Method for producing biopolymer

Abstract

To provide a method for increasing the purity of a biopolymer when producing the biopolymer with a microorganism.SOLUTION: 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.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Biopolymers produced from biomass are used in various applications as an alternative to petrochemical plastics. Many biopolymers have high biodegradability, and their demand has been increasing in recent years from the perspective of environmental protection. Representative biopolymers include starch, polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), and the like.

[0003] PHA, which is a type of biopolymer, can be produced by microbial fermentation. Since PHA is accumulated as granules inside cells, a downstream process for PHA recovery is required in PHA production. This downstream process in PHA production is costly and has become a factor hindering the commercialization of PHA. For PHA recovery, a method using an organic solvent such as chloroform is widely used. However, not only is a large amount of a toxic and volatile solvent required, but also a large amount of energy is needed, so there are concerns about the environmental impact (Non-Patent Document 1).

[0004] On the other hand, in order to recover PHA without using an organic solvent, a method using an enzyme has been studied for a long time. By decomposing cell components such as cell walls, proteins, sugars, nucleic acids, and lipids with an enzyme, effects such as promoting the release of PHA due to cell weakening and improving the purity of PHA due to decomposition of contaminants can be obtained. Since enzymes have high substrate specificity and mild reaction conditions, they have the advantages of causing little damage to PHA, requiring little energy and having a small drainage load, and having little environmental impact.

[0005] Among enzymes, proteases are particularly widely used for PHA recovery, and enzymes such as alcalase, esperase, alkaline protease, and trypsin are preferably used (Non-Patent Documents 1 to 3, Patent Documents 1 and 2). Since they are often used in the presence of surfactants or under alkaline conditions, microbial-derived proteases that exhibit high activity and are inexpensive under these conditions are particularly utilized. However, industrial proteases used heretofore may not be sufficiently optimized for PHA recovery, and mutant proteases with improved performance in PHA recovery have not been reported heretofore. Since the enzyme cost is a major issue in PHA recovery using enzymes (Patent Document 3), there is a demand for proteases having higher performance than heretofore in PHA recovery in order to reduce the required amount of enzyme. Also, for the purpose of further improving the purity of PHA, proteases having high performance are demanded.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0008] The present invention relates to a method for increasing the purity of a biopolymer in the production of the biopolymer by a microorganism having biopolymer-producing ability. Means for Solving the Problems

[0009] The present inventors have found a protease having a higher residue reduction effect than the proteases conventionally widely used in the purification step of a biopolymer when used in the purification step of the biopolymer produced by a microorganism having biopolymer-producing ability, and have obtained a protease mutant having a higher residue reduction effect than the parent protease using the protease as the parent protease.

[0010] That is, the present invention relates to the following 1) to 3). 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 purification step of the biopolymer. 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, characterized in that at least one protease selected from the group consisting of the above a) and b) is used in the method for producing a biopolymer by a microorganism having biopolymer-producing ability. 3) An enzyme composition for biopolymer purification containing at least one protease selected from the group consisting of the above a) and b). Advantages of the Invention

[0011] According to the method of the present invention, in the method for producing a biopolymer by a microorganism having biopolymer-producing ability, the purity of the biopolymer can be improved. Detailed Description of the Invention

[0012] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety into this specification.

[0013] In this specification, "protease" means a group of enzymes (EC 3.4) having protease activity that hydrolyzes peptide bonds of protein molecules to produce peptides and amino acids. Protease activity can be measured by methods known in the art.

[0014] In this specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12 with Unit size to compare (ktup) set to 2.

[0015] In this specification, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 90% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more.

[0016] As used herein, the "corresponding position" on an amino acid sequence or nucleotide sequence can be determined by aligning the target sequence and a reference sequence (e.g., the amino acid sequence shown in SEQ ID NO: 1) to give the maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, the alignment can be carried out by using the Clustal W multiple alignment program (Thompson, J.D. et al, 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the Clustal website [www.clustal.org] operated by University College Dublin, the website of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), or 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 with any position of the reference sequence by the above alignment is regarded as the "corresponding position" to the said any position.

[0017] A person skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined in consideration of the similarity of the amino acid sequences and the frequency of inserted gaps. Here, the similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues in the full length. Similar amino acid residues mean amino acid residues that have similar properties to each other in terms of polarity and charge among the 20 kinds of amino acids constituting proteins, and cause so-called conservative substitutions. Groups consisting of such similar amino acid residues are well known to those skilled in the art. For example, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc. can be mentioned respectively, but are not limited thereto.

[0018] In this specification, "amino acid residue" means the 20 kinds of amino acid residues constituting 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).

[0019] In this specification, the description of the position of amino acids and variants is represented as follows using the recognized one-letter amino acid abbreviations of IUPAC. The amino acid at a predetermined position is represented as [amino acid, position]. For example, serine at position 16 is indicated as "S16". Regarding the "substitution" of an amino acid, it is represented by [original amino acid, position, substituted amino acid]. For example, the substitution of serine at position 16 with valine is denoted as "S16V". Variants containing multiple modifications are represented by the addition symbol ("+"). For example, "S16V+T65P" represents the substitution of serine at position 16 with valine and the substitution of threonine at position 65 with proline, respectively.

[0020] In this specification, "upstream" and "downstream" with respect 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 present on the 3' side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5' side of the gene in the DNA sense strand.

[0021] In this specification, the "parent" polypeptide of a given mutant polypeptide refers to the polypeptide that becomes the mutant polypeptide when a predetermined mutation is made to its amino acid residues. In other words, the "parent" polypeptide is the polypeptide before the mutation is added to the mutant polypeptide.

[0022] The protease (referred to as "the protease of the present invention") used in the method for producing a biopolymer and the method for purifying a biopolymer of the present invention is a polypeptide having at least one 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, b) A protease consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8.

[0023] The protease of the present invention is preferably a mutant polypeptide having at least one proteolytic (protease) activity selected from the group consisting of the following a') and b') from the viewpoint of the residue reduction effect. The protease of the present invention may be used alone or in combination of two or more. a’) It consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and has 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, protease. b’) It consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and has 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, protease.

[0024] The protease consisting of the amino acid sequence of SEQ ID NO: 1 is the 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 by processing from an alkaline protease precursor containing the prepro sequence consisting of the amino acid sequence of SEQ ID NO: 2 and the mature enzyme region of alkaline protease KP43 (Japanese Patent Application Laid-Open No. 2021-97605).

[0025] The protease consisting of the amino acid sequence of SEQ ID NO: 4 is a mature enzyme of the alkaline protease KP43 variant consisting of an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 1, the 16th amino acid residue is substituted with valine, the 65th amino acid residue is substituted with proline, the 83rd amino acid residue is substituted with alanine, the 273rd amino acid residue is substituted with isoleucine, the 359th amino acid residue is substituted with serine, the 387th amino acid residue is substituted with alanine, the 132nd amino acid residue is substituted with threonine, the 166th amino acid residue is substituted with glycine, the 167th amino acid residue is substituted with valine, the 195th amino acid residue is substituted with glutamine, the 294th amino acid residue is substituted with threonine, and the 369th amino acid residue is substituted with asparagine. The identity between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 4 is about 97%.

[0026] As an example of the parent protease of the protease a') of the present invention, a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 1 of the protease a) of the present invention can be mentioned. As another example of the parent protease of the protease a') of the present invention, a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 4 of the protease a) of the present invention can be mentioned. The protease consisting of the amino acid sequence of SEQ ID NO: 4 is also the protease a) of the present invention, is also the parent protease of the protease a') of the present invention, and is also the protease a') of the present invention having the protease consisting of the amino acid sequence of SEQ ID NO: 1 as the parent protease.

[0027] The protease a') of the present invention consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 or 4, and, with reference to the numbering of SEQ ID NO: 1, is a protease having at least one of the 12 amino acid residues 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, preferably having 3 or more, more preferably 6 or more, still more preferably 10 or more, and still more preferably all 12 of the 12 amino acid residues.

[0028] The protease consisting of the amino acid sequence of SEQ ID NO: 5 is the 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 by processing 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.).

[0029] The protease consisting of the amino acid sequence of SEQ ID NO: 8 is the mature enzyme of an alkaline protease K16 variant having an amino acid sequence 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 in the amino acid sequence of SEQ ID NO: 5. The identity between the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 8 is about 99%.

[0030] As an example of the parent protease of protease b’) of the present invention, a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 5 can be mentioned. As another example of the parent protease of protease b) of the present invention, a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8 can be mentioned. The protease consisting of the amino acid sequence of SEQ ID NO: 8 is also protease b) of the present invention, and is also the parent protease of protease b’) of the present invention, and is also protease b’) of the present invention having the protease consisting of the amino acid sequence of SEQ ID NO: 5 as the parent protease.

[0031] Protease b’) of the present invention consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 5 or 8, and with the numbering of SEQ ID NO: 5, it is a protease having at least one of the four amino acid residues 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, preferably a protease having two or more of the four amino acid residues, more preferably three or more, and even more preferably all four.

[0032] The protease of the present invention may be microbiologically produced or chemically synthesized. In the microbiological production of the protease of the present invention, it is preferable that the protease of the present invention is expressed as a protease precursor (proprotein) containing a pro sequence and a mature enzyme region or a preproprotein containing a signal sequence (pre sequence), a pro sequence, and a mature enzyme region.

[0033] The pro-sequence of a protease is involved in the folding of the protease. In a protease precursor containing a pro-sequence, the pro-sequence is located on the N-terminal side of the mature enzyme region of the protease precursor. The pro-sequence acts as an intramolecular chaperonin and is an essential region for the mature protease to adopt an accurate three-dimensional structure before and after passing through the cell membrane. The pro-sequence is finally cleaved by the mature protease and further degraded into smaller peptides. Examples of the pro-sequence include a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence at 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 at positions 28 to 111 of SEQ ID NO: 6. For example, the pro-sequences of the above-mentioned alkaline protease KP43 and alkaline protease K16 can be mentioned.

[0034] In the preproprotein of a protease, the signal sequence is located on the N-terminal side of the pro-sequence. 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 signal peptidase to generate a proprotein. Examples of the signal sequence include a sequence consisting of an amino acid sequence having at least 90% identity with the amino acid sequence at 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 at positions 1 to 27 of SEQ ID NO: 6. For example, the signal sequences of the above-mentioned alkaline protease KP43 and alkaline protease K16 can be mentioned.

[0035] Therefore, 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 the 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.

[0036] The protease precursor of protease a') of the present invention comprises a pro sequence and a mature enzyme region, and consists of the amino acid sequence from positions 32 to 640 of SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the amino acid sequences of positions 32 to 206 of SEQ ID NO: 2 and SEQ ID NO: 4, and is a polypeptide 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 according to the numbering of SEQ ID NO: 1. Alternatively, the protease precursor of protease a') of the present invention comprises a signal sequence, a pro sequence, and a mature enzyme region, and consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the amino acid sequences of positions 1 to 206 of SEQ ID NO: 2 and SEQ ID NO: 4, and is a polypeptide 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 according to the numbering of SEQ ID NO: 1.

[0037] The protease precursor of protease b) of the present invention includes a pro sequence and a mature enzyme region, and may be a polypeptide consisting of the amino acid sequence from positions 28 to 380 of SEQ ID NO: 6 or an amino acid sequence having at least 90% identity with the amino acid sequence from 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 includes a signal sequence, a pro sequence, and a mature enzyme region, and may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% identity with the amino acid sequence from positions 1 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8.

[0038] The protease precursor of protease b’) of the present invention includes a pro sequence and a mature enzyme region, and consists of an amino acid sequence having at least 90% identity with the amino acid sequence from positions 28 to 380 of SEQ ID NO: 6 or the amino acid sequence from positions 28 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8, and has 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 includes a signal sequence, a pro sequence, and a mature enzyme region, and consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence from positions 1 to 111 of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 8, and has 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.

[0039] 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 protease precursor of interest by a conventional method, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the protease precursor of interest, a corresponding nucleotide sequence can be chemically synthesized and used as the polynucleotide encoding the protease precursor of interest.

[0040] Alternatively, protease precursors can be produced using various mutagenesis techniques known in the art. For example, a polynucleotide encoding the amino acid residue to be substituted within the parental protease gene (reference protease gene) encoding its reference amino acid sequence is mutated to a polynucleotide encoding the amino acid residue after substitution, and further, a mutant polypeptide can be produced by expressing the mutant gene.

[0041] As a means of mutating the amino acids of the protease precursor, various mutagenesis techniques known in the art can be used. For example, a polynucleotide encoding the amino acid sequence (parental protease sequence) to which a mutation is to be introduced (hereinafter also referred to as the parental gene) can be changed to a polynucleotide encoding the mutated amino acid sequence (hereinafter also referred to as the mutant gene), and a protease variant having the desired mutation can be expressed from the mutant gene.

[0042] The introduction of mutations into the parental gene can basically be carried out using various site-directed mutagenesis methods well-known to those skilled in the art. The site-directed mutagenesis method can be carried out by any method such as the inverse PCR method or the annealing method, for example. Commercial site-directed mutagenesis kits (for example, Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0043] Site-directed mutagenesis of the parental gene can most commonly be carried out using a mutagenic primer containing the nucleotide mutation to be introduced. The mutagenic primer anneals to a region containing the nucleotide sequence encoding the amino acid residue to be mutated in the parental gene and is designed to contain a nucleotide sequence (codon) encoding the amino acid residue after mutation instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. The nucleotide sequences (codons) encoding the amino acid residues before and after mutation can be appropriately recognized and selected by those skilled in the art based on ordinary textbooks and the like. Alternatively, site-directed mutagenesis can also be carried out using a method in which two complementary primers containing the nucleotide mutation to be introduced are separately used to amplify DNA fragments on the upstream and downstream sides of the mutation site, respectively, and then ligated together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0044] The template DNA containing the parental gene can be prepared by extracting genomic DNA from a strain producing the parental protease (e.g., Bacillus sp. KSM-KP43, Bacillus sp. KSM-K16) and the like by a conventional method, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the parental protease, the corresponding nucleotide sequence can be chemically synthesized and used as the template DNA. If necessary, the parental gene may be codon-optimized according to the type of transformant expressing the protease precursor. Information on the codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0045] The parental gene preferably encodes a protease proprotein containing a pro sequence and a mature enzyme region, or a preproprotein of a protease containing a signal sequence, a pro sequence, and a mature enzyme region. Examples of the parental gene of the protease a') of the present invention include a polynucleotide encoding the amino acid sequence at positions 32 to 640 of SEQ ID NO: 2, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, etc. Examples of the parental gene of the protease b') of the present invention include a polynucleotide encoding the amino acid sequence at positions 28 to 380 of SEQ ID NO: 6, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, etc.

[0046] The polynucleotide encoding the protease precursor may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acids. The DNA, cDNA, and RNA may be chemically synthesized. Further, the polynucleotide of the present invention may include the nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). Also, the polynucleotide may be codon-optimized according to the species of the transformant for producing the protease precursor.

[0047] The polynucleotide encoding the obtained protease precursor can be incorporated into a vector. The vector can be prepared by inserting the polynucleotide into any vector by a conventional method. The type of the vector is not particularly limited and may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector, etc. Further, the vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in bacteria of the genus Bacillus (e.g., Bacillus subtilis or its mutant strain), and more preferably an expression vector capable of inducing the expression of the transgene in bacteria of the genus Bacillus. Among them, a shuttle vector, which is a vector capable of replicating in both bacteria of the genus Bacillus and other organisms, can be preferably used for the recombinant production of the variant of the present invention. Examples of preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60: 235-243), pAC3 (Nucleic Acids Res, 1988, 16: 8732), etc. shuttle vectors; pUB110 (J Bacteriol, 1978, 134: 318-329), pTA10607 (Plasmid, 1987, 18: 8-15), etc. plasmid vectors available for the transformation of bacteria of the genus Bacillus, etc. Also, plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can be used.

[0048] When recombinantly producing a protease precursor, the vector is preferably an expression vector. The expression vector may contain, as necessary, various elements essential for expression in the host, such as a transcription promoter, a terminator, and a ribosome binding site; cis elements such as a polylinker and an enhancer; a polyA addition signal; a ribosome binding sequence (SD sequence); a selection marker gene such as a drug (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloramphenicol, etc.) resistance gene, and other useful sequences. Alternatively, the polynucleotide encoding the protease precursor may contain the above useful sequences.

[0049] By introducing a polynucleotide encoding a protease precursor or a vector containing the same into a host, a transformant containing the polynucleotide encoding the protease precursor or the vector containing the same can be obtained.

[0050] Examples of the host of the transformant include bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Clostridium, yeast, etc. Among them, bacteria of the genus Bacillus are preferred, and Bacillus subtilis or its mutant strain is more preferred. Therefore, the transformant is preferably a recombinant bacterium of the genus Bacillus, and more preferably a recombinant of Bacillus subtilis or its mutant strain. Examples of the Bacillus subtilis mutant strain include a strain in which aprX and a gene selected from aprE, nprB, nprE, bpr, vpr, mpr, epr, and wprA are deleted (Japanese Patent Laid-Open No. 2006-174707).

[0051] For the introduction of polynucleotides and vectors into host cells, well-known transformation techniques such as the calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method, etc. can be applied. For example, methods applicable to Bacillus subtilis or its mutants include the competent cell transformation method (J Bacteriol, 1967, 93: 1925-1937), electroporation method (FEMS Microbiol Lett, 1990, 55: 135-138), protoplast transformation method (Mol Gen Genet, 1979, 168: 111-115), Tris-PEG method (J Bacteriol, 1983, 156: 1130-1134), etc.

[0052] If the transformant is cultured in an appropriate medium, the protease precursor can be expressed. The expressed protease precursor becomes the mature enzyme of the protease through folding and cleavage of the pro sequence. Furthermore, when the protease precursor is a preproprotein having a signal sequence, the mature enzyme is secreted and produced extracellularly.

[0053] The cultivation of the transformant for protease production can be carried out according to the general methods in the art. For example, when the transformant is Bacillus subtilis or its mutant strain, the medium for its cultivation contains a carbon source necessary for the growth of Bacillus subtilis, and an inorganic nitrogen source or an organic nitrogen source. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol, etc. Examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. If necessary, the medium may contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, erythromycin, etc.). The culture conditions, such as temperature, aeration and agitation conditions, pH of the medium, and culture time, etc., can be appropriately selected according to the species and traits of the microorganism, the culture scale, etc.

[0054] After cultivation, the mature enzyme of protease is recovered from the obtained culture by conventional methods. For example, the culture is recovered, and if necessary, cell disruption treatment such as ultrasonic wave or pressurization is carried out, and the mature enzyme can be recovered from the culture by appropriately combining filtration, centrifugation, ultrafiltration, salting out, dialysis, chromatography, etc. When the protease precursor has a signal sequence and the mature enzyme is secreted and produced extracellularly, the mature enzyme of protease 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 its crude separation and purification product can be obtained as a composition containing the mature enzyme.

[0055] As shown in the following examples, when the protease of the present invention is used in the purification step of producing a biopolymer by a microorganism having the ability to produce a biopolymer, compared with alkaline protease and Esperase (both from Novozymes), which are proteases commonly used in the purification step, and in addition, when the protease of the present invention is a protease mutant, it has a higher residue reduction effect compared with the parent protease, can efficiently decompose the components derived from the cells of microorganisms other than the biopolymer, and can greatly contribute to improving the purity of the biopolymer. In addition, since the required amount of enzyme is small, it also leads to cost reduction in the method for producing a biopolymer. Therefore, the protease of the present invention is useful as an enzyme for purifying a biopolymer, particularly as an enzyme for purifying a biopolymer used in the purification step of a method for producing a biopolymer by a microorganism having the ability to produce a biopolymer, and can be an active ingredient of an enzyme composition for purifying a biopolymer, particularly an enzyme composition for purifying a biopolymer used in the purification step of a method for producing a biopolymer by a microorganism having the ability to produce a biopolymer.

[0056] In one aspect, the present invention provides an enzyme composition for purifying a biopolymer 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 be appropriately blended with a surfactant, a chelating agent, a water-soluble polymer, an alkaline agent, an organic acid or its salt, an enzyme other than the protease of the present invention, an enzyme stabilizer, an antioxidant, a solubilizing agent, a pH adjuster, a buffer, a preservative, a fragrance, etc.

[0057] 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 at which the protease of the present invention exhibits 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 1 kg of the enzyme composition.

[0058] The protease of the present invention is used for purifying a biopolymer in a method for producing a biopolymer by a microorganism having the ability to produce a biopolymer, either alone or in the form of an enzyme composition containing the same. Therefore, in another aspect, the present invention provides a method for producing a biopolymer by a microorganism having the ability to produce a biopolymer, characterized in that the protease of the present invention is used in the purification step. Further, in 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 by a microorganism having the ability to produce a biopolymer. Hereinafter, these methods are collectively referred to as "the method of the present invention".

[0059] The "microorganism having the ability to produce a biopolymer" in the method of the present invention is a microorganism having the ability to fermentatively produce a biopolymer and accumulate it in the cells. The microorganism is not particularly limited, and may be either a wild-type microorganism or a mutant microorganism (mutant) in which mutations such as insertion, substitution, or deletion of a nucleotide sequence have occurred by various genetic manipulations, or a genetically modified microorganism to which a desired biopolymer production ability has been imparted by known artificial modifications.

[0060] The method for producing a biopolymer by a microorganism having the ability to produce a biopolymer is a method for fermentatively producing a biopolymer by a microorganism having the ability to produce a biopolymer, and includes steps generally performed in the art, typically, a step of culturing a microorganism having the ability to produce 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 step of purifying the biopolymer.

[0061] The "biopolymer" produced by the method for producing a biopolymer by a microorganism having the ability to produce a biopolymer in the method of the present invention refers to a polymer produced by fermentation by a microorganism and accumulated in the cells among polymers produced using biomass as a raw material. Examples of such biopolymers include polyhydroxyalkanoate (PHA).

[0062] PHA is a polyester with hydroxyalkanoic acid as the monomer unit, which is produced by microorganisms having the ability to produce PHA using saccharides, alcohols, fatty acids, oils and fats, etc. as carbon sources and accumulated in the microbial cells. Examples of hydroxyalkanoic acids include (R)-3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), 3-hydroxypropionic acid (3HP), (R)-3-hydroxyvaleric acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), (R)-3-hydroxyoctanoic acid (3HO), (R)-3-hydroxydecanoic acid (3HD), (R)-3-hydroxydodecanoic acid (3HDD), (R)-3-hydroxytetradecanoic acid (3HTD), etc. PHA may be a homopolymer composed of one kind of monomer unit or a copolymer composed of two or more kinds of monomer units.Examples of PHA include poly[(R)-3-hydroxybutyric acid] {P(3HB)}, poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid] {P(3HB-co-3HV)}, poly[(R)-3-hydroxybutyric acid-co-(R)-4-hydroxybutyric acid] {P(3HB-co-4HB)}, poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyhexanoic acid] {P(3HB-co-3HHx)}, poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxydecanoic] {P(3HB-co-3HD)}, and the like. The composition ratio of monomer units in the copolymer is not particularly limited, and PHA with various composition ratios can be obtained according to differences such as the type of microorganism having PHA production ability used, the type of carbon source, and the culture method.

[0063] As the microorganism having PHA production ability, it may be a wild-type microorganism originally having PHA production ability, a mutant microorganism whose PHA production ability is modified such as by modifying the gene encoding PHA polymerase, or a genetically modified microorganism whose PHA production ability is imparted such as by introducing a gene encoding PHA polymerase from the outside. Examples of such microorganisms include microorganisms belonging to the genus Aeromonas, Bacillus, Cupriavidus, Escherichia, Pseudomonas, etc. Among them, from the viewpoint of PHA productivity, microorganisms belonging to the genus 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 (former name Ralstonia eutropha), Cupriavidus metallidurans, etc., and Cupriavidus necator is preferred among them.

[0064] The culturing step of the microorganism having biopolymer production ability is a step of fermentatively producing a biopolymer by the microorganism. This step can be carried out under general conditions employed for the production of biopolymers by microorganisms having biopolymer production ability. For example, the culture medium for culturing may be either a synthetic medium or a natural medium as long as it contains nutrients necessary for the production of biopolymers by microorganisms having biopolymer production ability, such as a carbon source, a nitrogen source, and inorganic salts.

[0065] As the carbon source, any carbon source that can be assimilated by microorganisms having the ability to produce biopolymers may be used, such as sugars like glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolyzates, molasses; alcohols like ethanol; fatty acids such as dodecanoic acid (lauric acid), tridecanoic acid, tetradecanoic acid (myristic acid), pentadecanoic acid (pentadecyl acid), hexadecanoic acid (palmitic acid), hexadecenoic acid, heptadecanoic acid (margaric acid), octadecanoic acid (stearic acid), octadecenoic acid, octadecadienoic acid, octadecatrienoic acid, nonadecanoic acid, eicosanoic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid; and oils and fats such as coconut oil, palm oil, palm kernel oil, olive oil, rapeseed oil, rice bran oil, soybean oil, castor oil, and macadamia oil. These carbon sources can be used alone or in combination of two or more, and can be added to the medium by any method such as all at once, divided addition, or continuous addition.

[0066] As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolyzate can be mentioned.

[0067] Examples of inorganic salts include disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron(III) chloride, calcium chloride, cobalt chloride, copper sulfate, nickel chloride, etc. Furthermore, vitamins and the like can be added to the medium as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.

[0068] Cultivation is preferably carried out under aerobic conditions, and general methods such as aerated agitation cultivation and shaking cultivation can be applied. Also, any cultivation format such as batch cultivation, semi-batch cultivation, and continuous cultivation can be selected. The cultivation temperature is preferably 10 - 50°C, more preferably 20 - 42°C, and even more preferably 25 - 35°C. The initial pH (30°C) of the medium is preferably 6 - 9, and more preferably 7 - 8. The cultivation time is preferably 24 - 200 hours, and more preferably 50 - 100 hours.

[0069] The microorganism having the ability to produce biopolymer in the culture after cultivation contains the biopolymer produced in the cells. The culture is subjected to a biopolymer purification step, but before that, the microorganism having the ability to produce biopolymer may be inactivated as needed. Inactivation of the microorganism having the ability to produce biopolymer can be carried out, for example, by heat-treating the microorganism at about 50 - 80°C for about 10 - 60 minutes. Also, before the biopolymer purification step, the culture medium components may be removed as needed to recover the microorganism having the ability to produce biopolymer.

[0070] The biopolymer purification step is a step of decomposing the cell-derived components other than the biopolymer of the microorganism having the ability to produce biopolymer containing the biopolymer (hereinafter referred to as "biopolymer-containing microorganism"), separating the biopolymer from the biopolymer-containing microorganism, and increasing the purity of the biopolymer. This step is carried out using the protease of the present invention. The cell-derived components other than the biopolymer are cell walls, proteins, sugars, nucleic acids, lipids, etc. derived from the cells of the biopolymer-containing microorganism other than the biopolymer, and are impurities that are desired to reduce contamination of the biopolymer.

[0071] In a preferred embodiment, the protease of the present invention is allowed to act on a biopolymer-containing microorganism or its disrupted cells. As a means for allowing the protease of the present invention to act on the biopolymer-containing microorganism or its disrupted cells, any means known in the art may be used, and it is preferable to add the protease of the present invention to a suspension containing the biopolymer-containing microorganism or its disrupted cells. The reaction conditions between the biopolymer-containing microorganism or its disrupted cells and the protease of the present invention are not particularly limited as long as the protease of the present invention is not inactivated. Appropriate reaction conditions can be appropriately determined by those skilled in the art according to the type, shape and amount of the microorganism having biopolymer-producing ability, the type, shape and amount of the biopolymer, the type and amount of the protease of the present invention, and the like. Examples of the reaction conditions of the method of the present invention are described below.

[0072] The amount of the protease of the present invention used in the reaction is appropriately determined depending on the type, shape and amount of the microorganism having biopolymer-producing ability, and the like. 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, still more preferably 0.1 to 3% by mass, based on 100% by mass of the mass of the microorganism having biopolymer-producing ability or its disrupted cells in terms of dry mass.

[0073] The pH condition of the reaction is preferably pH 8 or higher, more preferably pH 9 or higher, and preferably pH 13 or lower, more preferably pH 12 or lower, from the viewpoint of improving the purification efficiency of the biopolymer. Also, it is preferably pH 8 to 13, more preferably pH 9 to 12, still more preferably pH 10. The pH can be adjusted by adding an appropriate acid or base.

[0074] The temperature condition of the reaction is preferably 30 to 80°C, more preferably 40 to 70°C, still more preferably 40 to 60°C, from the viewpoint of improving the purification efficiency of the biopolymer.

[0075] The reaction time is preferably 30 minutes to 24 hours, more preferably 1 to 12 hours, still more preferably 1 to 5 hours, from the viewpoint of improving the purification efficiency of the biopolymer.

[0076] From the viewpoint of improving the purification efficiency of the biopolymer, the reaction is preferably carried out in the presence of a surfactant. Examples of the surfactant include one or a combination of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant, with an anionic surfactant being preferred. Examples of the anionic surfactant include sulfate esters of alcohols having 10 to 18 carbon atoms, sulfate esters of alkoxylates of alcohols having 8 to 20 carbon atoms, alkylbenzene sulfonates, paraffin sulfonates, α-olefin sulfonates, internal olefin sulfonates, α-sulfo fatty acid salts, α-sulfo fatty acid alkyl ester salts, or fatty acid salts. In particular, one or more anionic surfactants selected from sulfate esters of alcohols having 10 to 14 carbon atoms in the alkyl chain and linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain are preferred. As the counter ion, alkali metal salts and amines are preferred, and particularly sodium and / or potassium, monoethanolamine, and diethanolamine are preferred. In particular, sodium dodecyl sulfate is preferred.

[0077] In the bio-polymer purification process, in addition to the treatment with the protease of the present invention described above, other treatments such as mechanical treatment, heat treatment, alkali treatment, surfactant treatment, oxidant treatment, etc. may be performed. These other treatments can be carried out under general conditions employed in the production of bio-polymers by microorganisms having bio-polymer production ability. Mechanical treatment means cell disruption treatment by ultrasonic waves, pressurization, etc. Examples of the alkali include sodium hydroxide, sodium sesquicarbonate, sodium hydrogen carbonate, etc. Examples of the surfactant include one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, but an anionic surfactant is preferred. Examples of the anionic surfactant include sulfate esters of alcohols having 10 to 18 carbon atoms, sulfate esters of alkoxylates of alcohols having 8 to 20 carbon atoms, alkylbenzene sulfonates, paraffin sulfonates, α-olefin sulfonates, internal olefin sulfonates, α-sulfo fatty acid salts, α-sulfo fatty acid alkyl ester salts, or fatty acid salts. In particular, one or more anionic surfactants selected from sulfate esters of alcohols having 10 to 14 carbon atoms in the alkyl chain and linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain are preferred, and as the counter ion, alkali metal salts and amines are preferred, and in particular, sodium and / or potassium, monoethanolamine, and diethanolamine are preferred. In particular, sodium dodecyl sulfate is preferred. Examples of the oxidant include hydrogen peroxide, percarbonate, perborate, persulfate, sodium peroxide, ozone, chlorite, chlorate, perchlorate, perchloric acid, etc. These other treatments can be carried out one or more kinds of treatments simultaneously and in parallel with or independently of the treatment with the protease of the present invention. Also, in the bio-polymer purification process, in addition to the treatment with the protease of the present invention described above, treatment with an enzyme other than the protease of the present invention may be performed. These treatments with enzymes other than the protease of the present invention can be carried out under general conditions employed in the production of bio-polymers by microorganisms having bio-polymer production ability.Examples of the enzyme other than the protease of the present invention include enzymes that can hydrolyze components derived from microbial cells other than biopolymers, such as lysozyme, muramidase, glycosidase, cellulase, nuclease, lipase, amylase, cutinase, laccase, and proteases other than the protease of the present invention. The treatment with the enzyme other than the protease of the present invention can be carried out simultaneously or independently with the treatment with the protease of the present invention using one or more enzymes.

[0078] The solid content of the reaction solution obtained after the purification step of the biopolymer contains the biopolymer. Therefore, the biopolymer is recovered by drying the solid content of the reaction solution in the drying step of the biopolymer. This step can be carried out under general conditions employed for the production of biopolymers by microorganisms having the ability to produce 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 a 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. By this step, a powder of the biopolymer can be obtained.

[0079] The biopolymer thus produced has less contamination with residues, which are components derived from microbial cells having the ability to produce biopolymers, and is much higher in purity than biopolymers produced by conventional methods. The purity of the biopolymer can be measured, for example, using as an index the ratio of the dry weight of the residues mixed in the powder of the biopolymer to the weight of the powder of the biopolymer according to the method shown in Example 1(4) described below. The dry weight of the residues mixed in the powder of the biopolymer can be measured by adding chloroform to the powder of the biopolymer to dissolve the biopolymer, then removing the chloroform, and drying the remaining residues. It can be determined that the higher the purity of the biopolymer powder is, the lower the ratio of the dry weight of the residues is. The biopolymer produced by the method of the present invention can preferably have a ratio of the dry weight of the residues of 20% or less, more preferably 18% or less.

[0080] As an exemplary embodiment of the present invention, the following compositions, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[0081] 〔1〕A method for producing a biopolymer by a microorganism having the ability to produce a biopolymer, characterized in that at least one protease selected from the group consisting of the following a) and b) is used in the purification step of the biopolymer. 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, characterized in that at least one protease selected from the group consisting of the above a) and b) is used in the method for producing a biopolymer by a microorganism having the ability to produce a biopolymer. 〔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 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’) It consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and has at least one amino acid residue selected from the group consisting of glutamic acid at the position corresponding to the 9th position, aspartic acid at the position corresponding to the 253rd position, tryptophan at the position corresponding to the 255th position, and glutamic acid at the position corresponding to the 256th position, numbered according to SEQ ID NO: 5, protease 〔4〕The method according to any one of 〔1〕~〔3〕, wherein the protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and has valine at the position corresponding to the 16th position, proline at the position corresponding to the 65th position, alanine at the position corresponding to the 83rd position, isoleucine at the position corresponding to the 273rd position, serine at the position corresponding to the 359th position, alanine at the position corresponding to the 387th position, threonine at the position corresponding to the 132nd position, glycine at the position corresponding to the 166th position, valine at the position corresponding to the 167th position, glutamine at the position corresponding to the 195th position, threonine at the position corresponding to the 294th position, and asparagine at the position corresponding to the 369th position, numbered according to SEQ ID NO: 1 〔5〕The method according to any one of 〔1〕~〔3〕, wherein the protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and has glutamic acid at the position corresponding to the 9th position, aspartic acid at the position corresponding to the 253rd position, tryptophan at the position corresponding to the 255th position, and glutamic acid at the position corresponding to the 256th position, numbered according to SEQ ID NO: 5 〔6〕The method according to any one of 〔1〕~〔5〕, wherein the protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell mass thereof 〔7〕The method according to any one of 〔1〕~〔6〕, wherein the protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell mass thereof under the conditions of pH 8 or higher, preferably pH 9 or higher, and pH 13 or lower, preferably pH 12 or lower, or pH 8~13, preferably pH 9~12, more preferably pH 10 The method according to any one of [1] to [7], wherein the protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell mass thereof in the presence of a surfactant, preferably in the presence of sodium dodecyl sulfate. The method according to any one of [1] to [8], wherein the biopolymer is polyhydroxyalkanoic acid (PHA). The method according to any one of [1] to [9], wherein the microorganism having the ability to produce a biopolymer belongs to the genus Cupriavidus, preferably Cupriavidus necator. The method according to any one of [1] to

[10] , wherein the method for producing a biopolymer by a microorganism having the ability to produce a biopolymer includes a step of culturing the microorganism having the ability to produce a biopolymer, a step of purifying the biopolymer, and a step of drying the biopolymer.

[0082] An enzyme composition for purifying a biopolymer, containing at least one protease selected from the group consisting of the above a) and b). The enzyme composition according to

[12] , wherein the protease is at least one protease selected from the group consisting of the above a') and b'). 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 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. 〔15〕The protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and has glutamic acid at the position corresponding to the 9th position, aspartic acid at the position corresponding to the 253rd position, tryptophan at the position corresponding to the 255th position, and glutamic acid at the position corresponding to the 256th position in the numbering of SEQ ID NO: 5, the enzyme composition according to 〔12〕 or 〔13〕. 〔16〕The enzyme composition according to any one of 〔12〕 to 〔15〕, wherein the biopolymer is PHA. 〔17〕The enzyme composition according to any one of 〔12〕 to 〔16〕, which is used in a method for producing a biopolymer by a microorganism having the ability to produce a biopolymer.

[0083] 〔18〕Use for the production of an enzyme composition for purifying a biopolymer of at least one protease selected from the group consisting of the above a) and b). 〔19〕Use as an enzyme for purifying a biopolymer of at least one protease selected from the group consisting of the above a) and b). 〔20〕The use according to 〔18〕 or 〔19〕, wherein the protease is at least one protease selected from the group consisting of the above a’) and b’). 〔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 with SEQ ID NO: 1 or 4, and has valine at the position corresponding to the 16th position, proline at the position corresponding to the position corresponding to the 65th position, alanine at the position corresponding to the 83rd position, isoleucine at the position corresponding to the 273rd position, serine at the position corresponding to the 359th position, alanine at the position corresponding to the 387th position, threonine at the position corresponding to the 132nd position, glycine at the position corresponding to the 166th position, valine at the position corresponding to the 167th position, glutamine at the position corresponding to the 195th position, threonine at the position corresponding to the 294th position, and asparagine at the position corresponding to the 369th position in the numbering of SEQ ID NO: 1. 〔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 with SEQ ID NO: 5 or 8, and has 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. 〔23〕The use according to any one of 〔18〕 to 〔22〕, wherein the biopolymer is PHA. 〔24〕A method of using at least one protease selected from the group consisting of the above a) and b) 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 the above a’) and b’). 〔26〕The method according to 〔24〕 or 〔25〕, wherein the protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or 4, and has 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. 〔27〕The method according to 〔24〕 or 〔25〕, wherein the protease consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 5 or 8, and has 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. 〔28〕The method according to any one of 〔24〕 to 〔27〕, wherein the biopolymer is PHA.

Example

[0084] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0085] Example 1 (1) Construction of protease expression plasmid For the expression of KP43, the plasmid pHY-KP43 described in JP-A-2021-97605 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 the mature KP43 is shown in SEQ ID NO: 1. For introducing mutations into the protease, a site-directed mutagenesis method by PCR using a complementary primer pair was used (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115-e115.).

[0086] (2) Preparation of protease solution The protease expression plasmid was introduced into a Bacillus subtilis strain by the protoplast method and cultured in a 2×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; % is (w / v)%) at 30°C for 3 days. Then, the culture supernatant containing the protease was recovered by centrifugation. The one subjected to buffer exchange to 20 mM Tris-HCl (pH 7.5) containing 2 mM CaCl2 using Amicon 10K was used as the protease solution. Alcalase (SIGMA, 126741) and Esperase (SIGMA, P5860) were used as reagents as the protease solution. A DC Protein Assay Kit (Bio-Rad) was used for measuring the concentration of the protease solution. BSA Standard Solution (WAKO) was used as the standard solution for calculating the protein amount.

[0087] (3) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) NBRC 102504 strain was inoculated into LB liquid medium and cultured with shaking at 30 °C for 24 hours. 1 mL of this culture solution 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 at 30 °C for 24 hours. After 24 hours, 9 g of coconut oil was added and the culture was continued for another 48 hours. The cell culture solution was heated at 60 °C for 20 minutes to inactivate the cells. The inactivated culture solution was dispensed into 50 mL tubes at 15 mL each and centrifuged at 8000 rpm for 10 minutes to obtain pellets of PHA-containing cells.

[0088] (4) PHA Recovery Using Protease 1 An aqueous solution of 1 mM sodium hydroxide (pH 11) containing 0.2% by weight of sodium dodecyl sulfate was added in 15 mL portions per tube containing the pellet obtained in (3) and suspended, and the mixture was incubated at 50 °C for 1 hour. The cells were disrupted using a sonicator Bioruptor II (Sonic Bio Co., Ltd.) (output: High, 10 cycles of 30-second disruption and 30-second pause). Distilled water was added to the disrupted solution in 15 mL portions, and after centrifugation at 10,000 rpm for 10 minutes, the supernatant was discarded in 15 mL portions. An aqueous solution of 1 mM sodium hydroxide (pH 11) was added in 15 mL portions, and after centrifugation at 10,000 rpm for 20 minutes, the supernatant was removed. Again, an aqueous solution of 1 mM sodium hydroxide (pH 11) was added in 15 mL portions, and after centrifugation at 10,000 rpm for 20 minutes, the supernatant was removed. The pellet was suspended in 15 mL of an aqueous solution of sodium dodecyl sulfate with a final concentration of 0.2% by weight adjusted to pH 10 by adding sodium hydroxide. 200 μg of each protease was added thereto and mixed well. After reacting at 50 °C for 2 hours with shaking and stirring, centrifugation was performed at 10,000 rpm for 20 minutes, and the obtained pellet was washed 3 times with an aqueous solution of 1 mM sodium hydroxide (pH 11). The obtained pellet was freeze-dried to obtain a powder containing PHA. The dried powder obtained above was put into a glass test tube whose weight had been measured, and the weight was measured. 2 mL of chloroform was added thereto in portions and stirred well. The test tube was centrifuged to remove the chloroform. Again, 1 mL of chloroform was added in portions, stirred well, then centrifuged to remove the chloroform. Again, 2 mL of chloroform was added in portions, stirred well, then centrifuged to remove the chloroform. After drying the residue remaining in the test tube, the weight was measured, and the ratio of the weight of the residue to the weight of the powder before PHA dissolution was determined (Table 1). Since PHA is soluble in chloroform, this residue is an impurity derived from the cells. The KP43 wild type and the KP43 mutant S16V+T65P+N83A+V273I+T359S+S387A+A132T+N166G+G167V+Y195Q+A294T+D369N had a greater residue reduction effect compared to alkaline protease and esperase. In particular, the KP43 mutant S16V+T65P+N83A+V273I+T359S+S387A+A132T+N166G+G167V+Y195Q+A294T+D369N significantly reduced the residue even compared to the KP43 wild type.

[0089]

Table 1

[0090] (5) PHA recovery using protease 2 PHA recovery was performed in the same manner as in (4) except that sonication was not performed, and the residue amount 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 residue compared to the KP43 wild type.

[0091]

Table 2

[0092] Example 2 (1) Construction of protease expression plasmid Using the plasmid for expressing VHH of SEQ ID NO: 26 containing the promoter derived from the Bacillus subtilis spoVG gene described in International Publication No. 2021 / 153129 as a template, the K16 gene (polynucleotide of SEQ ID NO: 7, encoding the K16 preproprotein of SEQ ID NO: 6) artificially gene-synthesized by seamless cloning was placed on the full-length ORF containing the VHH gene to construct the K16 expression plasmid pHY-K16. The amino acid sequence of the K16 mature form is shown in SEQ ID NO: 5. For the introduction of mutations into the protease, a site-directed mutagenesis method by PCR using a complementary primer pair was used (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115-e115.).

[0093] (2) Preparation of protease solution A protease solution was prepared in the same manner as in Example 1(2).

[0094] (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 using alkaline protease was determined (Table 3). A positive value indicates that the residue reduction effect is greater than that of alkaline protease. The K16 wild type had a greater residue reduction effect compared to alkaline protease, and furthermore, the mutants S9E, L256E, and S9E+N253D+N255W+L256E had a greater residue reduction effect compared to the K16 wild type.

[0095]

Table 3

Claims

1. A method for producing a biopolymer using a microorganism having the ability to produce a biopolymer, characterized in that at least one protease selected from the group consisting of the following a) and b) is used in the purification step of the biopolymer. 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, characterized in that at least one protease selected from the group consisting of the following a) and b) is used in the method for producing a biopolymer using a microorganism having the ability to produce a biopolymer. 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 claim 1 or 2, wherein a protease is allowed to act on a microorganism containing a biopolymer or a disrupted cell mass thereof.

5. The method according to claim 1 or 2, wherein the biopolymer is polyhydroxyalkanoic acid (PHA).

6. The method according to claim 1 or 2, 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, 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

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

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