Method for producing biopolymer

Abstract

Provided is a method for increasing the purity of a biopolymer when producing the biopolymer with a microorganism. The method for producing a biopolymer with a microorganism is characterized by using at least one protease selected from the group consisting of proteases (a) and (b) in a purification step: (a) a protease comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4; and (b) a protease comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8.

Description

Biopolymer manufacturing method

[0001] The present invention relates to a method for producing a biopolymer.

[0002] Biopolymers, which are produced from biomass as raw materials, are used in a variety of applications as alternatives to petrochemical plastics. Many biopolymers are highly biodegradable, and demand for them has been expanding in recent years from the perspective of environmental conservation. Representative biopolymers include starch, polylactic acid (PLA), and polyhydroxyalkanoic acid (PHA).

[0003] PHA, a type of biopolymer, can be produced by microbial fermentation. Because PHA accumulates as granules within cells, downstream processing for PHA recovery is necessary in PHA production. This downstream processing is costly, making commercialization of PHA difficult. PHA recovery is commonly performed using organic solvents such as chloroform, but this requires large amounts of toxic and volatile solvents as well as large amounts of energy, raising concerns about the environmental impact (Non-Patent Document 1).

[0004] Meanwhile, methods using enzymes have long been studied to recover PHA without using organic solvents. By using enzymes to decompose cell components such as cell walls, proteins, sugars, nucleic acids, and lipids, it is possible to obtain effects such as promoting PHA release by weakening the cells and improving PHA purity by decomposing impurities. Because enzymes have high substrate specificity and mild reaction conditions, they have the advantages of causing little damage to PHA, requiring less energy and wastewater, and having a small environmental impact.

[0005] Among enzymes, proteases are particularly widely used for PHA recovery, with Alcalase, Esperase, alkaline protease, trypsin, and the like being preferred (Non-Patent Documents 1-3, Patent Documents 1 and 2). Because proteases are often used in the presence of surfactants or under alkaline conditions, inexpensive microbial proteases that exhibit high activity even under these conditions are particularly popular. However, industrial proteases used to date may not be fully optimized for PHA recovery, and no mutant proteases with improved performance in PHA recovery have been reported to date. Since enzyme costs are a major issue in enzymatic PHA recovery (Patent Document 3), proteases with higher performance than conventional ones are needed to reduce the amount of enzyme required. Furthermore, proteases with higher performance are needed to further improve the purity of PHA.

[0006] (Patent Document 1) China Patent No. 1070534 (Patent Document 2) International Publication No. 2023 / 021878 (Patent Document 3) China Patent Application Publication No. 109504715 (Non-Patent Document 1) Gonzalez, K., et al. Current microbiology 78 (2021): 1-10. (Non-Patent Document 2) Neves, A. and Mueller, J. Biotechnology progress 28.6 (2012): 1575-1580. (Non-patent document 3) Kapritchkoff, FM, et al. Journal of biotechnology 122.4 (2006): 453-462.

[0007] The present invention relates to the following 1) to 5): 1) A method for producing a biopolymer using a microorganism capable of producing a biopolymer, characterized in that at least one protease selected from the group consisting of a) and b) below is used in the biopolymer purification step: a) a protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4; b) a protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8; 2) A method for purifying a biopolymer using a microorganism capable of producing a biopolymer, characterized in that at least one protease selected from the group consisting of a) and b) above is used; 3) An enzyme composition for biopolymer purification, containing at least one protease selected from the group consisting of a) and b) above; 4) Use of at least one protease selected from the group consisting of a) and b) above for producing an enzyme composition for biopolymer purification; and 5) Use of at least one protease selected from the group consisting of a) and b) above as an enzyme for biopolymer purification. Detailed Description of the Invention

[0008] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0009] As used herein, the term "protease" refers to a group of enzymes (EC 3.4) that have protease activity and hydrolyze peptide bonds in protein molecules to produce peptides and amino acids. Protease activity can be measured by methods known in the art.

[0010] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity is calculated by performing an analysis using the Search homology program in the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0011] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0012] As used herein, a "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 1) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 and Clustal omega, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" that position.

[0013] Those skilled in the art can further fine-tune the amino acid sequence alignment obtained above to optimize it. Such optimal alignment is preferably determined taking into account the similarity of the amino acid sequences and the frequency of inserted gaps. Here, amino acid sequence similarity refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both aligned amino acid sequences relative to the total number of amino acid residues in the two sequences. Similar amino acid residues refer to amino acid residues among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Such groups of similar amino acid residues are well known to those skilled in the art, and include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; and leucine and isoleucine.

[0014] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0015] As used herein, amino acid positions and variants are denoted using the recognized IUPAC single-letter amino acid abbreviations, as follows: An amino acid at a given position is denoted as [amino acid, position]. For example, serine at position 16 is designated as "S16." Amino acid "substitutions" are designated as [original amino acid, position, substituted amino acid]. For example, a substitution of serine at position 16 with valine is designated as "S16V." Variants containing multiple modifications are designated by a plus sign ("+"). For example, "S16V+T65P" represents a substitution of serine at position 16 with valine and a substitution of threonine at position 65 with proline, respectively.

[0016] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0017] As used herein, a "parent" polypeptide of a given mutant polypeptide refers to a polypeptide that has a predetermined mutation in an amino acid residue thereof that results in the mutant polypeptide. In other words, a "parent" polypeptide is a polypeptide before the mutation is introduced into the mutant polypeptide.

[0018] The present invention relates to providing a method for increasing the purity of a biopolymer produced by a microorganism capable of producing the biopolymer.

[0019] The present inventors have discovered a protease that, when used in the purification process of a biopolymer produced by a microorganism capable of producing a biopolymer, has a greater effect of reducing the amount of residue than the proteases that have conventionally been widely used in the purification process.Furthermore, using this protease as a parent protease, they have obtained a protease variant that has a greater effect of reducing the amount of residue than the parent protease.

[0020] According to the method of the present invention, the purity of a biopolymer can be improved in a method for producing a biopolymer using a microorganism capable of producing a biopolymer.

[0021] The protease used in the biopolymer production method and biopolymer purification method of the present invention (hereinafter referred to as "the protease of the present invention") is a polypeptide having at least one type of proteolytic (protease) activity selected from the group consisting of the following a) and b): a) a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and b) a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8.

[0022] In terms of the residue-reducing effect, the protease of the present invention is preferably a mutant polypeptide having at least one type of proteolytic (protease) activity selected from the group consisting of the following a') and b'). The proteases of the present invention may be used alone or in combination of two or more types: a') a protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4, and having at least one amino acid residue selected from the group consisting of valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369 in the numbering of SEQ ID NO: 1; b') A protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at a position corresponding to position 9, aspartic acid at a position corresponding to position 253, tryptophan at a position corresponding to position 255, and glutamic acid at a position corresponding to position 256 in the numbering of SEQ ID NO: 5.

[0023] The protease consisting of the amino acid sequence of SEQ ID NO: 1 is a mature enzyme of alkaline protease KP43 derived from Bacillus sp. KSM-KP43 (FERM BP-6532), and is obtained by cleaving and removing the prepro sequence from an alkaline protease precursor containing a prepro sequence consisting of the amino acid sequence of SEQ ID NO: 2 and the mature enzyme region of alkaline protease KP43 by processing (JP 2021-97605 A).

[0024] The protease having the amino acid sequence of SEQ ID NO:4 is a mature enzyme of an alkaline protease KP43 mutant having the amino acid sequence of SEQ ID NO:1, in which the amino acid residue at position 16 is substituted with valine, the amino acid residue at position 65 with proline, the amino acid residue at position 83 with alanine, the amino acid residue at position 273 with isoleucine, the amino acid residue at position 359 with serine, the amino acid residue at position 387 with alanine, the amino acid residue at position 132 with threonine, the amino acid residue at position 166 with glycine, the amino acid residue at position 167 with valine, the amino acid residue at position 195 with glutamine, the amino acid residue at position 294 with threonine, and the amino acid residue at position 369 with asparagine. The amino acid sequences of SEQ ID NO:1 and SEQ ID NO:4 share approximately 97% identity.

[0025] An example of a parent protease of protease a') of the present invention is a protease consisting of an amino acid sequence at least 90% identical to the amino acid sequence of protease a) of the present invention shown in SEQ ID NO: 1. Another example of a parent protease of protease a') of the present invention is a protease consisting of an amino acid sequence at least 90% identical to the amino acid sequence of protease a) of the present invention shown in SEQ ID NO: 4. A protease consisting of the amino acid sequence of SEQ ID NO: 4 is protease a') of the present invention, a parent protease of protease a') of the present invention, and also protease a') of the present invention whose parent protease is a protease consisting of the amino acid sequence of SEQ ID NO: 1.

[0026] The protease a') of the present invention consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or 4, and has at least one of the 12 amino acid residues in the numbering of SEQ ID NO: 1: valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369. Preferably, the protease a') of the present invention has three or more, more preferably six or more, even more preferably ten or more, and even more preferably all twelve of the 12 amino acid residues.

[0027] The protease consisting of the amino acid sequence of SEQ ID NO:5 is a mature enzyme of alkaline protease K16 derived from Bacillus sp. KSM-K16 (FERM BP-3376), and is obtained by cleaving and removing the prepro sequence from an alkaline protease precursor containing the prepro sequence consisting of the amino acid sequence of SEQ ID NO:6 and the mature enzyme region of alkaline protease K16 (Kobayashi, T., et al. Applied Microbiology and Biotechnology 43 (1995): 473-481.).

[0028] The protease consisting of the amino acid sequence of SEQ ID NO:8 is a mature enzyme of an alkaline protease K16 mutant consisting of the amino acid sequence of SEQ ID NO:5, in which the amino acid residue at position 9 is substituted with glutamic acid, the amino acid residue at position 253 is substituted with aspartic acid, the amino acid residue at position 255 is substituted with tryptophan, and the amino acid residue at position 256 is substituted with glutamic acid. The amino acid sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:8 share approximately 99% identity.

[0029] An example of a parent protease of protease b') of the present invention is a protease consisting of an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5. Another example of a parent protease of protease b) of the present invention is a protease consisting of an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 8. A protease consisting of the amino acid sequence of SEQ ID NO: 8 is protease b) of the present invention, a parent protease of protease b') of the present invention, and also protease b') of the present invention in which the parent protease is a protease consisting of the amino acid sequence of SEQ ID NO: 5.

[0030] The protease b') of the present invention is a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 5 or 8, and having at least one of the four amino acid residues, i.e., glutamic acid at position 9, aspartic acid at position 253, tryptophan at position 255, and glutamic acid at position 256 in the numbering of SEQ ID NO: 5, and preferably has two or more, more preferably three or more, and even more preferably all four of the four amino acid residues.

[0031] The protease of the present invention may be produced microbiologically or chemically synthesized. In the microbiological production of the protease of the present invention, it is preferable to express the protease as a protease precursor (proprotein) comprising a prosequence and a mature enzyme domain, or as a protease precursor (preproprotein) comprising a signal sequence (presequence), a prosequence, and a mature enzyme domain.

[0032] The prosequence of a protease is involved in folding of the protease. In a protease precursor containing a prosequence, the prosequence is located on the N-terminal side of the mature enzyme region of the protease precursor. The prosequence acts as an intramolecular chaperonin and is an essential region for the mature protease to assume the correct three-dimensional structure before and after passing through the cell membrane. The prosequence is ultimately cleaved by the mature protease and further degraded into smaller peptides. Examples of such prosequences include a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of positions 32 to 206 of SEQ ID NO: 2 and a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of positions 28 to 111 of SEQ ID NO: 6, and examples thereof include the prosequence of the alkaline protease KP43 and the prosequence of the alkaline protease K16 described above.

[0033] In a protease preproprotein, the signal sequence is located on the N-terminal side of the prosequence. The signal sequence is involved in the extracellular secretion of the protease. When the preproprotein passes through the cell membrane, the signal sequence is cleaved by a signal peptidase to produce a proprotein. Examples of such signal sequences include a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of positions 1 to 31 of SEQ ID NO: 2, and a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of positions 1 to 27 of SEQ ID NO: 6, and examples thereof include the signal sequence of alkaline protease KP43 and the signal sequence of alkaline protease K16 described above.

[0034] Thus, the protease precursor of protease a) of the present invention may be a polypeptide comprising a pro-sequence and a mature enzyme region, and consisting of an amino acid sequence of positions 32 to 640 of SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the amino acid sequence of positions 32 to 206 of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4. Alternatively, the protease precursor of protease a) of the present invention may be a polypeptide comprising a signal sequence, a pro-sequence, and a mature enzyme region, and consisting of an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the amino acid sequence of positions 1 to 206 of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4.

[0035] The protease precursor of protease a') of the present invention may be a polypeptide comprising a pro-sequence and a mature enzyme region, and consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of positions 32 to 640 of SEQ ID NO:2 or the amino acid sequence of positions 32 to 206 of SEQ ID NO:2 and SEQ ID NO:4, and having at least one amino acid residue selected from the group consisting of valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369 in the numbering of SEQ ID NO:1. Alternatively, the protease precursor of protease a') of the present invention may be a polypeptide comprising a signal sequence, a pro-sequence, and a mature enzyme region, and consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of positions 1 to 206 of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4, and having at least one amino acid residue selected from the group consisting of valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369 in the numbering of SEQ ID NO: 1.

[0036] The protease precursor of protease b) of the present invention may be a polypeptide comprising a pro-sequence and a mature enzyme region, and consisting of an amino acid sequence of positions 28 to 380 of SEQ ID NO: 6, or an amino acid sequence having at least 90% identity with the amino acid sequence of positions 28 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8. Alternatively, the protease precursor of protease b) of the present invention may be a polypeptide comprising a signal sequence, a pro-sequence, and a mature enzyme region, and consisting of an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90% identity with the amino acid sequence of positions 1 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8.

[0037] The protease precursor of protease b') of the present invention may be a polypeptide comprising a pro-sequence and a mature enzyme region, and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of positions 28 to 380 of SEQ ID NO: 6 or the amino acid sequence of positions 28 to 111 of SEQ ID NO: 6 and SEQ ID NO: 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at the position corresponding to position 9, aspartic acid at the position corresponding to position 253, tryptophan at the position corresponding to position 255, and glutamic acid at the position corresponding to position 256 in the numbering of SEQ ID NO: 5. Alternatively, the protease precursor of protease b') of the present invention may be a polypeptide comprising a signal sequence, a pro-sequence, and a mature enzyme region, and consisting of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% identity with positions 1 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at position 9, aspartic acid at position 253, tryptophan at position 255, and glutamic acid at position 256 in the numbering of SEQ ID NO: 5.

[0038] A protease precursor can be produced by expressing a polypeptide from a polynucleotide encoding the protease precursor. The polynucleotide can be prepared by extracting genomic DNA from a microorganism that produces the desired protease precursor using standard methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence can be chemically synthesized based on the amino acid sequence of the desired protease precursor and used as a polynucleotide encoding the desired protease precursor.

[0039] Alternatively, protease precursors can be produced using various mutagenesis techniques known in the art, for example, by mutating a polynucleotide encoding an amino acid residue to be substituted in a parent protease gene (reference protease gene) encoding the reference amino acid sequence to a polynucleotide encoding the substituted amino acid residue, and then expressing a mutant polypeptide from the mutant gene.

[0040] Various mutagenesis techniques known in the art can be used to mutate amino acids in protease precursors. For example, a polynucleotide (hereinafter also referred to as a parent gene) encoding the amino acid sequence to be mutated (parent protease sequence) can be changed to a polynucleotide (hereinafter also referred to as a mutant gene) encoding the mutated amino acid sequence, and a protease mutant having the desired mutation can be expressed from the mutant gene.

[0041] Mutations can be introduced into a parent gene essentially using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit) can also be used.

[0042] Site-specific mutagenesis of a parent gene can most commonly be performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer anneals to a region of the parent gene containing a nucleotide sequence encoding the amino acid residue to be mutated, and is designed to contain a nucleotide sequence having a nucleotide sequence (codon) encoding the mutated amino acid residue in place of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. Those skilled in the art can recognize and select the nucleotide sequences (codons) encoding the amino acid residues before and after mutation as appropriate, based on standard textbooks, etc. Alternatively, site-specific mutagenesis can be performed using a method in which DNA fragments upstream and downstream of the mutation site are amplified separately using two complementary primers containing the nucleotide mutation to be introduced, and the resulting fragments are then ligated together using SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): pp. 61-68).

[0043] Template DNA containing the parent gene can be prepared by extracting genomic DNA from a strain that produces the parent protease (e.g., Bacillus sp. KSM-KP43, Bacillus sp. KSM-K16) using standard methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence can be chemically synthesized based on the amino acid sequence of the parent protease and used as template DNA. If necessary, the parent gene may be codon-optimized to match the species of the transformant that will express the protease precursor. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0044] The parent gene preferably encodes a protease proprotein comprising a prosequence and a mature enzyme region, or a protease preproprotein comprising a signal sequence, a prosequence, and a mature enzyme region. Examples of the parent gene for protease a') of the present invention include a polynucleotide encoding the amino acid sequence from positions 32 to 640 of SEQ ID NO: 2, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3. Examples of the parent gene for protease b') of the present invention include a polynucleotide encoding the amino acid sequence from positions 28 to 380 of SEQ ID NO: 6, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7.

[0045] The polynucleotide encoding the protease precursor may comprise single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acid. The DNA, cDNA, and RNA may be chemically synthesized. The polynucleotide of the present invention may also comprise a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide may also be codon-optimized for the species of the transformant used to produce the protease precursor.

[0046] The resulting polynucleotide encoding the protease precursor can be incorporated into a vector. Such a vector can be prepared by inserting the polynucleotide into any vector using standard methods. The type of vector is not particularly limited, and may be any vector, such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, or shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. In particular, shuttle vectors, which are replicable in both Bacillus bacteria and other organisms, are suitable for recombinant production of the mutant of the present invention. Preferred examples of the vector include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R, or pASP64 (Japanese Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used to transform bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0047] When a protease precursor is recombinantly produced, the vector is preferably an expression vector. The expression vector may contain useful sequences as needed, such as various elements essential for expression in a host, such as a transcription promoter, terminator, and ribosome binding site; cis elements, such as a polylinker and enhancer; a polyA addition signal; a ribosome binding sequence (SD sequence); and a selection marker gene, such as a drug (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloramphenicol, etc.) resistance gene. Alternatively, a polynucleotide encoding the protease precursor may contain the above-mentioned useful sequences.

[0048] By introducing a polynucleotide encoding a protease precursor or a vector containing the same into a host, a transformant containing the polynucleotide encoding a protease precursor or a vector containing the same can be obtained.

[0049] Examples of hosts for the transformant include bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Clostridium, and yeast, among which bacteria of the genus Bacillus are preferred, and Bacillus subtilis or a mutant thereof is more preferred. Therefore, the transformant is preferably a recombinant Bacillus bacterium, more preferably a recombinant of Bacillus subtilis or a mutant thereof. Examples of Bacillus mutant strains include strains lacking aprX and a gene selected from aprE, nprB, nprE, bpr, vpr, mpr, epr, and wprA (JP 2006-174707 A).

[0050] Polynucleotides or vectors can be introduced into host cells by well-known transformation techniques, such as the calcium phosphate method, electroporation, lipofection, particle gun method, and PEG method. For example, methods that can be applied to Bacillus subtilis or its mutants include competent cell transformation (J Bacteriol, 1967, 93:1925-1937), electroporation (FEMS Microbiol Lett, 1990, 55:135-138), protoplast transformation (Mol Gen Genet, 1979, 168:111-115), and Tris-PEG method (J Bacteriol, 1983, 156:1130-1134).

[0051] The transformant can be cultured in an appropriate medium to express the protease precursor. The expressed protease precursor undergoes folding and cleavage of the prosequence to become a mature protease enzyme. Furthermore, if the protease precursor is a preproprotein having a signal sequence, the mature enzyme is secreted outside the cell.

[0052] The transformant can be cultured for protease production according to methods commonly used in the art. For example, when the transformant is Bacillus subtilis or a mutant thereof, the culture medium contains a carbon source and an inorganic or organic nitrogen source necessary for Bacillus subtilis growth. Carbon sources include, for example, glucose, dextran, soluble starch, sucrose, and methanol. Inorganic or organic nitrogen sources include, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If necessary, the medium may contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins, and antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, and erythromycin). Culture conditions, such as temperature, aeration and agitation conditions, medium pH, and culture time, can be appropriately selected depending on the species and characteristics of the microorganism, the culture scale, and the like.

[0053] After cultivation, the mature protease enzyme is recovered from the resulting culture by conventional methods. For example, the culture is recovered, and if necessary, the cells are disrupted by ultrasound or pressure, and the mature enzyme can be recovered from the culture by an appropriate combination of filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, and the like. When the protease precursor has a signal sequence and the mature enzyme is secreted outside the cells, the mature protease enzyme can be recovered without disrupting the cells. The degree of purification of the mature enzyme is not particularly limited. For example, the culture supernatant or a crudely separated and purified product thereof can be obtained as a composition containing the mature enzyme.

[0054] As shown in the Examples below, when used in the purification process of a biopolymer produced by a microorganism capable of producing a biopolymer, the proteases of the present invention are more effective in reducing residues than Alcalase and Esperase (both manufactured by Novozymes), which are proteases commonly used in the purification process, and, in the case of protease variants, are more effective than the parent proteases. These proteases can efficiently degrade microbial components other than the biopolymer, thereby significantly contributing to improving the purity of the biopolymer. Furthermore, the reduced amount of enzyme required also contributes to cost reduction in the biopolymer production process. Therefore, the proteases of the present invention are useful as enzymes for biopolymer purification, particularly as enzymes for biopolymer purification used in the purification process of biopolymer production methods using microorganisms capable of producing a biopolymer. They can also serve as an active ingredient in enzyme compositions for biopolymer purification, particularly enzyme compositions for biopolymer purification used in the purification process of biopolymer production methods using microorganisms capable of producing a biopolymer.

[0055] In one aspect, the present invention provides an enzyme composition for biopolymer purification containing the protease of the present invention. The enzyme composition of the present invention may be a solid composition such as a powder, or a liquid composition. In addition to the protease of the present invention, the enzyme composition may contain, as appropriate, surfactants, chelating agents, water-soluble polymers, alkaline agents, organic acids or salts thereof, enzymes other than the protease of the present invention, enzyme stabilizers, antioxidants, solubilizing agents, pH adjusters, buffers, preservatives, fragrances, and the like.

[0056] The content of the protease of the present invention in the enzyme composition of the present invention is not particularly limited as long as it is an amount that allows the protease of the present invention to exhibit activity, but is preferably 0.01 to 500 g, more preferably 0.1 to 200 g, and even more preferably 1 to 100 g per kg of the enzyme composition.

[0057] The protease of the present invention, either alone or in the form of an enzyme composition containing the protease, is used to purify a biopolymer in a method for producing a biopolymer using a microorganism capable of producing a biopolymer. Thus, in another aspect, the present invention provides a method for producing a biopolymer using a microorganism capable of producing a biopolymer, characterized in that the protease of the present invention is used in the purification step. In yet another aspect, the present invention provides a method for purifying a biopolymer, characterized in that the protease of the present invention is used in a method for producing a biopolymer using a microorganism capable of producing a biopolymer. Hereinafter, these methods are collectively referred to as the "method of the present invention."

[0058] The "microorganism capable of producing a biopolymer" in the method of the present invention is a microorganism capable of fermentatively producing a biopolymer and accumulating it within its cells. The microorganism is not particularly limited and may be either a wild-type microorganism or a mutant microorganism (mutant) in which a mutation such as an insertion, substitution, or deletion of a nucleotide sequence has been caused by various genetic manipulations, or may be a genetically modified microorganism that has been imparted with the ability to produce a desired biopolymer by known artificial modification.

[0059] The method for producing a biopolymer using a microorganism capable of producing a biopolymer is a method for fermentatively producing a biopolymer using a microorganism capable of producing a biopolymer, and includes steps commonly performed in the field, typically a step of culturing a microorganism capable of producing a biopolymer, a step of purifying the biopolymer, and a step of drying the biopolymer. The protease of the present invention is applied to the biopolymer purification step.

[0060] The "biopolymer" produced by the method of producing a biopolymer using a microorganism capable of producing a biopolymer according to the present invention refers to a polymer produced by fermentation by a microorganism and accumulated within the cells of the microorganism, among polymers produced using biomass as a raw material. Examples of such biopolymers include polyhydroxyalkanoates (PHAs).

[0061] PHA is a polyester whose monomer unit is hydroxyalkanoic acid, and is produced by microorganisms capable of producing PHA using sugars, alcohols, fatty acids, oils and fats as carbon sources, and is accumulated within the cells of the microorganisms. Hydroxyalkanoic acids include (R)-3-hydroxybutanoic acid ((R)-3-hydroxybutyric acid: 3HB), 4-hydroxybutanoic acid (4-hydroxybutyric acid: 4HB), 3-hydroxypropionic acid (3-hydroxypropionic acid: 3HP), (R)-3-hydroxypentanoic acid ((R)-3-hydroxyvaleric acid, (R)-3-hydroxyvaleric acid: 3HV), (R)-3-hydroxyhexanoic acid ((R)-3-hydroxyhexanoic acid: 3HHx), and (R)-3-hydroxyoctanoic acid ((R)-3-hydroxyoctanoic acid: 3HHx). acid: 3HO), (R)-3-hydroxydecanoic acid ((R)-3-hydroxydecanoic acid: 3HD), (R)-3-hydroxydodecanoic acid ((R)-3-hydroxydodecanoic acid: 3HDD), (R)-3-hydroxytetradecanoic acid ((R)-3-hydroxytetradecanoic acid: 3HTD), and the like. PHA may be a homopolymer composed of one type of monomer unit, or a copolymer composed of two or more types of monomer units.Examples of PHA include poly[(R)-3-hydroxybutanoic acid] {poly[(R)-3-hydroxybutyric acid]: P(3HB)}, poly[(R)-3-hydroxybutanoic acid-co-(R)-3-hydroxypentanoic acid] {poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid]: P(3HB-co-3HV)}, and poly[(R)-3-hydroxybutanoic acid-co-(R)-4-hydroxybutanoic acid] {poly[(R)-3-hydroxybutyric acid-co-(R)-4-hydroxybutyric acid]: P(3HB-co-3HV)}. acid]:P(3HB-co-4HB)}, poly[(R)-3-hydroxybutanoic acid-co-(R)-3-hydroxyhexanoic acid] {poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyhexanoic acid]:P(3HB-co-3HHx)}, poly[(R)-3-hydroxybutanoic acid-co-(R)-3-hydroxydecanoic acid] {poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxydecanoic acid]:P(3HB-co-3HD)}, and the like. The composition ratio of the monomer units in the copolymer is not particularly limited, and PHAs with various composition ratios can be obtained depending on the type of microorganism capable of producing PHA, the type of carbon source, the culture method, etc.

[0062] Microorganisms capable of producing PHA may be wild-type microorganisms that inherently have the ability to produce PHA, mutant microorganisms in which the PHA-producing ability has been modified by, for example, modifying a gene encoding a PHA synthase, or genetically modified microorganisms to which the PHA-producing ability has been imparted by, for example, introducing a gene encoding a PHA synthase from outside. Examples of such microorganisms include microorganisms belonging to the genera Aeromonas, Bacillus, Cupriavidus, Escherichia, and Pseudomonas. Among these, from the viewpoint of PHA productivity, microorganisms belonging to the genera Aeromonas, Cupriavidus, and Escherichia are preferred, and microorganisms belonging to the genus Cupriavidus are more preferred. Examples of microorganisms belonging to the genus Cupriavidus include Cupriavidus necator (formerly known as Ralstonia eutropha) and Cupriavidus metallidurans, with Cupriavidus necator being particularly preferred.

[0063] The step of culturing a microorganism capable of producing a biopolymer is a step of fermenting a biopolymer using the microorganism. This step can be carried out under general conditions employed for the production of a biopolymer using a microorganism capable of producing a biopolymer. For example, the culture medium may be either a synthetic medium or a natural medium, as long as it contains nutrients necessary for the production of a biopolymer by the microorganism capable of producing a biopolymer, such as a carbon source, a nitrogen source, and inorganic salts.

[0064] The carbon source may be any that can be assimilated by a microorganism capable of producing a biopolymer, and examples thereof include sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysates, and molasses; alcohols such as ethanol; fatty acids such as dodecanoic acid (lauric acid), tridecanoic acid, tetradecanoic acid (myristic acid), pentadecanoic acid (pentadecylic acid), hexadecanoic acid (palmitic acid), hexadecenoic acid, heptadecanoic acid (margaric acid), octadecanoic acid (stearic acid), octadecenoic acid, octadecadienoic acid, octadecanetridecenoic acid, nonadecanoic acid, eicosanoic acid, eicosadienoic acid, eicosatrienoic acid, and eicosatetraenoic acid; and fats and oils such as coconut oil, palm oil, palm kernel oil, olive oil, rapeseed oil, rice bran oil, soybean oil, castor oil, and mahua oil. These carbon sources can be used alone or in combination of two or more, and can be added to the medium in any manner, such as all at once, in portions, or continuously.

[0065] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0066] Examples of inorganic salts include disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron(III) chloride, calcium chloride, cobalt chloride, copper sulfate, and nickel chloride. Furthermore, vitamins can be added to the medium as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0067] Cultivation is preferably carried out under aerobic conditions, and common methods such as aeration culture and shaking culture can be applied. Any culture method can be selected from batch culture, semi-batch culture, continuous culture, etc. The culture temperature is preferably 10 to 50°C, more preferably 20 to 42°C, and even more preferably 25 to 35°C. The initial pH of the medium (30°C) is preferably 6 to 9, more preferably 7 to 8. The culture time is preferably 24 to 200 hours, more preferably 50 to 100 hours.

[0068] After the culture, the microorganisms capable of producing a biopolymer in the culture contain the biopolymer produced intracellularly. The culture is then subjected to a biopolymer purification step, but prior to this step, the microorganisms capable of producing a biopolymer may be inactivated, if necessary. The microorganisms capable of producing a biopolymer can be inactivated, for example, by heat-treating the microorganisms at about 50 to 80°C for about 10 to 60 minutes. Furthermore, prior to the biopolymer purification step, if necessary, the medium components may be removed and the microorganisms capable of producing a biopolymer may be recovered.

[0069] The biopolymer purification process involves decomposing components derived from the cells of a microorganism capable of producing a biopolymer containing a biopolymer (hereinafter referred to as a "biopolymer-containing microorganism") other than the biopolymer, separating the biopolymer from the biopolymer-containing microorganism, and increasing the purity of the biopolymer. This process is carried out using the protease of the present invention. The components derived from the cells other than the biopolymer include cell walls, proteins, sugars, nucleic acids, lipids, etc. derived from the cells of the biopolymer-containing microorganism other than the biopolymer, and are impurities whose contamination in the biopolymer is desired to be reduced.

[0070] In a preferred embodiment, the protease of the present invention is allowed to act on a biopolymer-containing microorganism or a disrupted cell product thereof. Any method known in the art may be used to allow the protease of the present invention to act on a biopolymer-containing microorganism or a disrupted cell product thereof. Preferably, the protease of the present invention is added to a suspension containing the biopolymer-containing microorganism or a disrupted cell product thereof. The reaction conditions for the biopolymer-containing microorganism or a disrupted cell product thereof with the protease of the present invention are not particularly limited, as long as the conditions do not inactivate the protease of the present invention. Appropriate reaction conditions can be determined by those skilled in the art depending on the type, shape, and amount of the microorganism capable of producing a biopolymer, the type, shape, and amount of the biopolymer, and the type and amount of the protease of the present invention. Examples of reaction conditions for the method of the present invention are described below.

[0071] The amount of the protease of the present invention used in the reaction is appropriately determined depending on the type, shape, amount, etc. of the microorganism capable of producing a biopolymer. For example, the amount of the protease of the present invention used is preferably 0.1 to 10% by mass, more preferably 0.1 to 5% by mass, and even more preferably 0.1 to 3% by mass, based on 100% by mass of the microorganism capable of producing a biopolymer or its disrupted cell mass, in terms of dry mass.

[0072] From the viewpoint of improving the purification efficiency of the biopolymer, the pH conditions for the reaction are preferably pH 8 or higher, more preferably pH 9 or higher, and preferably pH 13 or lower, more preferably pH 12 or lower. The pH is also preferably 8 to 13, more preferably pH 9 to 12, and even more preferably pH 10. The pH can be adjusted by adding an appropriate acid or base.

[0073] The temperature conditions for the reaction are preferably 30 to 80°C, more preferably 40 to 70°C, and even more preferably 40 to 60°C, from the viewpoint of improving the efficiency of purifying the biopolymer.

[0074] The reaction time is preferably 30 minutes to 24 hours, more preferably 1 to 12 hours, and even more preferably 1 to 5 hours, from the viewpoint of improving the efficiency of biopolymer purification.

[0075] The reaction is preferably carried out in the presence of a surfactant to improve the purification efficiency of the biopolymer. Examples of surfactants include anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, either singly or in combination. Anionic surfactants are preferred. Examples of anionic surfactants include sulfate salts of alcohols having 10 to 18 carbon atoms, sulfate salts of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzenesulfonates, paraffin sulfonates, α-olefin sulfonates, internal olefin sulfonates, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts, and fatty acid salts. Particularly preferred are one or more anionic surfactants selected from sulfate salts of alcohols having an alkyl chain containing 10 to 14 carbon atoms and linear alkylbenzenesulfonates having an alkyl chain containing 10 to 14 carbon atoms. The counterion is preferably an alkali metal salt or an amine, with sodium and / or potassium, monoethanolamine, or diethanolamine being particularly preferred. Sodium dodecyl sulfate is particularly preferred.

[0076] In the biopolymer purification process, in addition to the treatment with the protease of the present invention, other treatments such as mechanical treatment, heat treatment, alkali treatment, surfactant treatment, and oxidizing agent treatment may be performed. These other treatments can be performed under conditions generally used in the production of biopolymers by microorganisms capable of producing biopolymers. Mechanical treatment refers to disruption of bacterial cells using ultrasound or pressure. Examples of alkali include sodium hydroxide, sodium sesquicarbonate, and sodium bicarbonate. Examples of surfactants include anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, or a combination thereof. Anionic surfactants are preferred. Examples of anionic surfactants include sulfate salts of C10-C18 alcohols, sulfate salts of alkoxylated C8-C20 alcohols, alkylbenzenesulfonates, paraffin sulfonates, α-olefin sulfonates, internal olefin sulfonates, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts, and fatty acid salts. In particular, one or more anionic surfactants selected from sulfate salts of alcohols having an alkyl chain containing 10 to 14 carbon atoms and linear alkylbenzene sulfonates having an alkyl chain containing 10 to 14 carbon atoms are preferred. As counterions, alkali metal salts and amines are preferred, with sodium and / or potassium, monoethanolamine, and diethanolamine being particularly preferred. Sodium dodecyl sulfate is particularly preferred. Examples of oxidizing agents include hydrogen peroxide, percarbonate, perborate, persulfate, sodium peroxide, ozone, chlorite, chlorate, perchlorate, and perchloric acid. One or more of these other treatments can be carried out simultaneously with or independently of the treatment with the protease of the present invention. Furthermore, in the biopolymer purification process, in addition to the treatment with the protease of the present invention, treatment with an enzyme other than the protease of the present invention may be carried out. Treatment with these enzymes other than the protease of the present invention can be carried out under conditions generally employed in the production of biopolymers using microorganisms capable of producing biopolymers.The enzyme other than the protease of the present invention may be any enzyme capable of hydrolyzing components derived from fungal cells other than biopolymers, and examples thereof include lysozyme, muramidase, glycosidase, cellulase, nuclease, lipase, amylase, cutinase, laccase, proteases other than the protease of the present invention, etc. Treatment with an enzyme other than the protease of the present invention can be carried out by treating with one or more enzymes simultaneously with or independently of the treatment with the protease of the present invention.

[0077] The solid content of the reaction solution obtained after the biopolymer purification step contains the biopolymer. Therefore, the biopolymer is recovered by drying the solid content of the reaction solution in the biopolymer drying step. This step can be carried out under general conditions employed in the production of biopolymers using microorganisms capable of producing biopolymers. For example, this step can be carried out by subjecting the reaction solution to solid-liquid separation by centrifugation or the like to obtain the solid content, washing the solid content as necessary, and then drying the solid content by spray drying, evaporation to dryness, freeze drying, or the like. This step can produce a biopolymer powder.

[0078] The biopolymers produced in this manner have much higher purity than biopolymers produced by conventional methods, with less contamination by residues derived from the cells of microorganisms capable of producing biopolymers. The purity of a biopolymer can be measured, for example, according to the method shown in Example 1(4) below, using the ratio of the dry weight of the residue mixed in the biopolymer powder to the weight of the biopolymer powder as an indicator. The dry weight of the residue mixed in the biopolymer powder can be measured by adding chloroform to the biopolymer powder to dissolve the biopolymer, then removing the chloroform and drying the remaining residue. The lower the dry weight of the residue, the higher the biopolymer purity of the biopolymer powder can be determined. The biopolymers produced by the methods of the present invention may have a dry weight of residue of preferably 20% or less, more preferably 18% or less.

[0079] The following compositions, manufacturing methods, uses, and methods are further disclosed herein as exemplary embodiments of the present invention, but the present invention is not limited to these embodiments.

[0080] [1] A method for producing a biopolymer using a microorganism capable of producing a biopolymer, characterized in that at least one protease selected from the group consisting of the following a) and b) is used in the biopolymer purification step: a) a protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4 b) a protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8 [2] A method for purifying a biopolymer using a microorganism capable of producing a biopolymer, characterized in that at least one protease selected from the group consisting of the above a) and b) is used. [3] The method according to [1] or [2], wherein the protease is at least one protease selected from the group consisting of the following a') and b'):a') A protease consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4, and having at least one amino acid residue selected from the group consisting of valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369 in the numbering of SEQ ID NO: 1. b') A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at a position corresponding to position 9, aspartic acid at a position corresponding to position 253, tryptophan at a position corresponding to position 255, and glutamic acid at a position corresponding to position 256 in the numbering of SEQ ID NO: 5. [4] The method of any one of [1] to [3], wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4, and has, in the numbering of SEQ ID NO: 1, a valine at a position corresponding to position 16, a proline at a position corresponding to position 65, an alanine at a position corresponding to position 83, an isoleucine at a position corresponding to position 273, a serine at a position corresponding to position 359, an alanine at a position corresponding to position 387, a threonine at a position corresponding to position 132, a glycine at a position corresponding to position 166, a valine at a position corresponding to position 167, a glutamine at a position corresponding to position 195, a threonine at a position corresponding to position 294, and an asparagine at a position corresponding to position 369.[5] The method according to any one of [1] to [3], wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8, and has glutamic acid at position 9, aspartic acid at position 253, tryptophan at position 255, and glutamic acid at position 256 in the numbering of SEQ ID NO: 5. [6] The method according to any one of [1] to [5], wherein the protease is allowed to act on a microorganism containing a biopolymer or a cell lysate thereof. [7] The method according to any one of [1] to [6], wherein the protease is allowed to act on a microorganism containing a biopolymer or a cell lysate thereof under conditions of pH 8 or higher, preferably pH 9 or higher, and pH 13 or lower, preferably pH 12 or lower, or pH 8 to 13, preferably pH 9 to 12, more preferably pH 10. [8] The method according to any one of [1] to [7], wherein a protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell product thereof in the presence of a surfactant, preferably sodium dodecyl sulfate. [9] The method according to any one of [1] to [8], wherein the biopolymer is polyhydroxyalkanoic acid (PHA).

[10] The method according to any one of [1] to [9], wherein the microorganism capable of producing a biopolymer is a microorganism belonging to the genus Cupriavidus, preferably Cupriavidus necator.

[11] The method according to any one of [1] to

[10] , wherein the method for producing a biopolymer using a microorganism capable of producing a biopolymer comprises the steps of culturing the microorganism capable of producing a biopolymer, purifying the biopolymer, and drying the biopolymer.

[0081]

[12] An enzyme composition for biopolymer purification, comprising at least one protease selected from the group consisting of a) and b).

[13] The enzyme composition according to

[12] , wherein the protease is at least one protease selected from the group consisting of a') and b').

[14] The enzyme composition according to

[12] or

[13] , wherein the protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and has, in the numbering of SEQ ID NO: 1, valine at position 16, proline at position 65, alanine at position 83, isoleucine at position 273, serine at position 359, alanine at position 387, threonine at position 132, glycine at position 166, valine at position 167, glutamine at position 195, threonine at position 294, and asparagine at position 369.

[15] The enzyme composition according to

[12] or

[13] , wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8, and has glutamic acid at a position corresponding to position 9, aspartic acid at a position corresponding to position 253, tryptophan at a position corresponding to position 255, and glutamic acid at a position corresponding to position 256 in the numbering of SEQ ID NO: 5.

[16] The enzyme composition according to any one of

[12] to

[15] , wherein the biopolymer is a PHA.

[17] The enzyme composition according to any one of

[12] to

[16] , which is used in a method for producing a biopolymer using a microorganism capable of producing a biopolymer.

[0082]

[18] Use of at least one protease selected from the group consisting of a) and b) above for producing an enzyme composition for biopolymer purification.

[19] Use of at least one protease selected from the group consisting of a) and b) above as an enzyme for biopolymer purification.

[20] The use according to

[18] or

[19] above, wherein the protease is at least one protease selected from the group consisting of a') and b') above.

[21] The use according to any one of

[18] to

[20] , wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4, and has, in the numbering of SEQ ID NO: 1, a valine at a position corresponding to position 16, a proline at a position corresponding to position 65, an alanine at a position corresponding to position 83, an isoleucine at a position corresponding to position 273, a serine at a position corresponding to position 359, an alanine at a position corresponding to position 387, a threonine at a position corresponding to position 132, a glycine at a position corresponding to position 166, a valine at a position corresponding to position 167, a glutamine at a position corresponding to position 195, a threonine at a position corresponding to position 294, and an asparagine at a position corresponding to position 369.

[22] The use according to any one of

[18] to

[20] , wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8, and has glutamic acid at a position corresponding to position 9, aspartic acid at a position corresponding to position 253, tryptophan at a position corresponding to position 255, and glutamic acid at a position corresponding to position 256 in the numbering of SEQ ID NO: 5.

[23] The use according to any one of

[18] to

[22] , wherein the biopolymer is PHA.

[24] A method using at least one protease selected from the group consisting of a) and b) above as an enzyme for purifying a biopolymer.

[25] The method according to

[24] , wherein the protease is at least one protease selected from the group consisting of a') and b') above.

[26] The method of

[24] or

[25] , wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 4, and has, in the numbering of SEQ ID NO: 1, a valine at a position corresponding to position 16, a proline at a position corresponding to position 65, an alanine at a position corresponding to position 83, an isoleucine at a position corresponding to position 273, a serine at a position corresponding to position 359, an alanine at a position corresponding to position 387, a threonine at a position corresponding to position 132, a glycine at a position corresponding to position 166, a valine at a position corresponding to position 167, a glutamine at a position corresponding to position 195, a threonine at a position corresponding to position 294, and an asparagine at a position corresponding to position 369.

[27] The method of

[24] or

[25] , wherein the protease consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 5 or 8, and has, in the numbering of SEQ ID NO: 5, glutamic acid at a position corresponding to 9, aspartic acid at a position corresponding to 253, tryptophan at a position corresponding to 255, and glutamic acid at a position corresponding to 256.

[28] The method of any one of

[24] to

[27] , wherein the biopolymer is PHA.

[0083] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0084] Example 1 (1) Construction of Protease Expression Plasmid For expression of KP43, the plasmid pHY-KP43 described in JP 2021-97605 A was used. pHY-KP43 has a gene encoding the KP43 preproprotein (polynucleotide of SEQ ID NO: 3, encoding the amino acid sequence of SEQ ID NO: 2). The amino acid sequence of mature KP43 is shown in SEQ ID NO: 1. For mutation introduction into the protease, site-directed mutagenesis by PCR using complementary primer pairs was used (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14(2004):e115-e115.).

[0085] (2) Preparation of Protease Solution. Protease expression plasmids were introduced into Bacillus subtilis strains by the protoplast method and cultured in 2x L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % (w / v)%) at 30°C for 3 days. The culture supernatant containing the protease was then collected by centrifugation. The buffer was exchanged using an Amicon 10K to 20 mM Tris-HCl (pH 7.5) containing 2 mM CaCl2, and the resulting solution was used as a protease solution. For Alcalase (SIGMA, 126741) and Esperase (SIGMA, P5860), reagents were used as protease solutions. The concentration of the protease solution was measured using a DC Protein Assay Kit (Bio-Rad), and BSA Standard Solution (WAKO) was used as the standard solution for calculating the protein amount.

[0086] (3) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) NBRC 102504 strain was inoculated into LB liquid medium and cultured with shaking for 24 hours at 30 ° C. 1 mL of this culture was inoculated into a 500 mL baffled flask containing 100 mL of PHA production medium (1.1% disodium hydrogen phosphate dodecahydrate, 0.19% potassium dihydrogen phosphate, 1.29% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, 0.5%, 80 ppm iron (III) chloride hexahydrate, 50 ppm calcium chloride dihydrate, 1 ppm cobalt chloride hexahydrate, 0.8 ppm copper sulfate pentahydrate, 0.6 ppm nickel chloride hexahydrate, 1% coconut oil) and cultured with shaking for 24 hours at 30 ° C. After 24 hours, 9 g of coconut oil was added, and the culture was continued for another 48 hours. The bacterial culture was inactivated by heating at 60°C for 20 minutes. 15 mL of the inactivated culture was dispensed into 50 mL tubes and centrifuged at 8000 rpm for 10 minutes to obtain a pellet of PHA-containing bacterial cells.

[0087] (4) PHA Recovery Using Protease 1 15 mL of 1 mM sodium hydroxide aqueous solution (pH 11) containing 0.2 wt% sodium dodecyl sulfate was added to each tube containing the pellets obtained in (3) and suspended, followed by incubation at 50 °C for 1 hour. The cells were disrupted using an ultrasonicator, Bioruptor II (Sonic Bio Co., Ltd.) (output: High, 10 cycles of 30 seconds of disruption and 30 seconds of rest). 15 mL of distilled water was added to each disrupted solution, and the mixture was centrifuged at 10,000 rpm for 10 minutes, after which 15 mL of the supernatant was discarded. 15 mL of 1 mM sodium hydroxide aqueous solution (pH 11) was added to each tube, and the mixture was centrifuged at 10,000 rpm for 20 minutes, after which the supernatant was removed. 15 mL of 1 mM sodium hydroxide aqueous solution (pH 11) was added again, and the mixture was centrifuged at 10,000 rpm for 20 minutes, after which the supernatant was removed. The pellet was suspended in 15 mL of aqueous sodium dodecyl sulfate solution (final concentration: 0.2 wt%) adjusted to pH 10 with sodium hydroxide. 200 μg of each protease was added and mixed thoroughly. After 2 hours of reaction at 50°C with shaking and stirring, the mixture was centrifuged at 10,000 rpm for 20 minutes, and the resulting pellet was washed three times with 1 mM aqueous sodium hydroxide solution (pH 11). The resulting pellet was freeze-dried to obtain a powder containing PHA. The dried powder obtained above was placed in a weighed glass test tube and weighed. 2 mL of chloroform was added to each tube and thoroughly stirred. The test tube was centrifuged to remove the chloroform. 1 mL of chloroform was added again, thoroughly stirred, and centrifuged to remove the chloroform. 2 mL of chloroform was added again, thoroughly stirred, and centrifuged to remove the chloroform. The residue remaining in the test tube was dried and weighed, and the ratio of the weight of the residue to the weight of the powder before dissolution of PHA was calculated (Table 1). Since PHA dissolves in chloroform, this residue is an impurity derived from the bacterial cells. KP43 wild-type and KP43 mutant S16V + T65P + N83A + V273I + T359S + S387A + A132T + N166G + G167V + Y195Q + A294T + D369N showed a greater residue reduction effect than Alcalase and Esperase.In particular, the KP43 mutant S16V+T65P+N83A+V273I+T359S+S387A+A132T+N166G+G167V+Y195Q+A294T+D369N significantly reduced residues compared to the wild-type KP43.

[0088]

[0089] (5) PHA Recovery Using Protease 2 PHA was recovered in the same manner as in (4) except that ultrasonic disruption was not performed, and the amount of residue after each protease treatment was measured (Table 2). The KP43 mutant S16V + T65P + N83A + V273I + T359S + S387A + A132T + N166G + G167V + Y195Q + A294T + D369N significantly reduced the amount of residue compared to the wild-type KP43.

[0090]

[0091] Example 2 (1) Construction of Protease Expression Plasmid Using a VHH expression plasmid of SEQ ID NO: 26 containing the Bacillus subtilis spoVG gene-derived promoter described in WO 2021 / 153129 as a template, the K16 gene (polynucleotide of SEQ ID NO: 7, encoding the K16 preproprotein of SEQ ID NO: 6) artificially synthesized by seamless cloning was replaced with the full-length ORF containing the VHH gene to construct the K16 expression plasmid pHY-K16. The amino acid sequence of the mature K16 is shown in SEQ ID NO: 5. Mutations were introduced into the protease by site-directed mutagenesis using PCR with complementary primer pairs (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14(2004):e115-e115.).

[0092] (2) Preparation of Protease Solution A protease solution was prepared in the same manner as in Example 1(2).

[0093] (3) PHA Recovery Using Protease 3 PHA recovery was performed in the same manner as in Example 1 (5), and the amount of residue after each protease treatment was measured. The ratio (%) of the weight of the residue to the powder weight before PHA dissolution was calculated, and the difference from the residue (%) when Alcalase was used was determined (Table 3). Positive values ​​indicate a greater residue reduction effect than Alcalase. K16 wild-type had a greater residue reduction effect than Alcalase, and furthermore, mutant S9E, mutant L256E, and mutant S9E + N253D + N255W + L256E had a greater residue reduction effect than K16 wild-type.

[0094]

Claims

1. A method for producing a biopolymer by a microorganism having biopolymer-producing ability, characterized in that at least one protease selected from the group consisting of the following a) and b) is used in the biopolymer purification step. a) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4 b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8 2. A method for purifying a biopolymer in a method for producing a biopolymer by a microorganism having biopolymer-producing ability, characterized in that at least one protease selected from the group consisting of the following a) and b) is used. a) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4 b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8 3. The method according to claim 1 or 2, wherein the protease is at least one protease selected from the group consisting of the following a') and b'). a') A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and having at least one amino acid residue selected from the group consisting of valine at the position corresponding to position 16, proline at the position corresponding to position 65, alanine at the position corresponding to position 83, isoleucine at the position corresponding to position 273, serine at the position corresponding to position 359, alanine at the position corresponding to position 387, threonine at the position corresponding to position 132, glycine at the position corresponding to position 166, valine at the position corresponding to position 167, glutamine at the position corresponding to position 195, threonine at the position corresponding to position 294, and asparagine at the position corresponding to position 369 in the numbering of SEQ ID NO: 1 b') A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at the position corresponding to position 9, aspartic acid at the position corresponding to position 253, tryptophan at the position corresponding to position 255, and glutamic acid at the position corresponding to position 256 in the numbering of SEQ ID NO: 5 4. The method according to any one of claims 1 to 3, wherein a protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell mass thereof.

5. The method according to any one of claims 1 to 4, wherein the biopolymer is polyhydroxyalkanoic acid (PHA).

6. The method according to any one of claims 1 to 5, wherein the microorganism having the ability to produce a biopolymer belongs to the genus Cupriavidus.

7. An enzyme composition for purifying a biopolymer, containing at least one protease selected from the group consisting of the following a) and b): a) a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4; b) a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8.

8. The enzyme composition according to claim 7, wherein the protease is at least one protease selected from the group consisting of the following a') and b'): a') a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and having at least one amino acid residue selected from the group consisting of valine at the position corresponding to position 16, proline at the position corresponding to position 65, alanine at the position corresponding to position 83, isoleucine at the position corresponding to position 273, serine at the position corresponding to position 359, alanine at the position corresponding to position 387, threonine at the position corresponding to position 132, glycine at the position corresponding to position 166, valine at the position corresponding to position 167, glutamine at the position corresponding to position 195, threonine at the position corresponding to position 294, and asparagine at the position corresponding to position 369 in the numbering of SEQ ID NO: 1; b') a protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at the position corresponding to position 9, aspartic acid at the position corresponding to position 253, tryptophan at the position corresponding to position 255, and glutamic acid at the position corresponding to position 256 in the numbering of SEQ ID NO:

5.

9. The enzyme composition according to claim 7 or 8, wherein the biopolymer is PHA.

10. Use for the production of an enzyme composition for biopolymer purification, which comprises at least one protease selected from the group consisting of the following a) and b). a) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4. b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8.

11. Use as an enzyme for biopolymer purification, which comprises at least one protease selected from the group consisting of the following a) and b). a) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4. b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8.

12. The use according to claim 10 or 11, wherein the protease is at least one protease selected from the group consisting of the following a') and b'). a) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and having at least one amino acid residue selected from the group consisting of valine at the position corresponding to position 16, proline at the position corresponding to position 65, alanine at the position corresponding to position 83, isoleucine at the position corresponding to position 273, serine at the position corresponding to position 359, alanine at the position corresponding to position 387, threonine at the position corresponding to position 132, glycine at the position corresponding to position 166, valine at the position corresponding to position 167, glutamine at the position corresponding to position 195, threonine at the position corresponding to position 294, and asparagine at the position corresponding to position 369, numbered according to SEQ ID NO:

1. b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and having at least one amino acid residue selected from the group consisting of glutamic acid at the position corresponding to position 9, aspartic acid at the position corresponding to position 253, tryptophan at the position corresponding to position 255, and glutamic acid at the position corresponding to position 256, numbered according to SEQ ID NO:

5.

13. The use according to any one of claims 10 to 12, wherein the biopolymer is PHA.

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