 6% protein suspension of either soy bean meal or wheat gluten. Incubation was done in a Deep Well Plate (DWP) with shaking overnight. After incubation the samples were diluted in MQ water and analysed using the OPA assay (Assay V).
Soy bean meal: Protein content: 52% w/w on soy bean meal
Wheat gluten: Protein content: 73.3% w/w on wheat gluten
Alcalase AF 2.4 L: 0.4% on protein
Flavourzyme 1000 L: 0.8% on protein
Exo-peptidase candidate: 1.5-2 mg enzymeprotein/g protein
DWP: 1 ml DPW, wide-bottomed to ensure mixing; incubation in Eppendorf Thermomixer
pH as is: 6.0-6.5
Time: overnight, 18-19 hrs
DPW assay: Suspensions of the substrates were made giving a final protein concentration of 6.5% based on the protein content given above, and considering the further dilution of the substrate when enzymes were added. Total volume in the wells were 285 ul. All enzymes were diluted as appropriate before addition. Substrates were pretreated using Alcalase and Flavourzyme to ensure that dispensing into the wells were possible. Alcalase and Flavourzyme were therefore added to a larger portion of the substrate and the mixture incubated at 55° C. for 50-60 min. 245 ul substrate per well was then transferred to the DWP and 40 ul of the exo-peptidase added.
The outer column and row of the deep well plate were not used due to temperature fluctuations. Samples were run in quadruplicate and a sample holding Alcalase and Flavourzyme included as a reference. Plates were left overnight in a Thermomixer. After incubation the samples were diluted in MQ water (e.g., 15 ul in 985 ul, DWP) and the degree of hydrolysis determined using an OPA assay (Assay V). Samples were analysed within 20 min, since no inactivation of the samples were done.
Results are given in the tables 4-5 below. DH of the sample having only Alcalase and Flavourzyme were set as 100% and the response of the samples with exo-peptidase on top of Alcalase and Flavourzyme compared to this. Benchmark enzymes included were SEQ ID NO: 14 a carboxypeptidase from A. oryzae with preference for hydrophobic amino acids and SEQ ID NO: 15 a carboxypeptidase from A. oryzae with preference for basic amino acids but also activity on hydrophobic amino acids.
As seen the top candidate in both soy and gluten were enzyme sample SEQ ID NO: 2, characterised as a carboxypeptidase with a high activity on hydrophobic amino acids in general, and with a high activity on Pro in particular. Table 1: Final DH obtained in soy hydrolysates relative to the DH of the sample with background enzymes (Alcalase+Flavourzyme) set to 100. Dosages of exo-candidates were 2 mg enzyme protein/g protein except for SEQ ID NO: 4 and SEQ ID NO: 6 where dosages were 1.5 and 1.8 mg ep/g protein respectively.
Further small scale tests in the DWP set-up were done combining carboxy-peptidase and aminopeptidase (AP2) in soy and using either 0.5% Alcalase and 1% Flavourzyme as a background or 0.5% Alcalase alone. Dosage of exo-peptidase were 1 mg ep/g protein both when used alone or in combinations. Results are shown in table 6.
When using single candidates, results showed that SEQ ID NO: 2 was clearly superior giving the highest DH both when combined with Alcalase alone (DH 37) or with Alcalase and Flavourzyme (DH 46). When added on top of Alcalase only, the performance of SEQ ID NO: 2 was better than when using 1% FZ (37 vs. 28) on top of Alcalase. Performance of SEQ ID NO: 15 and SEQ ID NO: 16 were comparable both when added on top of Alc+Fz and when used with Alc only. The combined treatment showed the highest final DH value.
Dose response of the best candidate, carboxypeptidase SEQ ID NO: 2 was done in soy on a background of 0.8% Alcalase+1.6% Flavourzyme and in gluten on a background of 0.4% Alcalase+0.8% Flavourzyme. Test were run in the DWP set-up. Results are given in table www and show a significant effect on DH also at the lowest dosages.
25 g scale: Substrate suspensions were prepared as above and Alcalase and Flavourzyme added for pretreatment. Compared to the DWP assay dosages of Alcalase and FZ was doubled (0.8% and 1.6%). After incubation for 50 min at 55° C., 21.5 g substrate was transferred to 50 ml blue cap flasks and 3.5 g of the diluted exo-peptidase added (1 mg enzyme protein/g protein). Combinations of a carboxypeptidase and an aminopeptidase were also tested. Dosages were 1+1 mg ep/g protein. Flasks were held in a heated water bath (55° C.) with magnetic stirring. After overnight incubation (20 hrs), 1.8 ml samples were withdrawn and enzymes inactivated in a Thermomixer at 95° C. for 10 min. Samples were diluted and analysed using OPA assay (Assay V).
Results are given in table 8 and show that the best performing candidate (SEQ ID NO: 2) identified in the smaller scale assay (DWP) also proved superior in larger scale.
Background or reference DH obtained in the larger scale assay was clearly higher than in the DWP assay, which could be expected since the background dosages of Alcalase and FZ were higher. In addition to higher enzyme dosage improved mixing in larger scale might also increase hydrolysis.
Looking at the effect of the single candidates in the 25 g scale experiment and the combined treatments using one carboxypeptdase+one aminopeptidase, overall effects on DH seemed to be more or less additive.
Results of analysing the amino acid composition of the hydrolysates are shown in table 9.
As seen in the table the sum of free amino acids increased when the exo-peptidases were added on top of Alcalase and Flavourzyme. Best performance was achieved when combining carboxy- and aminopeptidases. Of the single candidates SEQ ID NO: 2 was superior, showing 100% increase in total amount of liberated amino acids in soy and 35% in gluten.
A wheat gluten suspension was made having a final protein concentration of 10% based on a protein content of 73,3% w/w as is in the vital wheat gluten and considering that the substrate was diluted further when enzymes were added. Hydrolysis was carried out in 2 ml Eppendorf tubes holding 1.8 ml substrate, incubated in an Eppendorf thermomixer with shaking (1200 rpm). Duplicate determinations were made. Incubation temperature was 55° C., and incubation time 18 hours. pH was not adjusted. Enzymes were inactivated by holding the samples at 90° C. for 10 min, and % DH (OPA analysis) and free amino acids (HPLC) were analyzed.
Results of analysing the amino acid composition of the hydrolysate are shown in table 10.
As seen in the table, the sum of free amino acids increased significantly when the exo-peptidases were added on top of Alcalase. Best performance was achieved when combining carboxy- and aminopeptidases. Of the single candidates SEQ ID NO: 2 was superior, showing the highest amount of liberated amino acids, followed by SEQ ID NO: 16. For the carboxypeptidases (SEQ ID NO: 2 and SEQ ID NO: 6) especially levels of hydrophobic amino acids and Pro were high, while for the aminopeptidase (SEQ ID NO: 16) levels of polar uncharged, acidic and basic amino acids were high.
Testing of Best Candidates in Combination with Aminopeptidase (SEQ ID NO: 16) at Higher Protein Concentration on Top of Alcalase
Testing of the different carboxypetidases combined with aminopeptidase were done in a 10% wheat gluten protein suspension. The set-up was the same as the one used in Example 15.
As seen in the table the sum of free amino acids increased significantly when the combination of carboxy-peptidases and aminopeptidase were added on top of alcalase. Best performance was achieved with the carboxypeptidases SEQ ID NO: 2 and SEQ ID NO: 6. Especially levels of hydrophobic amino acids and pro were high in these samples.
Testing of Carboxy-Peptidases in Whey in Combination with Alcalase
Performance of the carboxy-peptidase (SEQ ID NO: 2) was compared to the benchmark enzymes CPY and CP1 (SEQ ID NO: 14 and SEQ ID NO: 15) when combined with Alcalase in a 6% whey protein solution at pH 7.5.
Whey Protein Isolate: Protein content: 90% w/w
Alcalase AF 2.4 L: 0.4% w/w on protein
Exo-peptidase candidate: 0.05-0.2 mg enzymeprotein/g protein
pH: 7.5
Incubation was done in 50 ml glass flasks with stirring. After incubation pH was readjusted to 7.5 using NaOH and enzymes inactivated by holding the samples at 95° C. for 15 min (total time including heat-up). Samples were then diluted in MQ water and analysed using an OPA assay (Assay V).
Results are given in the table 12 below. DH of the sample with Alcalase alone was 14. Dosing of the carboxypeptidases were adjusted to give a final DH of approximately 20 in all samples. As seen, performance of SEQ ID NO: 2 was clearly superior, when comparing on enzyme protein dosage level, giving a final DH of 20.3 at a dosage of 0.05 mg enzyme protein/g protein. The benchmark enzymes had to be dosed at 0.1 and 0.2 mg ep/g, respectively, for a similar performance. This clearly illustrates the better performance and higher specific activity of the SEQ ID NO: 2 carboxypeptidase.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/087723 | Jun 2017 | CN | national |
This application is a divisional of U.S. application Ser. No. 16/611,539 filed Nov. 7, 2019, now pending, which is a 35 U.S.C. 371 national application of international application no. PCT/CN2018/090425 filed Jun. 8, 2018, which claims priority or the benefit under 35 U.S.C. 119 of international application no. PCT/CN2017/087723 filed Jun. 9, 2017. The content of each application is fully incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16611539 | Nov 2019 | US |
| Child | 17571594 | US |
