Methods for producing biopolymers

Abstract

To provide an efficient biopolymer production method for improving the purity of biopolymers when producing biopolymers using microorganisms capable of biopolymer production. [Solution] A method for producing a biopolymer, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of a microorganism capable of producing biopolymers or a treated product thereof under conditions of pH greater than 7.0.

Description

[Technical Field]

[0001] This invention relates to a method for producing biopolymers. [Background technology]

[0002] Biopolymers, manufactured using biomass as a raw material, are being used in a variety of applications as an alternative to petrochemical plastics. Many biopolymers are highly biodegradable, and their demand has been expanding in recent years from an environmental protection perspective. Representative biopolymers include starch, polylactic acid (PLA), and polyhydroxyalkanoic acid (PHA).

[0003] In the industrial production of biopolymers produced by microbial fermentation, including PHA, it is necessary to purify the biopolymer from within the production microbial cells cultured at high density. For example, in biopolymer production, chicken egg white lysozyme is used to decompose peptidoglycans derived from production bacteria and improve the purity of the biopolymer (Patent Documents 1 and 2). In the biopolymer production process, it is preferable to adjust the pH of the treatment solution to the alkaline side in order to improve the efficiency of alkaline protease treatment, which inactivates cells and reduces unwanted proteins (Patent Documents 1 and 2). However, chicken egg white lysozyme treatment is often performed at a pH close to neutral (Patent Documents 1 and 2), which necessitates adjusting the pH at each step, making the production process complicated. Therefore, in order to improve the efficiency of biopolymer production, there is a need for lysozyme that maintains its biopolymer purity-improving effect even under alkaline conditions.

[0004] Lysozyme is classified into families such as GH22, GH23, GH24, and GH25 in the glycoside hydrolase (GH) family classification. Chicken egg white lysozyme is a lysozyme classified into the GH22 family. On the other hand, GH25 family lysozyme is a lysozyme classified into the GH25 family in the GH family classification. The lysozyme acm derived from Streptomyces globisporus, which is a GH25 family lysozyme, has a reported optimal pH of 5.3 (Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention relates to providing an efficient method for producing a biopolymer in improving the purity of the biopolymer when producing the biopolymer by a microorganism having the ability to produce a biopolymer.

Means for Solving the Problems

[0008] The inventors searched for lysozyme and found that when GH25 family lysozyme is used for the purification of biopolymers in the production of biopolymers by microorganisms having biopolymer-producing ability, unexpectedly, it can effectively decompose microbial-derived peptidoglycan even under alkaline conditions above pH 7.0, and maintains the effect of improving the purity of biopolymers. Further, it has been found that by using GH25 family lysozyme, the production process of biopolymers can be simplified by combining the lysozyme treatment and the treatment carried out under alkaline conditions.

[0009] The present invention relates to a method for producing a biopolymer, which includes a GH25 family lysozyme treatment step of allowing a GH25 family lysozyme to act on a cultured cell body of a microorganism having biopolymer-producing ability or a processed product thereof under conditions above pH 7.0. The present invention also relates to a method for purifying a biopolymer, which includes a GH25 family lysozyme treatment step of allowing a GH25 family lysozyme to act on a cultured cell body of a microorganism having biopolymer-producing ability or a processed product thereof under conditions above pH 7.0. The present invention also relates to an enzyme composition for biopolymer purification containing a GH25 family lysozyme. The present invention also relates to a method for purifying a target substance, which includes a GH25 family lysozyme treatment step of allowing a GH25 family lysozyme to act on a cultured cell body of a bacterium that produces a target substance in the cell or a processed product thereof under conditions above pH 7.0.

Effects of the Invention

[0010] According to the present invention, by using a GH25 family lysozyme for the purification of biopolymers in the production of biopolymers by microorganisms having biopolymer-producing ability, microbial-derived peptidoglycan can be effectively decomposed even under alkaline conditions. This ultimately leads to the simplification of the production process of biopolymers.

Brief Description of the Drawings

[0011] [Figure 1] A diagram showing the lytic effect on the cells of microorganisms capable of producing PHA after treatment with egg white lysozyme or mutanolicin (pH 9.5). [Figure 2] Profile of GLYCOSYL_HYDROL_F25_2(PS51904) in PROSITE. [Figure 3] Sequence similarity network (SSN) analysis diagram of the GH25 family lysozyme. [Figure 4] The phylogenetic tree of the GH25 Family Resort Team. [Modes for carrying out the invention]

[0012] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0013] In this specification, the identity of amino acid sequences or nucleotide sequences is calculated using the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the homology analysis (Search homology) using the genetic information processing software GENETYX Ver.12 with a Unit size to compare (ktup) of 2.

[0014] In this specification, "at least 60% identity" with respect to an amino acid sequence or nucleotide sequence means identity of 60% or more, preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 91% or more, even more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0015] In this specification, the "corresponding position" on an amino acid sequence or nucleotide sequence can be determined by aligning the target sequence with a reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 1) to give the greatest possible homology. Alignment of an amino acid sequence or nucleotide sequence can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 or Clustal Omega, can be used. Clustal W, Clustal W2, and Clustal omega can be used, 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 DNA Databank 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 alignment is considered to be the "corresponding position" to that arbitrary position.

[0016] Those skilled in the art can further fine-tune the amino acid sequence alignment obtained above to optimize it. Such an optimal alignment is preferably determined by considering factors such as the similarity of the amino acid sequences and the frequency of inserted gaps. Here, 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, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues among the 20 amino acids that make up a protein that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups consisting of such 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; leucine and isoleucine, respectively.

[0017] In this specification, "amino acid residue" means 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).

[0018] In this specification, "operable linkage" between a regulatory region and a gene means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0019] In this specification, "upstream" and "downstream" with respect to a gene refer to the upstream and downstream regions 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 in the DNA sense strand, and "upstream of a gene" means the 5' region of the gene in the DNA sense strand.

[0020] In this specification, "protein expression" means the synthesis of a protein based on the information of a protein-coding gene. Furthermore, in this specification, "protein production" means the accumulation of a protein by culturing a microorganism that has a protein-coding gene, and in particular, "extracellular protein production" means the accumulation of a protein outside the cell of a microorganism by culturing a microorganism that has a protein-coding gene.

[0021] In this specification, "intracellular" refers to the area inside the cytoplasmic membrane in Gram-positive bacteria and the area inside the outer membrane in Gram-negative bacteria, while "extracellular" refers to the area outside the cytoplasmic membrane in Gram-positive bacteria and the area outside the outer membrane in Gram-negative bacteria.

[0022] In this specification, "protein secretion" refers to the passage of a protein through a lipid bilayer. Furthermore, in this specification, "secretion signal" refers to a peptide that has the function of causing the secretion of a desired protein.

[0023] In this specification, a "clade" is a group of proteins that are clustered together based on homologous sequence features that trace back to a common ancestor, and consists of a common ancestor and all of its direct descendants. Generally, proteins belonging to the same clade have common structural and functional characteristics.

[0024] The present invention provides a method for producing biopolymers. The method is a method for producing biopolymers using microorganisms capable of producing biopolymers, and includes a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of microorganisms capable of producing biopolymers or a treated product thereof under conditions of pH greater than 7.0. Hereinafter, the method for producing biopolymers of the present invention will be simply referred to as the method of the present invention.

[0025] The "microorganism having biopolymer production ability" of the present invention refers to a microorganism that has the ability to ferment and produce biopolymers and accumulate them within its cells. The microorganism is not particularly limited and may be a wild-type microorganism or a mutant microorganism (mutant) in which mutations such as insertion, substitution, or deletion of base sequences have occurred through various genetic manipulations, or a genetically modified microorganism to which a desired biopolymer production ability has been conferred by known artificial modifications.

[0026] The "biopolymer" produced by the microorganisms having biopolymer production ability of the present invention refers to a polymer produced from biomass as a raw material, which is fermented by microorganisms and accumulated within the microbial cells. A preferred example of a biopolymer is a hydroxyalkanoate-containing polymer. A hydroxyalkanoate-containing polymer is a polymer that contains hydroxyalkanoate as a monomer unit. Examples of hydroxyalkanoate-containing polymers include polyhydroxyalkanoate (PHA), which is a polyester containing only hydroxyalkanoate as a monomer unit, and polyesteramides, which further contain carboxylic acids having amino groups such as amino acids in addition to hydroxyalkanoate as monomer units, and have ester bonds and amide bonds. The proportion of hydroxyalkanoate in the monomer units constituting the hydroxyalkanoate-containing polymer is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 100%. A hydroxyalkanoic acid-containing polymer in which hydroxyalkanoic acid accounts for 100% of the monomer units constituting the polymer is called PHA.

[0027] PHA is a polyester with hydroxyalkanoic acid as its monomer unit. It is produced by microorganisms capable of PHA production using sugars, alcohols, fatty acids, and oils as carbon sources, and accumulates within the cells of these microorganisms. Hydroxyalkanoic acids are not particularly limited, but examples include (R)-3-hydroxybutanoic acid (3HB), 4-hydroxybutanoic acid (4HB), 3-hydroxypropionic acid (3HP), (R)-3-hydroxypentanoic acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), (R)-3-hydroxyoctanoic acid (3HO), (R)-3-hydroxydecanoic acid (3HD), and (R)-3-hydroxydodecanoic acid (3HHx). Examples include (R)-3-hydroxytetradecanoic acid ((R)-3-hydroxytetradecanoic acid: 3HTD). PHA may be a homopolymer composed of one type of monomer unit, or a copolymer composed of two or more types of monomer units.PHAs include poly[(R)-3-hydroxybutyric acid]:P(3HB), poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxypentanoic acid]:P(3HB-co-3HV), poly[(R)-3-hydroxybutyric acid-co-(R)-4-hydroxybutyric acid]:P(3HB-co-4HB), and poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyhexanoic acid]:P(3HB-co-4HB). Examples include {(R)-3-hydroxybutyric acid-co-(R)-3-hydroxydecanoic acid}:P(3HB-co-3HD)} and {(R)-3-hydroxybutyric acid-co-(R)-3-hydroxydecanoic acid}:P(3HB-co-3HD)}. The composition ratio of monomer units in the copolymer is not particularly limited, and it is possible to obtain PHA with various composition ratios depending on the type of microorganism capable of producing PHA used, the type of carbon source, the culture method, etc.

[0028] Microorganisms capable of producing PHA may be wild-type microorganisms that inherently possess PHA-producing ability, mutant microorganisms whose PHA-producing ability has been modified by altering the gene encoding PHA polymerase, or genetically modified microorganisms into which PHA-producing ability has been conferred by introducing a gene encoding PHA polymerase from an external source. Examples of such microorganisms include those 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.

[0029] Cultured cells of microorganisms capable of producing biopolymers can be obtained by culturing the microorganisms. Therefore, the method of the present invention may further include a culture step of culturing microorganisms capable of producing biopolymers. Biopolymers are produced within the cells of microorganisms capable of producing biopolymers. The produced biopolymers usually exist in the form of biopolymer granules. Examples of cultured cells of microorganisms capable of producing biopolymers include the culture solution of the microorganisms during and after cultivation, cells isolated from the culture of the microorganisms, and suspensions of the isolated cells. A processed product of cultured cells of microorganisms capable of producing biopolymers refers to a processed product in which the cultured cells of the microorganisms are subjected to a treatment that decomposes and / or removes at least a portion of the cell-derived components. Examples of processed products of cultured cells of microorganisms capable of producing biopolymers include a processed product in which the cultured cells of the microorganisms are subjected to at least one treatment selected from the following: heat treatment, physical crushing treatment, alkaline treatment, enzyme treatment, surfactant treatment, oxidizing agent treatment, centrifugation, membrane filtration, and washing. The treated product of cultured cells of microorganisms capable of producing biopolymers may contain not only biopolymers but also components derived from the microorganisms themselves (e.g., cell walls).

[0030] The cultivation of microorganisms capable of producing biopolymers can be carried out under the general conditions used for the production of biopolymers by microorganisms capable of producing biopolymers. For example, the culture medium can be either a synthetic medium or a natural medium, as long as it contains the nutrients necessary for the production of biopolymers by microorganisms capable of producing biopolymers, such as a carbon source, a nitrogen source, and inorganic salts.

[0031] As a carbon source, any carbon source that can be utilized by microorganisms capable of producing biopolymers is acceptable, including 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, octadecantridecaenoic acid, nonadecanoic acid, eicosanoic acid, eicosadienoic acid, eicosatrienoic acid, and 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 mahua oil. These carbon sources can be used individually or in combination of two or more, and can be added to the culture medium by any method, such as all at once, in divided additions, or continuously.

[0032] Nitrogen sources include nitrogen compounds such as ammonia, ammonium sulfate, ammonium chloride, ammonium heptamolybdate, ammonium salts and amines, peptone, and natural nitrogen sources such as soy hydrolysates.

[0033] 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, zinc sulfate, iron sulfate, sodium tetraborate, and manganese sulfate. Furthermore, vitamins and other substances can be added to the culture medium as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0034] Culturing is preferably carried out under aerobic conditions, and common methods such as aerated stirring culture and shaking culture can be applied. In addition, any culture format can be selected from batch culture, semi-batch culture, and continuous culture. 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 culture medium (at 30°C) is preferably 6 to 9, more preferably 7 to 8. The culture time is preferably 24 to 200 hours, and more preferably 50 to 100 hours.

[0035] In the GH25 family lysozyme treatment process, GH25 family lysozyme is applied to cultured cells of biopolymer-producing microorganisms or their treated products, thereby degrading the peptidoglycan that constitutes the cell wall of biopolymer-producing microorganisms, even under alkaline conditions with a pH above 7.0. In other words, it can be lysed. The peptidoglycan degradation products can be further degraded by enzymatic treatment or other means as needed, and then removed when recovering the biopolymer. Furthermore, the degradation of peptidoglycan facilitates the removal of cell-derived components other than peptidoglycan from biopolymer-producing microorganisms. Therefore, the GH25 family lysozyme treatment process can greatly contribute to improving the purity of the final biopolymer. Thus, the GH25 family lysozyme treatment process is a biopolymer purification process that degrades cell-derived components other than the biopolymer from biopolymer-producing microorganisms containing biopolymers, thereby separating the biopolymer and increasing its purity.

[0036] Here, "lysozyme" refers to an enzyme also called a lytic enzyme, which possesses β-1,4-N-acetylmuramidase activity that cleaves the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan sugar chain backbone that makes up the cell wall of bacteria. "Lysozyme activity" refers to the β-1,4-N-acetylmuramidase activity that cleaves the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. Lysozyme activity is usually measured by the decrease in turbidity of the bacterial suspension accompanying lysis, using bacterial cells such as Micrococcus luteus as a substrate. In the Carbohydrate-Active Enzymes database (CAZy, www.cazy.org) (Elodie Drula et al. Nucleic Acids Res. 2022, 50: D571-D577), lysozymes are classified into families such as GH22, GH23, GH24, and GH25. Chicken egg white lysozyme, which is widely used in industry, is classified into the GH22 family.

[0037] "GH25 family lysozyme" refers to a polypeptide that satisfies the following conditions: it is classified as belonging to the GH25 family in the GH family classification of the CAZy database, and / or contains an amino acid sequence (GH25 domain) predicted as GLYCOSYL_HYDROL_F25_2(PS51904) by the ScanProsite tool of PROSITE (https: / / prosite.expasy.org / ) (Christian JA Sigrist et al. Nucleic Acids Res. 2013, 41: D344-D347, Epub 2012). A GH25 family lysozyme may preferably be a polypeptide classified as belonging to the GH25 family in the GH family classification of the CAZy database, or preferably a polypeptide containing a GH25 domain, and more preferably a polypeptide containing a GH25 domain. Whether or not any polypeptide possesses a GH25 domain can be determined using the PROSITE scanning tool ps_scan program ver 1.90 (available from, for example, https: / / ftp.expasy.org / databases / prosite / ps_scan / , https: / / github.com / sib-swiss / pftools3, etc.) and the PS51904 profile (obtained from https: / / prosite.expasy.org / PS51904 on February 2, 2025, Figure 2). The GH25 family is a family composed solely of lysozyme and is widely distributed in bacteria, viruses, and eukaryotes. The GH25 family is structurally unrelated to the GH22, GH23, and GH24 families and adopts a characteristic β / α barrel structure. Furthermore, lysozymes are known to be classified into four groups from a functional standpoint, represented by chicken egg white lysozyme (HEWL), goose egg white lysozyme (GEWL), bacteriophage T4 lysozyme (T4L), and Chalaropsis lysozyme. The GH25 family lysozyme of the present invention is preferably a Chalaropsis (CH) type lysozyme.Unlike chicken egg white lysozyme, several CH-type lysozymes exhibit activity in both β-1,4-N-acetylmuramidase and β-1,4-N,6-O-diacetylmuramidase, and have been reported to be able to degrade 6-O-acetylated peptidoglycans present in some bacteria. Sequences classified as the GH25 family are evolutionarily related, and several functionally important amino acid residues are conserved. For example, the amino acid residues of the active site of GH25 family lysozymes, as defined by accession No. cd00599 in the preserved domain database (Wang J. et al., Nucleic Acids Res. 2023, 51(D1): D384-D388), are located at positions 9, 33, 62, 64, 98, 100, 138, 161, 183, and 198 in Sequence ID No. 1. In Sequence ID No. 1, position 9 is D, position 33 is K, position 62 is Y, position 64 is F, position 98 is D, position 100 is E, position 138 is Y, position 161 is W, position 183 is Q, and position 198 is D. Alternatively, the amino acid residues of the active site of CH type lysozyme in the GH25 family, as defined by accession No. cd06312 in the preserved domain database, are the amino acid residues at positions 9, 33, 36, 62, 64, 98, 100, 138, 161, 183, and 198 in the numbering of Sequence ID No. 1. In Sequence ID No. 1, position 9 is D, position 33 is K, position 36 is E, position 62 is Y, position 64 is F, position 98 is D, position 100 is E, position 138 is Y, position 161 is W, position 183 is Q, and position 198 is D. In particular, the amino acid residues at positions 9, 98, and 100 in the numbering of Sequence ID No. 1 (position 9 is D, position 98 is D, and position 100 is E) are highly conserved in the GH25 family lysozyme.In a preferred embodiment of the present invention, the GH25 family lysozyme has D at the position corresponding to the 9th position in the numbering of SEQ ID NO: 1, D at the position corresponding to the 98th position, and E at the position corresponding to the 100th position; more preferably, it has D at the position corresponding to the 9th position in the numbering of SEQ ID NO: 1, Y at the position corresponding to the 62nd position, D at the position corresponding to the 98th position, and E at the position corresponding to the 100th position; more preferably, it has D at the position corresponding to the 9th position in the numbering of SEQ ID NO: 1, Y at the position corresponding to the 62nd position, D at the position corresponding to the 98th position, E at the position corresponding to the 100th position, Y at the position corresponding to the 138th position, and Q at the position corresponding to the 183rd position; still more preferably, it has D at the position corresponding to the 9th position in the numbering of SEQ ID NO: 1, K at the position corresponding to the 33rd position, E at the position corresponding to the 36th position, Y at the position corresponding to the 62nd position, D at the position corresponding to the 98th position, E at the position corresponding to the 100th position, Y at the position corresponding to the 138th position, and Q at the position corresponding to the 183rd position. In another preferred embodiment of the present invention, the GH25 family lysozyme has DX 1 EX 2 NP(X 3 ) n G motif (SEQ ID NO: 117) in the region corresponding to positions 98 to 105 in the numbering of SEQ ID NO: 1. In the motif, D and E correspond to D at the 98th position and E at the 100th position which are the active centers in SEQ ID NO: 1, and G corresponds to G at the 105th position in the numbering of SEQ ID NO: 1. Here, X 1 , X 2 , and X 3 each independently represent an arbitrary amino acid residue. n represents an integer, which is 1 or more and 5 or less, preferably 3 or less, and more preferably 1. When n is 1, that is, when the motif is DX 1 EX 2 NPX 3 G (SEQ ID NO: 118), X 3 is preferably S, N or Y, and the motif is DX 1 EX 2It is represented as NP[S / N / Y]G (Sequence ID 119). In a more preferred embodiment of the present invention, the GH25 family lysozyme has D at position 9, K at position 33, E at position 36, Y at position 62, D at position 98, E at position 100, Y at position 138, and Q at position 183, and DX at positions 98 to 105 in the Sequence ID numbering. 1 EX 2 It has an NP[S / N / Y]G motif.

[0038] The GH25 family lysozyme used in the present invention is not particularly limited, but is preferably a GH25 family lysozyme derived from bacteria or eukaryotes, more preferably a GH25 family lysozyme derived from bacteria or fungi, and even more preferably a GH25 family lysozyme derived from bacteria. Preferred examples of GH25 family lysozymes derived from bacteria of the genus Streptomyces, GH25 family lysozyme derived from bacteria of the genus Actinokineospora, GH25 family lysozyme derived from bacteria of the genus Actinoallomurus, GH25 family lysozyme derived from bacteria of the genus Spelaeicoccus, GH25 family lysozyme derived from bacteria of the genus Janibacter, GH25 family lysozyme derived from bacteria of the genus Actinopolyspora, GH25 family lysozyme derived from bacteria of the genus Kibdelosporangium, GH25 family lysozyme derived from bacteria of the genus Umezawaea, and Sacc Examples include GH25 family lysozymes derived from bacteria of the genus *Harothrix*, *Peterkaempfera*, *Wenjunlia*, *Allokutzneria*, *Kribbellaceae*, *Propionibacteriaceae*, *Mycobacteriales*, and *Pseudonocardiaceae*, with a more preferred example being *Streptomyces*.Polypeptides having lysozyme activity include those comprising either the amino acid sequence of the GH25 domain of acm, a GH25 family lysozyme derived from globisporus (SEQ ID NO: 57), or the amino acid sequences of the GH25 domain of GH25 family lysozymes derived from bacteria listed in Table 1 below (SEQ ID NOs: 5-41, 58). More preferably, polypeptides comprising acm (SEQ ID NO: 1) and the amino acid sequences of putative mature proteins of GH25 family lysozymes derived from bacteria listed in Table 1 below (SEQ ID NOs: 59-96) are included. Even more preferably, polypeptides comprising the amino acid sequence shown in any of SEQ ID NOs: 1, 59-63, 66-79, 81-83, and 88-96 are included. Even more preferably, polypeptides comprising the amino acid sequence shown in any of SEQ ID NOs: 1, 59, 78, 89, and 91 are included. Preferred examples of fungal GH25 family lysozymes include GH25 family lysozymes derived from Aspergillus species and GH25 family lysozymes derived from Acremonium species, with more preferred examples being GH25 family lysozyme derived from Aspergillus fumigatus (Uniptot id: A4DA29, SEQ ID NO: 2) and GH25 family lysozyme derived from Acremonium alcalophilum as described in WO2023 / 110957A1 (SEQ ID NO: 3 or 4).

[0039] [Table 1]

[0040] The GH25 family lysozyme used in the present invention may be a polypeptide having equivalent functionality to the GH25 family lysozyme derived from bacteria or fungi described above. Preferred examples of polypeptides having equivalent functionality to bacterial GH25 family lysozyme include polypeptides having an amino acid sequence that is at least 60% identical to the amino acid sequence of the bacterial GH25 family lysozyme described above and having lysozyme activity. More preferred examples include polypeptides having an amino acid sequence that is at least 60% identical to the amino acid sequence shown in any of SEQ ID NOs. 5-41, 57, and 58 and having lysozyme activity. Even more preferred examples include polypeptides having an amino acid sequence that is at least 60% identical to the amino acid sequence shown in any of SEQ ID NOs. 1 and 59-96. A polypeptide comprising an amino acid sequence having at least 60% identity with the sequence and possessing lysozyme activity is an example; a more preferred example is a polypeptide comprising an amino acid sequence having at least 60% identity with the amino acid sequence shown in any of SEQ ID NOs: 1, 59-63, 66-79, 81-83, and 88-96 and possessing lysozyme activity; a further preferred example is a polypeptide comprising an amino acid sequence having at least 60% identity with the amino acid sequence shown in any of SEQ ID NOs: 1, 59, 78, 89, and 91 and possessing lysozyme activity. A preferred example of a polypeptide having equivalent function to fungal GH25 family lysozyme is a polypeptide comprising an amino acid sequence having at least 60% identity with the above fungal GH25 family lysozyme and possessing lysozyme activity; a more preferred example is a polypeptide comprising an amino acid sequence having at least 60% identity with the amino acid sequence shown in any of SEQ ID NOs: 2-4 and possessing lysozyme activity.

[0041] The GH25 family lysozyme used in this invention can be extracted or prepared from microorganisms containing it or from their cultures. For example, acm (SEQ ID NO: 1) can be extracted or prepared from Streptomyces globisporus strain ATCC 21553 or its culture, GH25 family lysozyme derived from Aspergillus fumigatus (SEQ ID NO: 2) can be extracted or prepared from Aspergillus fumigatus or its culture, and GH25 family lysozyme derived from Acremonium alcalophilum (SEQ ID NO: 3 or 4) can be extracted or prepared from Acremonium alcalophilum or its culture. The above microorganisms can be purchased from public microorganism storage institutions. The microorganisms containing the GH25 family lysozyme can be cultured under appropriate conditions using a medium containing assimilated carbon sources, nitrogen sources, metal salts, vitamins, etc. From the microorganisms or culture solution thus obtained, enzymes can be collected and prepared by general methods, and the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization, etc. For example, the recovery and preparation of enzymes from cultures can be carried out using conventional methods such as separation of microorganisms by centrifugation or filtration, precipitation of enzymes in the supernatant or filtrate by adding salts such as ammonium sulfate or organic solvents such as ethanol, concentration and desalting using ultrafiltration membranes, and purification using various chromatography methods such as ion exchange or gel filtration.

[0042] Alternatively, the GH25 family lysozyme used in the present invention may be a commercially available GH25 family lysozyme. Examples of commercially available GH25 family lysozymes include mutanolicin (SIGMA, product number: M9901), which has acm (sequence number 1) derived from Streptomyces globisporus as its main component.

[0043] Alternatively, the GH25 family lysozyme used in the present invention can be produced by chemical synthesis or microbiological methods. In the microbiological production of GH25 family lysozyme, only the mature protein region may be expressed, or it may be expressed as a proprotein of GH25 family lysozyme containing both the propeptide and the mature protein region.

[0044] The target GH25 family lysozyme mature protein or proprotein can be produced by expressing a polypeptide from a polynucleotide encoding the target GH25 family lysozyme mature protein or proprotein. This polynucleotide can be prepared according to conventional methods. For example, it can be prepared by extracting genomic DNA from a microorganism that naturally produces the target GH25 family lysozyme using conventional methods, or by extracting RNA and synthesizing cDNA by reverse transcription. For example, the polynucleotide encoding the acm proprotein, consisting of the amino acid sequence shown in SEQ ID NO: 52, can be prepared from Streptomyces globisporus (ATCC 21553 strain). This microorganism can be purchased from a public microorganism preservation institution.

[0045] The polynucleotides obtained by the above procedure may be further subjected to site-directed mutagenesis to prepare polynucleotides encoding the mature protein or proprotein of the target GH25 family lysozyme. Alternatively, the polynucleotide encoding the mature protein or proprotein of the target GH25 family lysozyme may be chemically synthesized based on the amino acid sequence of the mature protein or proprotein.

[0046] The polynucleotide encoding the mature protein or proprotein may be operably linked to a regulatory region. In this specification, “regulatory region” means a region that has the function of controlling the intracellular expression of a downstream gene, and preferably, constitutively expressing or overexpressing the downstream gene. More specifically, it can be defined as a region located upstream of the coding region of a gene, which has the function of controlling the transcription of the coding region through interaction with RNA polymerase. Preferably, in this specification, the regulatory region refers to a region of approximately 200 to 600 nucleotides upstream of the coding region of a gene. The regulatory region includes a transcription initiation regulatory region and / or a translation initiation regulatory region, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation regulatory region is the region containing the promoter and transcription start site, while the translation initiation regulatory region is the region corresponding to the Shine-Dalgarno (SD) sequence, which, along with the start codon, forms the ribosome binding site (Shine, J., Dalgarno, L., Proc. Natl. Acad. Sci. USA., 1974, 71:1342-1346).

[0047] Preferred examples of such regulatory regions include, but are not limited to, regulatory regions that function in host microorganisms, such as regulatory regions of α-amylase genes, protease genes, aprE genes, or spoVG genes derived from Bacillus bacteria, regulatory regions of the cellulase gene of Bacillus sp. KSM-S237 strain (JP 2000-210081 A), regulatory regions of the cellulase gene of Bacillus sp. KSM-64 strain (JP 2011-10387 A), and regulatory regions of kanamycin resistance genes or chloramphenicol resistance genes derived from Staphylococcus aureus (see JP 2009-089708 A for both).

[0048] Furthermore, the polynucleotide encoding the mature protein or proprotein is preferably operably linked to a sequence encoding a secretory signal (referred to as a secretory signal sequence) from the viewpoint of the extracellular production efficiency of the expressed mature protein or proprotein. Preferred examples of the secretory signal sequence include secretory signal sequences that function in Bacillus bacteria, such as secretory signal sequences derived from Bacillus bacteria. Preferred examples of secretory signal sequences derived from Bacillus bacteria include the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain, the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-64 strain, and the secretory signal sequence of the Bacillus subtilis amylase gene amyE.

[0049] Therefore, the polynucleotide encoding the mature protein or proprotein may include a nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). For example, the polynucleotide may include the secretion signal sequence described above, or it may include the promoter, secretion signal sequence, and terminator described above. Furthermore, the polynucleotide may be codon-optimized to suit the species of host microorganism for the production of the proprotein.

[0050] The introduction of the polynucleotide encoding the mature protein or proprotein into a host microorganism can be carried out according to standard procedures. For example, the polynucleotide encoding the mature protein or proprotein, or a vector containing the same, can be introduced into a host microbial cell to incorporate the polynucleotide into the genome of the host microbial cell. Alternatively, an expression vector containing the polynucleotide may be introduced into the host microbial cell.

[0051] For the introduction of polynucleotides and vectors into host microbial cells, well-known transformation techniques such as the protoplast method, calcium phosphate method, electroporation method, lipofection method, particle gun method, and PEG method can be applied.

[0052] A vector containing a polynucleotide encoding the mature protein or proprotein can be constructed by conventionally inserting and ligating the polynucleotide encoding the mature protein or proprotein, and optionally a regulatory region or secretory signal sequence, into any vector. 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 a vector that can be amplified in host cells, and more preferably an expression vector. Preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R, or pASP64 (Patent No. 3492935), shuttle vectors such as 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 plasmids usable for transforming Bacillus bacteria such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmids derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0053] Microorganisms into which the polynucleotide encoding the mature protein or proprotein is introduced include bacteria and filamentous fungi, and it is preferable to use a microorganism of a different species from the microorganism from which the target GH25 family lysozyme is derived. Bacteria are preferred as such microorganisms, and Gram-positive bacteria are preferred as well. Gram-positive bacteria are not particularly limited, but include bacteria of the families Bacillaceae, Corynebacteriaceae, Clostridaceae, Enterococcusae, Lactobacillaceae, Streptococcusae, Staphylococcusae and Streptomycetaceae, with Bacillaceae, Lactobacillaceae or Corynebacteriaceae being preferred, and Bacillaceae bacteria being more preferred. Bacillaceae bacteria are not particularly limited, but Bacillus bacteria are more preferred.Bacillus bacteria include Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis (also called Brevibacillus brevis or Brevibacillus choshinensis), Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, and Bacillus megatherium. The bacterium may be any Bacillus species, including Bacillus megatherium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis, but Bacillus licheniformis, Bacillus megatherium, Bacillus subtilis, or their mutant strains are preferred. As mutant strains of Bacillus licheniformis, Bacillus megatherium, or Bacillus subtilis, strains lacking extracellular proteases are preferred from the viewpoint of avoiding host toxicity due to proprotein maturation by the host and the resulting expression of lysozyme activity. As mutant strains of Bacillus licheniformis, Bacillus licheniformis mutant strains in which apr, the gene encoding the extracellular protease of Bacillus licheniformis, is deleted or inactivated are preferred. The amino acid sequence of apr is shown in SEQ ID NO: 106, and the nucleotide sequence is shown in SEQ ID NO: 116.As Bacillus subtilis mutants, preferred Bacillus subtilis mutants are those in which at least one of the genes encoding extracellular proteases of Bacillus subtilis—epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX—is deleted or inactivated, and Bacillus subtilis mutants in which all of these genes are deleted or inactivated are more preferred (Japanese Patent Publication No. 2006-174707). The amino acid sequences of epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX are shown in SEQ ID NOs: 97-105, and the nucleotide sequences are shown in SEQ ID NOs: 107-115. Genes that have the same function as the gene encoding the extracellular protease, and / or that have at least 80%, preferably 90%, more preferably 95%, even more preferably 96%, even more preferably 97%, even more preferably 98%, even more preferably 99%, and even more preferably 99.5% identity in nucleotide sequence with the gene encoding the extracellular protease, are also considered to be genes equivalent to the gene encoding the extracellular protease and are included in genes that can be deleted or inactivated.

[0054] By culturing a recombinant microorganism into which a polynucleotide encoding the mature protein or proprotein of the target GH25 family lysozyme, or a vector containing the same, has been introduced, in an appropriate medium, the target GH25 family lysozyme mature protein or proprotein can be expressed. The recombinant microorganism can be cultured according to a general culture method appropriate to the type of recombinant microorganism, and the culture medium used can be appropriately selected by a person skilled in the art, depending on the type of recombinant microorganism. For example, a culture medium for Bacillus bacteria contains a carbon source and a nitrogen source necessary for the growth of the bacteria. Examples of carbon sources include glucose, dextran, soluble starch, sucrose, methanol, etc. Examples of nitrogen sources include ammonium salts, nitrates, amino acids, corn slush liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. If necessary, the culture medium may also contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, and antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, erythromycin, etc.). Culture conditions, such as temperature, aeration and stirring conditions, pH of the medium, and culture time, can be appropriately selected depending on the bacterial species, characteristics, and culture scale.

[0055] The mature protein or proprotein of the expressed target GH25 family lysozyme is produced either inside or outside the host microorganism's cell. However, if a secretion signal sequence is operably ligated to the polynucleotide encoding the mature protein or proprotein of the GH25 family lysozyme, it is secreted and produced outside the host microorganism's cell. Furthermore, the expressed target GH25 family lysozyme proprotein is produced either as a proprotein or as a mature protein with the propeptide cleaved, depending on the host microorganism's ability to mature the target GH25 family lysozyme proprotein.

[0056] The mature protein or proprotein of the target GH25 family lysozyme produced can be recovered from the microorganism or culture by conventional methods, and the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization, etc. For example, the recovery of the enzyme from the culture can be carried out using conventional methods such as separation of the microorganism by centrifugation or filtration, precipitation of the enzyme in the supernatant or filtrate by adding a salt such as ammonium sulfate or by adding an organic solvent such as ethanol, concentration and desalting using an ultrafiltration membrane, purification using various chromatography methods such as ion exchange or gel filtration.

[0057] The target GH25 family lysozyme proprotein produced intracellularly or extracellularly from a host microorganism can be recovered as is or, if necessary, according to conventional methods, and converted into the target GH25 family lysozyme mature protein by treating it with a protease. Examples of proteases used to treat the proprotein include those used in the protease treatment described later.

[0058] In the method of the present invention, any one of the above GH25 family lysozymes may be used alone, or any two or more may be used in combination. Furthermore, in the method of the present invention, the above GH25 family lysozyme may be a mature protein, or a proprotein may be used as long as the environment is such that a protease can act on the proprotein, that is, as long as the proprotein and protease coexist at least at a certain point in time. If the environment is such that a protease can act on the proprotein, the proprotein will be converted into a mature protein by cleavage of the propeptide, and the converted mature protein can act on cultured cells of a microorganism capable of producing biopolymers or its processed product.

[0059] The means for treating cultured cells of a biopolymer-producing microorganism or its processed product with GH25 family lysozyme under conditions of pH greater than 7.0 are not particularly limited, as long as the enzymatic activity of GH25 family lysozyme on the cultured cells of the biopolymer-producing microorganism or its processed product is exerted under conditions of pH greater than 7.0. For example, such means include adjusting the pH of a suspension containing cultured cells of a biopolymer-producing microorganism or its processed product to greater than 7.0, and then adding GH25 family lysozyme; or adding GH25 family lysozyme to a suspension containing cultured cells of a biopolymer-producing microorganism or its processed product, and then adjusting the pH of the suspension to greater than 7.0. Note that if the pH of the suspension containing cultured cells of a biopolymer-producing microorganism or its processed product is already greater than 7.0, pH adjustment is unnecessary, and GH25 family lysozyme should be added to the suspension.

[0060] The reaction conditions between cultured cells of a microorganism capable of producing biopolymers or a processed product thereof and GH25 family lysozyme are not particularly limited, as long as the pH is above 7.0 and the GH25 family lysozyme is active.

[0061] The pH condition (at 25°C) should be above pH 7.0, and from the viewpoint of maintaining enzyme activity and improving the purity of the biopolymer, it is preferably pH 7.5 or higher, more preferably pH 8.0 or higher, even more preferably pH 8.5 or higher, even more preferably pH 9.0 or higher, even more preferably pH 9.5 or higher, even more preferably pH 10.0 or higher, and preferably pH 13.0 or lower, more preferably pH 12.5 or lower, even more preferably pH 12.0 or lower, even more preferably pH 11.5 or lower, and even more preferably pH 11.0 or lower. Furthermore, the pH condition is preferably above pH 7.0 and below pH 13.0, more preferably pH 7.5 to 13.0, even more preferably pH 8.0 to 13.0, even more preferably pH 8.0 to 12.5, even more preferably pH 8.5 to 12.0, even more preferably pH 9.0 to 12.0, even more preferably pH 9.5 to 12.0, and even more preferably pH 10.0 to 12.0. pH is measured using the glass electrode method.

[0062] Alkaline agents can be used to adjust the pH. Examples of alkaline agents include alkali metal hydroxides and alkaline earth metal hydroxides. Among these, alkali metal hydroxides are preferred. Alkali metal hydroxides are hydroxides of alkali metals such as potassium, lithium, and sodium, and specifically include potassium hydroxide, lithium hydroxide, and sodium hydroxide. When adjusting the pH, the alkaline agent may be used as is or dissolved in water and used in the form of an aqueous solution. The amount of alkaline agent used can be appropriately set so that the pH reaches the desired value.

[0063] The appropriate reaction conditions, excluding the pH condition, for the reaction between cultured cells or processed products of microorganisms capable of biopolymer production and GH25 family lysozyme can be appropriately determined by a person skilled in the art, taking into account the type, shape, and quantity of the microorganisms capable of biopolymer production, the type, shape, and quantity of the biopolymer, and the type and quantity of GH25 family lysozyme. Examples of reaction conditions are described below.

[0064] The amount of GH25 family lysozyme used in the reaction is appropriately determined depending on the type, shape, and quantity of microorganisms capable of producing biopolymers. For example, the amount of GH25 family lysozyme used is preferably 0.00001 to 1% by mass, more preferably 0.0001 to 0.5% by mass, and even more preferably 0.001 to 0.1% by mass, based on dry mass of the microorganisms capable of producing biopolymers or biopolymer granules.

[0065] The temperature conditions for this reaction are preferably 20-80°C, more preferably 30-70°C, and even more preferably 40-60°C, from the viewpoint of improving the purity of the biopolymer.

[0066] 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 purity of the biopolymer.

[0067] The method of the present invention may further include a protease treatment step, in which a protease is applied to cultured cells of a biopolymer-producing microorganism or a processed product thereof under conditions of pH greater than 7.0, from the viewpoint of improving the purity of the biopolymer. In the protease treatment step, proteins derived from the cells of the biopolymer-producing microorganism can be degraded by applying a protease to cultured cells of the biopolymer-producing microorganism or a processed product thereof under conditions of pH greater than 7.0. Protein degradation products can be removed when the biopolymer is recovered. Therefore, the protease treatment step can greatly contribute to improving the purity of the biopolymer finally obtained. Thus, the protease treatment step is a biopolymer purification step that degrades components derived from cells other than the biopolymer of the biopolymer-producing microorganism containing the biopolymer, separates the biopolymer, and increases the purity of the biopolymer.

[0068] Here, "protease" refers to a protein that possesses protease activity, which hydrolyzes the peptide bonds of protein molecules to produce peptides and amino acids.

[0069] The protease used in the method of the present invention is not particularly limited, but is preferably a protease that has protease activity in an alkaline environment, more preferably a serine protease (EC number 3.4.21), even more preferably a protease of the S8 peptidase family in the MEROPS classification system, and even more preferably a protease of the S8 peptidase family derived from bacteria of the genus Bacillus.Preferred examples of proteases from the S8 peptidase family include alkaline protease KP43 (SEQ ID NO: 125) derived from Bacillus sp. KSM-KP43 as described in Japanese Patent Application No. 2024-071822 and its variants (e.g., 12 variant, SEQ ID NO: 4 (SEQ ID NO: 126) of Japanese Patent Application No. 2024-071822), alkaline protease K16 (SEQ ID NO: 127) derived from Bacillus sp. KSM-K16 (FERM BP-3376) as described in Japanese Patent Application No. 2024-071822 and its variants (e.g., quadruple variant, SEQ ID NO: 8 (SEQ ID NO: 128) of Japanese Patent Application No. 2024-071822), and subtilisin BPN' (WO2016 / 087617) derived from Bacillus amyloliquefaciens. A1 SEQ ID NO: 2) and its variants, subtilisin Carlsberg (WO2016 / 087617 A1 SEQ ID NO: 5) (Alcalase®; Novonesys) and its variants derived from Bacillus licheniformis, subtilisin 309 (WO2016 / 087617 A1 SEQ ID NO: 1) (Savinase®; Novonesys) and its variants derived from Bacillus lentus, subtilisin 147 (WO2016 / 087617) derived from Bacillus lentus Examples include A1 (SEQ ID NO: 6) (Esperase®; Novonesys, Inc.) and its variants, AprE, Epr, AprX and their variants derived from Bacillus subtilis, HH844 (SEQ ID NO: 120) and its variants (SEQ ID NO: 121, 122), and LL147 variants (SEQ ID NO: 123, 124). Among these, KP43 variant (SEQ ID NO: 126), K16 variant (SEQ ID NO: 128), Alcalase, Savinase, Esperase, HH844 (SEQ ID NO: 120) and its variants (SEQ ID NO: 121, 122), and LL147 variants (SEQ ID NO: 123, 124) are preferred.The protease used in the method of the present invention can be prepared from a microorganism containing a protease or a culture thereof, similar to the case of GH25 family lysozyme described above, or produced by chemical synthesis or microbiological methods, or by enhancing the expression of protease in a microorganism capable of biopolymer production through transformation. Alternatively, the protease used in the present invention may be a commercially available protease. Examples of commercially available proteases include Protin SD-AY10, Protease P "Amano" 3SD (Amano Enzyme Co., Ltd.), Bioplase OP (Nagase Vita Co., Ltd.), Orientase 22BF (HBI Co., Ltd.), Aloase XA-10 (Yakult Pharmaceutical Co., Ltd.), Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novonesys Corporation), Preferenz (registered trademark; IFF Corporation) series, Lavergy (registered trademark; BASF Corporation), and preferably Alcalase and Esperase.

[0070] The above proteases may be used individually or in combination of two or more.

[0071] The means for reacting cultured cells of a microorganism capable of producing biopolymers or its treated product with a protease under conditions of pH greater than 7.0, and the reaction conditions between the cultured cells of a microorganism capable of producing biopolymers or its treated product and the protease, are the same as those for GH25 family lysozyme described above.

[0072] The order in which the GH25 family lysozyme treatment process and the protease treatment process are performed is not particularly limited, and the order in which the GH25 family lysozyme treatment process and the protease treatment process are performed does not matter. From the viewpoint of improving the purity of the biopolymer and increasing the efficiency of production, it is preferable to perform the GH25 family lysozyme treatment process and the protease treatment process simultaneously under conditions of pH 7.0 or higher. In particular, when a proprotein is used as the GH25 family lysozyme, it is preferable to perform the GH25 family lysozyme treatment process and the protease treatment process simultaneously under conditions of pH 7.0 or higher, in order to convert the proprotein into a mature protein by the protease. When the GH25 family lysozyme treatment process and the protease treatment process are performed simultaneously, from the viewpoint of maintaining the activity of the GH25 family lysozyme, it is preferable to use a GH25 family lysozyme that has enough protease resistance to maintain its activity in the presence of the protease. If the GH25 family lysozyme treatment step and the protease treatment step are not performed simultaneously, one treatment step may be performed under conditions of pH greater than 7.0, and then the other treatment step may be performed under the same pH conditions. Alternatively, one treatment step may be performed under pH conditions that optimize the effect of the enzyme used in the first treatment step within the pH range of pH greater than 7.0, and then the pH may be adjusted to optimize the effect of the enzyme used in the other treatment step within the pH range of pH greater than 7.0, and then the other treatment step may be performed.

[0073] In addition to the GH25 family lysozyme treatment step, or the GH25 family lysozyme treatment step and the protease treatment step described above, the method of the present invention may further include, in terms of improving the purity of the biopolymer, at least one step selected from a heat treatment step, a physical crushing step, an alkali treatment step, an enzyme treatment step, a surfactant treatment step, an oxidizing agent treatment step, a centrifugation step, a membrane filtration step, and a washing step, as a step for purifying the biopolymer.

[0074] The heat treatment step inactivates microorganisms capable of producing biopolymers, thereby facilitating the purification of the biopolymers. The heat treatment step can be carried out by maintaining the cultured cells of the biopolymer-producing microorganisms at a temperature of preferably 40-80°C, more preferably 50-80°C, and even more preferably 60-80°C. The treatment time is preferably 0.2-20 hours, more preferably 0.5-10 hours.

[0075] In the physical crushing process, the cultured cells of microorganisms capable of producing biopolymers can be crushed or weakened by mechanical force. It is expected that the cells can be crushed more efficiently and the biopolymer yield improved by combining the GH25 family lysozyme treatment process with the physical crushing process, or by combining the GH25 family lysozyme treatment process and the protease treatment process with the physical crushing process. The physical crushing process may be performed simultaneously with the GH25 family lysozyme treatment process. Examples of mechanical forces for physical crushing include ultrasonic crushing, high-pressure homogenation, shear force using an in-line mixer, and crushing.

[0076] In the alkaline treatment process, adjusting the pH of the treatment solution to alkaline conditions with a basic compound or an aqueous solution thereof can promote the weakening of bacterial cells and the solubilization of bacterial-derived components such as proteins. Furthermore, treating with an appropriate alkaline pH allows for the adjustment of the molecular weight of the biopolymer to an appropriate size, improving the processability of the PHA powder. Basic compounds are not particularly limited, but examples include hydroxides of alkali metals or alkaline earth metals such as sodium hydroxide and potassium hydroxide; metal carbonates such as sodium carbonate and potassium carbonate; and metal phosphates or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, and potassium hydrogen phosphate. It is preferable to adjust the pH of the treatment solution to 7.5 to 13.0 with a basic compound or an aqueous solution thereof, and more preferably to 8.0 to 12.0. In order to balance the adjustment of the molecular weight of the biopolymer with the weakening of bacterial cells, the treatment time is preferably 0.5 to 20 hours, more preferably 1 to 10 hours. The treatment temperature is preferably 40°C to 80°C.

[0077] In the enzyme treatment process, the purity of the biopolymer can be improved by applying enzymes other than GH25 family lysozymes and proteases to cultured cells of microorganisms capable of biopolymer production or to the treated product thereof. The enzymes other than GH25 family lysozymes and proteases can be any enzyme capable of hydrolyzing microbial components, such as glycosidases, cellulases, lipases, amylases, cutinases, laccases, nucleases, and lysozymes other than GH25 family lysozymes. One or more of these can be used in combination. The enzyme treatment process can be carried out under general conditions used in the production of biopolymers by microorganisms capable of biopolymer production, depending on the type of enzyme used.

[0078] In the surfactant treatment process, by contacting the cultured cells of microorganisms capable of producing biopolymers or the treated product thereof with a surfactant, impurities derived from the cells, particularly cell membranes, proteins, and nucleic acids, can be efficiently dispersed and dissolved, thereby improving the purity of the biopolymer. Examples of surfactants include anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, one or a combination thereof, but anionic surfactants are preferred in terms of impurity removal ability. Examples of anionic surfactants include sulfated salts of alcohols having 10 to 18 carbon atoms, sulfated salts of alkoxylated 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 ester salts of alcohols with 10 to 14 carbon atoms in the alkyl chain, or linear alkylbenzene sulfonates with 10 to 14 carbon atoms in the alkyl chain, 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 especially preferred. The surfactant treatment step can be carried out under general conditions used in the production of biopolymers by microorganisms capable of producing biopolymers, depending on the type of surfactant.

[0079] In the oxidizing agent treatment process, by contacting the cultured cells of microorganisms capable of producing biopolymers or the treated product thereof with an oxidizing agent, impurities derived from the cells are reduced to low molecular weight, improving the purity of the biopolymer, reducing the membrane filtration load in the membrane filtration process, and suppressing membrane clogging. Furthermore, by reducing the molecular weight of impurities such as nucleic acids, the viscosity of the treatment solution is reduced, improving the stirring efficiency in each process and the centrifugal efficiency in the centrifugation process. In addition, the decolorization of the biopolymer improves the chromaticity of the final product. An indicator of the chromaticity of the final product is, for example, the b* value of the CIELAB color coordinate, preferably a b* value of less than 15. By combining the GH25 family lysozyme treatment process and the oxidizing agent treatment process, or by combining the GH25 family lysozyme treatment process and the protease treatment process with the oxidizing agent treatment process, it is expected that the membrane filtration load in the membrane filtration process can be further reduced and the chromaticity of the final product can be further improved. The oxidizing agent is not particularly limited, but examples include hydrogen peroxide, ozone; other inorganic peroxides such as sodium peroxide, sodium perborate, sodium percarbonate, and sodium persulfate; similar halogen compounds such as chlorites, chlorates, metachloroperbenzoate perchlorate, perchloric acid, and chlorine dioxide; peracids such as performic acid and peracetic acid; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate; sodium bismuthate; and cerium(IV) compounds such as cerium ammonium nitrate and cerium sulfate. Sodium chlorite, hydrogen peroxide, or ozone are preferred. Sodium chlorite is preferred because it can reduce the molecular weight of bacterial-derived impurities while suppressing the reduction of biopolymer molecular weight, thereby improving color. Hydrogen peroxide or ozone is preferred because it reduces the molecular weight of bacterial-derived impurities, thereby reducing the load on membrane filtration. One or more oxidizing agents may be used in combination. The concentration of sodium chlorite in the treatment solution is not particularly limited, but from the viewpoint of improving color while suppressing molecular weight reduction, it is preferably 0.01 to 1.3% by weight, more preferably 0.05 to 1.0% by weight. The pH at which sodium chlorite is brought into contact with the biopolymer may be acidic, for example, pH 1.0 to 7.0, and preferably pH 3.0 to 5.0.The concentration of hydrogen peroxide in the treatment solution is not particularly limited, but is preferably 0.01 to 30% by weight, more preferably 0.1 to 15% by weight, and even more preferably 0.2 to 10% by weight. When using hydrogen peroxide, it may be brought into contact with the biopolymer granules under alkaline pH conditions, for example, pH 7.0 to 13.0, preferably 8.0 to 10.0. Furthermore, the action of hydrogen peroxide can be enhanced by using sodium bicarbonate in combination. In addition, the hydrogen peroxide solution can be stabilized by using a chelating agent in combination. Examples of chelating agents are not particularly limited, but include sodium silicate, EDTA, and trans-1,2-cyclohexanediaminetetraacetic acid monohydrate. In terms of improving color, sodium chlorate treatment and hydrogen peroxide treatment may be performed sequentially. The amount of ozone added is not particularly limited, but is preferably 0.01 to 0.1 g per 1 g of dry mass of cultured microorganisms capable of producing biopolymers or their processed product, preferably 0.02 to 0.08 g, and more preferably 0.02 to 0.07 g.

[0080] In the centrifugation process, the purity of the biopolymer can be improved by removing unwanted supernatant from the processing liquid containing the biopolymer. The method of centrifugation is not particularly limited, but a decanter-type centrifuge is preferably used. Decanter-type centrifuges include horizontal and vertical types, but a horizontal type is preferred from the viewpoint of being able to process a large amount of liquid.

[0081] In the membrane filtration process, impurities derived from bacterial cells can be removed by filtering the treatment solution through a membrane. The filtration method is not particularly limited, but cross-flow filtration and dead-end filtration are preferred, and cross-flow filtration is more preferred.

[0082] In the washing process, impurities derived from bacterial cells can be removed by washing the biopolymer with an aqueous solution, thereby improving the purity of the biopolymer. The washing method is not particularly limited, but in the centrifugation process, a solution may be added to the biopolymer after removing the unnecessary supernatant of the treatment solution, and the process of centrifugation and removal of the supernatant may be repeated. The solution added is preferably an alkaline solution.

[0083] The order in which at least one of the above processes is performed is not particularly limited, and multiple processes can be performed simultaneously.

[0084] The solid component of the processing solution obtained after the purification process described above contains a biopolymer, and the biopolymer can be recovered by recovering the solid component of the processing solution. Therefore, the method of the present invention may further include a recovery step for recovering the biopolymer from the processing solution. This step can be carried out under general conditions used in the production of biopolymers by microorganisms capable of producing biopolymers. For example, this step can be carried out by separating the processing solution into solid and liquid components by centrifugation or membrane filtration to obtain a solid component, washing the solid component as necessary, and then dehydrating and drying the solid component by a filter press, air blow, drum dryer, spray drying, evaporation to dryness, freeze-drying, etc. A biopolymer powder can be obtained by this step. For further improvement of purity, a GH25 family lysozyme treatment step, or a GH25 family lysozyme treatment step and a protease treatment step may be performed again after recovery.

[0085] According to the method of the present invention, by using GH25 family lysozyme for the purification of biopolymers when producing biopolymers using microorganisms capable of biopolymer production, unlike conventionally used chicken egg white lysozyme, it is possible to effectively decompose peptidoglycans derived from cultured microorganisms capable of biopolymer production even under alkaline conditions with a pH of over 7.0, ultimately producing a biopolymer of high purity. Furthermore, by using GH25 family lysozyme, the biopolymer production process can be simplified by combining lysozyme treatment with treatments carried out under alkaline conditions (e.g., protease treatment). It is known that chicken egg white lysozyme is highly active at neutral or below pH, and that the optimal pH for the acm of GH25 family lysozyme is 5.3 (Non-Patent Literature 1). However, while chicken egg white lysozyme shows a significant decrease in its biopolymer purity-improving effect when acted upon at pH above 7.0, no such decrease in biopolymer purity-improving effect was observed with GH25 family lysozyme. It was completely unexpected that GH25 family lysozyme maintains a sufficient biopolymer purity-improving effect under alkaline conditions above pH 7.0.

[0086] The GH25 family lysozyme treatment step described above is a step that decomposes the peptidoglycan constituting the cell wall of microorganisms capable of producing biopolymers even under alkaline conditions of pH greater than 7.0, and facilitates the removal of cell-derived components other than peptidoglycan. Therefore, the present invention also provides a method for purifying biopolymers. The method is a method for purifying biopolymers produced by microorganisms capable of producing biopolymers, and includes the step of reacting cultured cells of microorganisms capable of producing biopolymers or their treated products with GH25 family lysozyme under conditions of pH greater than 7.0. The method may include the protease treatment step described above, and may further include at least one step selected from the above-described heat treatment step, physical disruption step, alkaline treatment step, enzyme treatment step, surfactant treatment step, oxidizing agent treatment step, centrifugation step, membrane filtration step, and washing step. The details of each step are the same as in the method for producing biopolymers using microorganisms capable of producing biopolymers according to the present invention described above.

[0087] GH25 family lysozymes are useful as enzymes for purifying biopolymers, particularly as enzymes used for purification in the production of biopolymers by microorganisms capable of producing biopolymers, and can be active ingredients in enzyme compositions for purifying biopolymers, especially those used for purification in the production of biopolymers by microorganisms capable of producing biopolymers.

[0088] Therefore, the present invention also provides an enzyme composition for purifying biopolymers containing GH25 family lysozyme. The enzyme composition of the present invention may be a solid composition such as a powder or a liquid composition. In addition to GH25 family lysozyme, the enzyme composition may appropriately contain surfactants, chelating agents, water-soluble polymers, alkaline agents, organic acids or their salts, other enzymes other than the protease of the present invention, enzyme stabilizers, antioxidants, solubilizers, pH adjusters, buffers, preservatives, fragrances, and the like.

[0089] The amount of GH25 family lysozyme in the enzyme composition of the present invention is not particularly limited as long as the amount of GH25 family lysozyme exhibits activity, but is preferably 0.01 to 500 g per 1 kg of enzyme composition, more preferably 0.1 to 200 g, and even more preferably 1 to 100 g.

[0090] As described above, GH25 family lysozymes can decompose peptidoglycans, which constitute the cell walls of microorganisms capable of producing biopolymers, even under alkaline conditions with a pH above 7.0. Therefore, they can be used to purify target substances other than biopolymers that are produced within bacterial cells, similar to biopolymers. Here, the target substance refers to any substance produced by a bacterial host.

[0091] Therefore, the present invention also provides a method for purifying a target substance produced within the cells of bacteria. The method includes a GH25 family lysozyme treatment step in which GH25 family lysozyme is applied to cultured cells of bacteria that produce the target substance within their cells or a processed product thereof under conditions of pH greater than 7.0. If the purification of the target substance includes steps where alkaline treatment is desirable, such as a solubilization step of impurities with alkali or a protease treatment step, the lysozyme treatment and the treatment carried out under alkaline conditions can be combined to simplify the purification process. The bacteria are not particularly limited, but include bacteria commonly used for the fermentation production of the target substance and bacteria that have the ability to produce the target substance in nature. Examples of bacteria commonly used for the fermentation production of the target substance include Escherichia coli, Corynebacterium, Actinomycetes, and Bacillus subtilis. The target substances are not particularly limited, but include high molecular weight compounds such as proteins and peptides, oils and fats, pigments, terpenes (including terpenes and terpenoids), amino acids, sugars, fatty acids and their derivatives, and other low molecular weight compounds. Examples of proteins include enzymes such as polymerases and catalases, crystalline proteins such as Cry proteins, and fibrous proteins such as fibroin. When proteins are produced in cells as insoluble aggregates, methods for efficiently recovering the insoluble aggregates by disrupting the cells are known (Patent 7079525), and it is expected that the purification method of the present invention will further improve the purity and efficiently recover the target insoluble protein aggregates. Examples of terpenes and their derivatives include monoterpenes, sesquiterpenes, diterpenes, tetraterpenes, and their derivatives. Specific examples include valencene, an example of a sesquiterpene, and its oxidized forms such as nootkatone, patchulol, and santalol. Furthermore, carotenoids include β-carotene, lycopene, astaxanthin, and their cleavage products, apocarotenoids (β-ionone, damascone, damascenone, etc.). These terpenes and their derivatives generally accumulate insoluble or crystalline form as hydrophobic substances within cells, and residual components derived from the cell wall may inhibit purification; therefore, the purification method of the present invention is particularly effective.Furthermore, examples of pigments include insoluble pigments such as indigo, and their precursor, indican, and the purification method of the present invention can also be applied to these. Examples of the production of the target substance within cells include the production of fibroin-like protein by E. coli (JP 2023-153942 A), the production of catalase by Micrococcus sp. (JP Heisei 6-16704), the production of astaxanthin by Paracoccus carotinifaciens (Hayashi, Masahiro, et al. "Commercial production of astaxanthin with Paracoccus carotinifaciens." Carotenoids: Biosynthetic and biofunctional approaches. Singapore: Springer Singapore, 2021. 11-20.), and the production of β-carotene by E. coli (Zhao, Jing, et al. Metabolic engineering 17 (2013): 42-50.).

[0092] As exemplary embodiments of the present invention, the following compositions, manufacturing methods, uses, or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[0093] [1] A method for producing a biopolymer, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of a microorganism capable of producing a biopolymer or a treated product thereof under conditions of pH greater than 7.0. [2] The method according to [1], further comprising a protease treatment step of reacting a culture of a microorganism having biopolymer production ability or a processed product thereof with a protease under conditions of pH greater than 7.0. [3] The method according to [2], wherein the GH25 family lysozyme treatment step and the protease treatment step are performed simultaneously. [4] The method according to any one of [1] to [3], comprising the step of adjusting the pH of a suspension containing cultured cells of a microorganism capable of producing biopolymers or a processed product thereof to a pH greater than 7.0 with an alkaline agent. [5] The method according to any one of [1] to [4], further comprising a culture step of culturing microorganisms having biopolymer production ability prior to the GH25 family lysozyme treatment step. [6] The method according to any one of items [1] to [5], further comprising a recovery step for recovering the biopolymer.

[0094] [7] A method for purifying biopolymers, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of a microorganism capable of producing biopolymers or a processed product thereof under conditions of pH greater than 7.0. [8] The method according to [7], further comprising a protease treatment step of reacting a culture of a microorganism having biopolymer production ability or a processed product thereof with a protease under conditions of pH greater than 7.0. [9] The method according to [8], wherein the GH25 family lysozyme treatment step and the protease treatment step are performed simultaneously.

[10] A method for purifying a target substance, comprising a GH25 family lysozyme treatment step of reacting culture cells of a bacterium that produces the target substance within its cells or a processed product thereof with GH25 family lysozyme under conditions of pH greater than 7.0.

[11] The method according to

[10] , further comprising a protease treatment step of reacting a culture of the bacteria or a processed product thereof with a protease under conditions of pH greater than 7.0.

[12] The method according to

[11] , wherein the GH25 family lysozyme treatment step and the protease treatment step are performed simultaneously.

[0095]

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

[12] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 8.0 or higher.

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

[12] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 8.5 or higher.

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

[12] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 9.0 or higher.

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

[12] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 9.5 or higher.

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

[12] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 10.0 or higher.

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

[17] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 13.0 or less.

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

[17] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 12.5 or less.

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

[17] , wherein the pH condition in the GH25 family lysozyme treatment step is pH 12.0 or less.

[0096]

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

[20] , wherein the GH25 family lysozyme is a polypeptide having D at position 9, K at position 33, E at position 36, Y at position 62, D at position 98, E at position 100, Y at position 138, and Q at position 183.

[22] The GH25 family rizoteam has motif DX in the area corresponding to the 98th to 105th position in the numbering scheme of Sequence ID No. 1. 1 EX 2 NP(X 3 ) n G(X 1 , X 2 , and X 3 The method according to any one of [1] to

[21] , wherein each of the following independently represents an arbitrary amino acid residue, and n represents an integer from 1 to 5.

[23] The aforementioned motif is DX 1 EX 2 NP[S / N / Y]G(X 1 and X 2 The method described in

[22] , wherein each of the elements independently represents an arbitrary amino acid residue.

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

[23] , wherein the GH25 family lysozyme is at least one selected from the group consisting of polypeptides comprising amino acid sequences represented by any of SEQ ID NOs: 1 to 4 and 59 to 96, and polypeptides comprising amino acid sequences having at least 60% identity with the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 and 59 to 96, and having lysozyme activity, preferably at least one selected from the group consisting of polypeptides comprising amino acid sequences represented by any of SEQ ID NOs: 1 to 4, 59 to 63, 66 to 79, 81 to 83 and 88 to 96, and polypeptides comprising amino acid sequences having at least 60% identity with the amino acid sequences represented by any of SEQ ID NOs: 1 to 4, 59 to 63, 66 to 79, 81 to 83 and 88 to 96, and having lysozyme activity.

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

[23] , wherein the GH25 family lysozyme is at least one selected from the group consisting of polypeptides comprising an amino acid sequence represented by any one of SEQ ID NOs: 1, 59, 78, 89, and 91, and polypeptides comprising an amino acid sequence having at least 60% identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 59, 78, 89, and 91, and having lysozyme activity.

[26] The method according to

[24] or

[25] , wherein the amino acid sequence is identical by at least 70%.

[27] The method according to

[24] or

[25] , wherein the amino acid sequence is identical by at least 80%.

[28] The method according to

[24] or

[25] , wherein the amino acid sequence is identical by at least 90%.

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

[28] , wherein the GH25 family lysozyme is a GH25 family lysozyme derived from bacteria or fungi.

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

[28] , wherein the GH25 family lysozyme is a bacterial GH25 family lysozyme.

[0097]

[31] The method according to any one of [1] to [9] and

[13] to

[30] , wherein the biopolymer is a hydroxyalkanoic acid-containing polymer.

[32] The method according to

[31] , wherein the hydroxyalkanoic acid-containing polymer is polyhydroxyalkanoic acid (PHA), which is a polyester containing only hydroxyalkanoic acid as a monomer unit, or a polyesteramide containing hydroxyalkanoic acid and a carboxylic acid having an amino group as monomer units, and having ester bonds and amide bonds, and is preferably PHA.

[33] The method according to

[31] or

[32] , wherein the proportion of hydroxyalkanoic acid in the monomer units constituting the hydroxyalkanoic acid-containing polymer is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 100%.

[34] The method according to any one of [1] to [9] and

[13] to

[30] , wherein the biopolymer is PHA.

[35] The method according to any one of [1] to [9] and

[13] to

[34] , wherein the microorganism having biopolymer production ability is a microorganism belonging to the genera Aeromonas, Escherichia, or Cupriavidus.

[36] The method according to any one of [1] to [9] and

[13] to

[34] , wherein the microorganism having biopolymer production ability is a microorganism belonging to the genus Cupriavidus.

[37] The method according to

[35] or

[36] , wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus necator (formerly known as Ralstonia eutropha) or Cupriavidus metallidurans.

[38] The method according to

[35] or

[36] , wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus necator.

[0098]

[39] An enzyme composition for purifying biopolymers, containing GH25 family lysozyme.

[40] The enzyme composition for purifying biopolymers according to

[39] , wherein the GH25 family lysozyme is a polypeptide having D at position 9, D at position 98, and E at position 100 in the numbering of Sequence ID No. 1, preferably a polypeptide having D at position 9, Y at position 62, D at position 98, E at position 100, Y at position 138, and Q at position 183 in the numbering of Sequence ID No. 1, more preferably a polypeptide having D at position 9, K at position 33, E at position 36, Y at position 62, D at position 98, E at position 100, Y at position 138, and Q at position 183 in the numbering of Sequence ID No. 1.

[41] The GH25 family rizoteam has motif DX in the area corresponding to the 98th to 105th position in the numbering scheme of Sequence ID No. 1. 1 EX 2 NP(X 3 ) n G(X 1 , X 2 , and X 3 The enzyme composition for purifying biopolymers according to

[39] or

[40] , wherein each of the following independently represents an arbitrary amino acid residue, and n represents an integer from 1 to 5.

[42] The aforementioned motif is DX 1 EX 2 NP[S / N / Y]G(X 1 and X 2 The enzyme composition for purifying biopolymers according to

[41] , wherein each of the above independently represents an arbitrary amino acid residue.

[43] The enzyme composition for purifying biopolymers according to

[39] to

[42] , wherein the GH25 family lysozyme is at least one selected from the group consisting of polypeptides comprising amino acid sequences represented by any of SEQ ID NOs: 1 to 4 and 59 to 96, and polypeptides comprising amino acid sequences having at least 60% identity with the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 and 59 to 96, and having lysozyme activity, preferably at least one selected from the group consisting of polypeptides comprising amino acid sequences represented by any of SEQ ID NOs: 1 to 4, 59 to 63, 66 to 79, 81 to 83 and 88 to 96, and polypeptides comprising amino acid sequences having at least 60% identity with the amino acid sequences represented by any of SEQ ID NOs: 1 to 4, 59 to 63, 66 to 79, 81 to 83 and 88 to 96, and having lysozyme activity.

[44] The enzyme composition for purifying biopolymers according to any one of

[39] to

[42] , wherein the GH25 family lysozyme is at least one selected from the group consisting of polypeptides comprising an amino acid sequence represented by any one of SEQ ID NOs: 1, 59, 78, 89, and 91, and polypeptides comprising an amino acid sequence having at least 60% identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 59, 78, 89, and 91, and having lysozyme activity.

[45] The enzyme composition for purifying biopolymers according to

[43] or

[44] , wherein the amino acid sequence identity is at least 70%.

[46] The enzyme composition for purifying biopolymers according to

[43] or

[44] , wherein the amino acid sequence identity is at least 80%.

[47] The enzyme composition for purifying biopolymers according to

[43] or

[44] , wherein the amino acid sequence identity is at least 90%.

[48] ​​The enzyme composition for purifying biopolymers according to any one of

[39] to

[47] , wherein the GH25 family lysozyme is a lysozyme selected from bacterial GH25 family lysozymes and fungal GH25 family lysozymes.

[49] The enzyme composition for purifying biopolymers according to any one of

[39] to

[47] , wherein the GH25 family lysozyme is a bacterial GH25 family lysozyme.

[50] An enzyme composition for PHA purification, as described in any one of items

[39] to

[49] . Use of the enzyme composition described in any one of items

[51] ,

[39] , to

[50] for the production of biopolymers. Use of the enzyme composition described in any one of items

[52] ,

[39] , to

[50] for the purification of biopolymers. [Examples]

[0099] 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.

[0100] (1) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain NBRC 102504 was inoculated into LB liquid medium and cultured with shaking at 30°C for 24 hours. 0.2 mL of this culture solution was inoculated into a 500 mL ribbed flask containing 20 mL of PHA production medium (1.1% disodium hydrogen phosphate dodecahydrate, 0.19% potassium dihydrogen phosphate, 0.13% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, 16.2 ppm iron(III) chloride hexahydrate, 10.3 ppm calcium chloride dihydrate, 0.218 ppm cobalt chloride hexahydrate, 0.156 ppm copper sulfate pentahydrate, 0.118 ppm nickel chloride hexahydrate, 2% coconut oil; % is (w / v)%), and cultured with shaking at 30°C for 72 hours to obtain a PHA-containing bacterial suspension.

[0101] (2) Preparation of enzyme solution Mutanoricin (product number: M9901) from SIGMA was used, dissolved in 100 mM sodium citrate buffer (pH 6.0). Egg white lysozyme (chicken egg white lysozyme, Fujifilm Wako Pure Chemical Industries, product number: 127-06724) was used, dissolved in 100 mM sodium citrate buffer (pH 6.0). Alcalase (product number: 126741) from SIGMA was used. A DC Protein Assay Kit (Bio-Rad) was used to measure the concentration of the enzyme solution. BSA Standard Solution (WAKO) was used as the standard solution for calculating protein amount.

[0102] (3) PHA purification using lysozyme 1 A pH meter LAQUAact D-72 (HORIBA) was used to measure pH. The PHA-containing bacterial cell suspension obtained in (1) was heated at 70°C for 1 hour to inactivate the cells. After dispensing into four tubes, 1N sodium hydroxide aqueous solution was added to adjust the pH to 7.0, 8.0, 8.5, and 9.0 respectively, and 0.8 mL was dispensed into 2 mL tubes. The enzymes listed in Table 1 were added at the final concentrations listed and incubated at 50°C for 1 hour. 90 μL of 1M Tris-HCl (pH 9.0) was added to confirm that all samples were within the pH range of 9.0 ± 0.3. Alcalase at a final concentration of 30 ppm (total final concentration of 60 ppm for samples to which alcalase had already been added) was added and incubated at 50°C for 1 hour. 1 mL of 12 mM sodium hydroxide aqueous solution containing 1.2 (w / v)% SDS was added and mixed by inversion. The mixture was centrifuged at 12,000 rpm for 5 minutes, and 1 mL of the supernatant was removed. The appearance of the pellet was observed, and pellets with a volume equivalent to that of a sample treated with 0.3 ppm egg white lysozyme at pH 7.0 were recorded as +, while those with a volume greater than that of a sample treated with 0.3 ppm egg white lysozyme at pH 7.0 were recorded as ++ (Table 2). The action of lysozyme decomposes the peptidoglycan derived from the producing bacteria, resulting in a smaller volume of the purified PHA pellet. 1 mL of 1 mM sodium hydroxide aqueous solution was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes. The supernatant was then completely removed, and this procedure was repeated twice. 1 mL of deionized water was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes. The supernatant was then completely removed. The tube cap was opened and the tube was dried overnight at 50°C. The color of the dried pellet (purified PHA) was visually observed and recorded as × for translucent and cloudy pellets, and ○ for white pellets (Table 2). Due to the action of lysozyme, the peptidoglycan derived from the production bacteria is broken down, and the purified PHA pellet, with improved purity, becomes white. Egg white lysozyme showed a significant decrease in purity-improving effect when applied at pH levels higher than 7.0. To achieve the same effect as when 0.3 ppm egg white lysozyme was applied at pH 7.0, a concentration of 30 ppm was required at pH 9.0. On the other hand, mutanolicin showed an added effect at a concentration of 0.3 ppm at both pH 7.0 and pH 9.0. Furthermore, mutanolicin showed a similar effect even when applied in the presence of proteases.

[0103] [Table 2]

[0104] (4) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain NBRC 102504 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 Sakaguchi flask containing 100 mL of PHA production medium (1.1% disodium hydrogen phosphate dodecahydrate, 0.19% potassium dihydrogen phosphate, 0.13% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, 16.2 ppm iron(III) chloride hexahydrate, 10.3 ppm calcium chloride dihydrate, 0.218 ppm cobalt chloride hexahydrate, 0.156 ppm copper sulfate pentahydrate, 0.118 ppm nickel chloride hexahydrate, 2% coconut oil; % is (w / v)%), and cultured with shaking at 30°C for 72 hours to obtain a PHA-containing bacterial suspension.

[0105] (5) PHA purification using lysozyme 2 A pH meter LAQUAact D-72 (HORIBA) was used to measure pH. The PHA-containing bacterial cell suspension obtained in (4) was heated at 70°C for 1 hour to inactivate the cells. After dispensing into two tubes, 1N sodium hydroxide aqueous solution was added to adjust the pH to 7.0 and 9.0 respectively, and 0.9 mL was dispensed into 2 mL tubes. The enzymes listed in Table 2 were added at the final concentrations listed and incubated at 50°C for 1 hour. 100 μL of 1M Tris-HCl (pH 9.0) was added only to the sample adjusted to pH 7.0 to confirm that all samples were within the pH range of 9.0 ± 0.3. Alcalase at a final concentration of 30 ppm (total final concentration of 60 ppm for samples that already had alcalase added) was added and incubated at 50°C for 1 hour. 1 mL of 12 mM sodium hydroxide aqueous solution containing 1.2 (w / v)% SDS was added and mixed by inversion. The mixture was centrifuged at 12,000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide aqueous solution was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes. The supernatant was then completely removed, and this procedure was repeated twice. 1 mL of deionized water was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes. The supernatant was then completely removed. The tube cap was opened and the tube was dried overnight at 50°C. The color of the dried pellet (purified PHA) was visually observed and recorded as follows: translucent and cloudy was marked with ×, slightly whiter than that was marked with △, and even whiter was marked with ○ (Table 3). Egg white lysozyme showed a significant decrease in purity-improving effect when applied at pH levels higher than 7.0. To obtain the same effect as when 0.3 ppm egg white lysozyme was applied at pH 7.0, a concentration of 30 ppm was required at pH 9.0. On the other hand, mutanolicin showed a high additive effect at a concentration of 0.3 ppm at both pH 7.0 and pH 9.0, compared to both 0.3 ppm at pH 7.0 and 30 ppm at pH 9.0 for egg white lysozyme. Furthermore, mutanolicin showed a similar effect when applied in the presence of proteases.

[0106] [Table 3]

[0107] (6) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain H16 was inoculated into BD Difco® Nutrient Broth medium and cultured with shaking at 30°C for 24 hours. 1 mL of this culture solution was inoculated into a Sakaguchi flask containing 100 mL of PHA production medium (1.1% disodium hydrogen phosphate dodecahydrate, 0.19% potassium dihydrogen phosphate, 0.13% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, 16.2 ppm iron(III) chloride hexahydrate, 10.3 ppm calcium chloride dihydrate, 0.22 ppm cobalt chloride hexahydrate, 0.16 ppm copper sulfate pentahydrate, 0.12 ppm nickel chloride hexahydrate, 1.5% coconut oil, 2% fructose; % is (w / v)%), and cultured with shaking at 30°C for 72 hours to obtain a PHA-containing bacterial suspension.

[0108] (7) PHA purification using lysozyme 3 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (6) was centrifuged, and the PHA-containing bacteria were concentrated 10-fold by discarding 90% of the supernatant and resuspending it. The bacteria were inactivated by heating at 70°C for 1 hour. The pH was adjusted to 9.5 by adding 1N sodium hydroxide aqueous solution, and 0.3 mL was dispensed into 1.5 mL tubes. The enzymes shown in Figure 1 were added at the final concentrations shown, and incubated at 50°C for 30 minutes. The pH of the sample without enzyme added had decreased to 8.1 after incubation. In the concentrated bacterial cell suspension, the elution of intracellular components due to partial lysis by heating and alkali had a significant effect on the pH of the solution, which is thought to have caused the pH to decrease. After incubation, each sample was subjected to sonication using an ultrasonic disruptor BIORUPTOR II (Sonic Bio Co., Ltd.) (High, 30 sec ON, 30 sec OFF, 10 cycles). The supernatant was collected after centrifugation at 12000 rpm for 5 minutes. The obtained supernatant was mixed in equal volume with 2×Laemmli Sample Buffer (Bio-Rad) containing 100 mM DTT and incubated at 100°C for 30 seconds. Each sample was applied to Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-Rad), and electrophoresis was performed at a constant voltage of 200V. The post-electrophoresis gels were imaged using the Chemi Doc MP Imaging system (Bio-Rad) (Figure 1). The addition of lysozyme weakened the cells, leading to an increase in cell-derived proteins in the supernatant after sonication. A clear increase in cell-derived proteins was observed only at 20 ppm with egg white lysozyme. On the other hand, mutanoricin showed a slightly greater increase in cell-derived proteins at 2 ppm than at 20 ppm with egg white lysozyme, and a more significant increase at 20 ppm.

[0109] (8) Construction of enzyme expression plasmids Plasmid pHY-BlNucB was obtained by substituting the full-length ORF of plasmid pHY-BLP2, described in WO2019 / 142773, consisting of the S237 secretion signal sequence and the BLP proprotein, with the native secretion signal-containing nuclease (BlNucB) gene (encoding the polynucleotide of SEQ ID NO: 42 and the amino acid sequence of SEQ ID NO: 43). The amino acid sequence of the mature BlNucB is SEQ ID NO: 44.

[0110] (9) Preparation of nuclease solution Enzyme expression plasmids were introduced into Bacillus subtilis strains by protoplast, and the cultures were incubated in 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. The culture supernatant containing the enzyme was then collected by centrifugation. The buffer was changed to 20 mM Tris-HCl (pH 7.5) containing 2 mM CaCl2 using Amicon 10K. The DC Protein Assay Kit (Bio-Rad) was used to measure the concentration of the enzyme solution. BSA Standard Solution (WAKO) was used as the standard solution for calculating the protein amount.

[0111] (10) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain H16 was inoculated into LB liquid medium and cultured with shaking at 30°C for 24 hours. 1 mL of this culture medium was inoculated into a Sakaguchi flask containing 100 mL of PHA production medium (0.35% ammonium chloride, 0.175% potassium dihydrogen phosphate, 0.12% magnesium sulfate heptahydrate, 0.17% citric acid, 3.5% fructose, 0.0225 ppm zinc sulfate heptahydrate, 0.1 ppm ferrous sulfate heptahydrate, 0.02 ppm calcium chloride dihydrate, 0.0023 ppm sodium tetraborate heptahydrate, 0.001 ppm hexaammonium heptamolybdate tetrahydrate, 0.01 ppm copper sulfate pentahydrate, 0.006 ppm manganese sulfate pentahydrate, 35 (v / v) ppm hydrochloric acid; % is (w / v)) and cultured with shaking at 30°C for 72 hours to obtain a suspension of PHA-containing bacterial cells.

[0112] (11) PHA purification using lysozyme 4 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The pH of the PHA-containing bacterial cell suspension obtained in (10) was adjusted to 7.0 by adding 1N HCl. 1 mL was dispensed into two 2 mL tubes, and 30 mg / L nuclease (BlNucB) was added to one of the tubes. Each tube was heated at 70°C for 10 minutes, then lysozyme (mutanolysin, SIGMA M9901) was added at a final concentration of 20 mg / L and incubated at 50°C for 1 hour. After adjusting the pH to 9.0 by adding 1N sodium hydroxide aqueous solution, 30 mg / L alcalase was added and incubated at 50°C for 1 hour. 1 mL of 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide was added and mixed by inversion. The pH at this time was 9.4. Centrifuge was performed at 12000 rpm for 5 minutes. 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide aqueous solution was added and mixed by inversion, then centrifuged at 12000 rpm for 5 minutes, and the supernatant was completely removed. This procedure was repeated twice. The tube cap was opened and it was dried overnight at 60°C. The color of the dried pellet (purified PHA) was observed visually and it was white.

[0113] (12) PHA purification using lysozyme 5 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (10) was heated at 70°C for 30 minutes to inactivate the cells. 1N HCl was added to adjust the pH to 7.0. 1 mL was dispensed into two 2 mL tubes. Lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L was added to one tube, and lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and nuclease (BlNucB) at a final concentration of 30 mg / L were added to the other tube, and incubated at 50°C for 1 hour. After adjusting the pH to 9.0 by adding 1N sodium hydroxide aqueous solution, alcalase at a final concentration of 30 mg / L was added and incubated at 50°C for 1 hour. 1 mL of 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide was added and mixed by inversion. The pH was 9.4 at this time. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide solution was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes, followed by the removal of the entire supernatant. This procedure was repeated twice. The tube cap was opened and the mixture was dried overnight at 60°C. The color of the dried pellet (purified PHA) was visually observed and found to be white.

[0114] (13) PHA purification using lysozyme 6 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (10) was heated at 70°C for 30 minutes to inactivate the cells. 1N HCl was added to adjust the pH to 7.0. 1 mL was dispensed into two 2 mL tubes. One tube was filled with lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and alcalase at a final concentration of 30 mg / L, while the other tube was filled with lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L, alcalase at a final concentration of 30 mg / L, and nuclease (BlNucB) at a final concentration of 30 mg / L. The mixture was incubated at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 9.0, and the mixture was incubated again at 50°C for 1 hour. 1 mL of a 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide was added and mixed by inversion. The pH at this time was 9.4. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. This procedure was repeated twice, with 1 mL of 1 mM sodium hydroxide aqueous solution added, mixed by inversion, centrifuged at 12000 rpm for 5 minutes, and the entire supernatant removed. The tube cap was opened and the mixture was dried overnight at 60°C. Visual inspection of the dried pellet (purified PHA) revealed that it was white.

[0115] (14) PHA purification using lysozyme 7 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (10) was heated at 70°C for 30 minutes to inactivate the cells. pH was adjusted to 9.0 by adding 1N sodium hydroxide aqueous solution. After dispensing 1 mL each into two 2 mL tubes, lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and alcalase at a final concentration of 30 mg / L were added to one tube, and lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L, alcalase at a final concentration of 30 mg / L and nuclease (BlNucB) at a final concentration were added to the other tube, and incubated at 50°C for 1 hour. 1 mL of 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide was added and mixed by inversion. At this time, the pH was 9.6. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide solution was added, mixed by inversion, and centrifuged again at 12000 rpm for 5 minutes. This process of removing the entire supernatant was repeated twice. The tube cap was opened and the mixture was dried overnight at 60°C. Visual inspection of the dried pellet (purified PHA) revealed it to be white.

[0116] (15) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain NBRC 102504 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, 0.13% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, 16.2 ppm iron(III) chloride hexahydrate, 10.3 ppm calcium chloride dihydrate, 0.218 ppm cobalt chloride hexahydrate, 0.156 ppm copper sulfate pentahydrate, 0.118 ppm nickel chloride hexahydrate, 2% coconut oil; % is (w / v)%), and cultured with shaking at 30°C for 72 hours to obtain a PHA-containing bacterial suspension.

[0117] (16) PHA purification using lysozyme 8 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (15) was heated at 70°C for 30 minutes to inactivate the cells. pH was adjusted to 9.0 by adding 1N sodium hydroxide aqueous solution. After dispensing 1 mL each into two 2 mL tubes, lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and alcalase at a final concentration of 30 mg / L were added to one tube, and lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L, alcalase at a final concentration of 30 mg / L and nuclease (BlNucB) at a final concentration were added to the other tube, and incubated at 50°C for 1 hour. 1 mL of 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide was added and mixed by inversion. At this time, the pH was 11.3. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide solution was added, mixed by inversion, and centrifuged again at 12000 rpm for 5 minutes. This process of removing the entire supernatant was repeated twice. The tube cap was opened and the mixture was dried overnight at 60°C. Visual inspection of the dried pellet (purified PHA) revealed it to be white.

[0118] (17) Preparation of GH25 family lysozyme proproteins Gene synthesis of GH25 family lysozymes selected from public databases was performed after codon optimization for Bacillus subtilis (Table 4). Enzyme expression plasmids were obtained by replacing the full-length ORF containing the VHH gene of the VHH expression plasmid of SEQ ID NO: 26, which contains a Bacillus subtilis spoVG gene-derived promoter as described in WO2021 / 153129, with genes encoding the proproteins of each GH25 family lysozyme (Table 4), with the S237 secretion signal sequence (encoding the polynucleotide of SEQ ID NO: 45 and the amino acid sequence of SEQ ID NO: 46) ligated to the N-terminus. The enzyme expression plasmids were introduced into the Dpr9 strain, a 9-fold knockout strain of Bacillus subtilis extracellular protease (see Microbial cell factories 20.1(2021):1-13.) by protoplast. The resulting transformants were cultured in 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, after which the culture supernatant containing the GH25 family lysozyme proprotein was collected by centrifugation. ACM mature protein was prepared by adding a final concentration of 10 mg / L of alcalase to the culture supernatant containing the ACM proprotein and incubating overnight at 30°C. The concentrations of the proprotein and mature protein of GH25 family lysozyme expressed in Bacillus subtilis were measured by SDS-PAGE as follows. Each sample was mixed in equal volumes with 2×Laemmli Sample Buffer (Bio-Rad) containing 100 mM DTT and incubated at 100°C for 30 seconds. Each sample was applied to Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-Rad), and electrophoresis was performed at a constant voltage of 200V. The gel after electrophoresis was imaged using the Chemi Doc MP Imaging system (Bio-Rad), and the band intensity of the target protein was quantified. A calibration curve was created using the band intensity of mutanolicin (SIGMA M9901), and the concentration of the target protein in the culture supernatant was calculated. The concentration of mutanolicin (SIGMA M9901) was quantified using the DC Protein Assay Kit (Bio-Rad) with BSA Standard Solution (WAKO) as the standard.

[0119] [Table 4]

[0120] (18) PHA purification using lysozyme 9 The effects of the enzymes listed in Table 5 on PHA purification were evaluated using the same method as in Example (5). The GH25 family lysozyme proproteins are activated by cleavage of the propeptide upon contact with alcalase. All activated GH25 family lysozyme proproteins showed a higher purity improvement effect than egg white lysozyme at the same concentration at pH 9.0. Similarly, the pre-matured acm mature protein also showed a higher purity improvement effect than egg white lysozyme at the same concentration at pH 9.0.

[0121] [Table 5]

[0122] (19) PHA purification using lysozyme 10 PHA purification is performed in the same manner as in Example (5), except that the enzymes listed in Table 5 are used, and instead of alcalase, KP43 (SEQ ID NO: 125), KP43 mutant (SEQ ID NO: 126), K16 (SEQ ID NO: 127), K16 mutant (SEQ ID NO: 128), Esperase, Savinase, HH844 (SEQ ID NO: 120), HH844 mutant (SEQ ID NO: 121, 122), or LL147 mutant (SEQ ID NO: 123, 124) are used as the protease.

[0123] (20) Sequence analysis of the GH25 family lysozyme 1. Sequence Similarity Network (SSN) SSNs are a method for visualizing and analyzing sequence similarity relationships within protein families. Following the previously reported method (see Biochemistry. 2018 Aug 7; 57(31): 4651-4662.), SSNs for the GH25 family lysozyme were created. First, 27,610 sequences registered in UniprotKB as containing PS51904 (GLYCOSYL_HYDROL_F25_2) were downloaded from Uniprot (www.uniprot.org / ). These 27,610 sequences were clustered using MMseq2 (see Bioinformatics. 2016; 32(9): 1323-1330.) to obtain sequences with 50% or more identity. 41 sequences (sequence numbers 1-3, 5-41, and 58) were added to the 4,464 sequences obtained by clustering, resulting in 4,505 sequences. From the 4505 sequences, 4470 sequences were obtained by removing those with 100% identity using CD-HIT (see Bioinformatics. 2006; 22(13): 1658-1659). These 4470 sequences were then compiled into a database using makeblastdb in Blast+ (see BMC Bioinformatics. 2009; 10: 421). Using the 4470 sequences as a query, blastp was performed on the created database of 4470 sequences (all vs all blast). The E-value was set to 10e-80. The obtained results were visualized using Cytoscape software (Figure 3). In SSN, each protein is shown as a "node," and each node is connected to other nodes by "edges." In this case, nodes of proteins with an E-value of 10e-80 or less are connected by edges. Since groups of proteins with similar amino acid sequences form clusters, proteins within the same cluster are likely to be functionally related. All 41 sequences used in the examples, including acm, KaLys, Ssp12Lys, Ssp14Lys, and Asp3Lys (shown as black circles in Figure 3), belonged to Cluster 2. We compared the amino acid residues important for activity in the major clusters, Clusters 1-4.Of the 241 sequences belonging to Cluster 2, 222 sequences contained D (9D) at position 9, D (98D) at position 98, and E (100E) at position 100 in the numbering of sequence number 1, which is the active site. Of these, 204 sequences contained K (33K) at position 33, E (36E) at position 36, Y (62Y) at position 62, Y (138Y) at position 138, and Q (183Q) at position 183 in the numbering of sequence number 1. Of the 471 sequences belonging to Cluster 1, 452 sequences contained 9D, 98D, and 100E, and of these, 85 sequences contained 33K, 36E, 62Y, 138Y, and 183Q. No sequences in Cluster3 or Cluster4 contained all of 9D, 33K, 36E, 62Y, 98D, 100E, 138Y, and 183Q. Therefore, it is considered that 9D, 33K, 36E, 62Y, 98D, 100E, 138Y, and 183Q are sequence features found in almost all of Cluster2 and some of Cluster1. Also, there is a motif DX containing 98D and 100E. 1 EX 2 NP(X 3 ) n G(X 1 , X 2 , and X 3 Each of the symbols independently represents an arbitrary amino acid residue, and n represents an integer from 1 to 5. Sequence ID No. 117) was found in 142 of the 222 sequences in Cluster 2 that had 9D, 98D, and 100E, while GH25 family lysozymes other than Cluster 2 did not have this motif. Furthermore, acm, Kalys, Ssp12Lys, Ssp14Lys, and Asp3Lys used in the examples all had this motif.

[0124] 2.Phylogenetic analysis Further phylogenetic analysis was performed on sequences closely related to Cluster2. For sequences classified as Cluster2 in the above SSN analysis, all sequences were recovered before clustering by MMseq2, yielding 3467 sequences. These 3467 sequences were clustered using CD-HIT to obtain 1182 sequences with 80% or more identity. 41 sequences (sequences 1-3, 5-41, and 58) were added to this to make a total of 1223 sequences, and multiple sequence alignments were created using MAFFT (FFT-NS-2). Only the alignments between the N-terminal and C-terminal positions of the mature acm (sequence 1) were extracted, and further trimal (see Bioinformatics. 2009; 25(15): 1972-1973.) was used to remove positions where more than 10% of the sequence was a gap, resulting in alignments of length 199. The alignment of the remaining 1193 sequences, after excluding 30 sequences with gaps in 20 or more positions out of 199, was analyzed phylogenetically using the WAG model with fasttree (see PloS One. 2010; 5(3): e9490.). The phylogenetic tree is shown in Figure 4. In the examples, acm, Ssp12Lys, Ssp14Lys, and Asp3Lys belonged to Clade 1, while KaLys belonged to Clade 2. For sequences belonging to each clade, motif DX 1 EX 2 NP[S / N / Y]G(X 1 and X 2The presence or absence of the motif (SEQ ID NO: 119), which independently represents any amino acid residue, was checked. Of the 474 sequences belonging to Clade 1 and containing 9D, 98D, and 100E, 268 sequences contained this motif. Of the 298 sequences belonging to Clade 2 and containing 9D, 98D, and 100E, 278 sequences contained this motif. Of the 410 sequences belonging to Clade 3 and containing 9D, 98D, and 100E, 89 sequences contained this motif. Therefore, the motif DXEXNP[S / N / Y]G is a sequence feature that is particularly common in Clade 1 and Clade 2, which contain acm, KaLys, Ssp12Lys, Ssp14Lys, and Asp3Lys, rather than in Clade 3. acm, KaLys, Ssp12Lys, Ssp14Lys, and Asp3Lys also contain this motif.

[0125] 3. Sequence Identity For the examples acm, Ssp12Lys, Ssp14Lys, Asp3Lys, and KaLys, the sequence identity between each amino acid sequence was calculated (Table 6). Amino acid sequence identity was calculated using the homology analysis (Search homology) program of GENETYX Ver.12, with a unit size to compare (ktup) of 2.

[0126] [Table 6]

Claims

1. A method for producing a biopolymer, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of a microorganism capable of producing a biopolymer or a treated product thereof under conditions of pH greater than 7.

0.

2. The method according to claim 1, further comprising a protease treatment step of reacting a cultured cell of a microorganism having biopolymer production ability or a processed product thereof with a protease under conditions of pH greater than 7.

0.

3. The method according to claim 2, wherein the GH25 family lysozyme treatment step and the protease treatment step are performed simultaneously.

4. The method according to claim 1, wherein the pH conditions in the GH25 family lysozyme treatment step are pH 8.0 or higher.

5. The method according to claim 1, wherein the GH25 family lysozyme is a polypeptide having D at position 9, K at position 33, E at position 36, Y at position 62, D at position 98, E at position 100, Y at position 138, and Q at position 183.

6. The aforementioned GH25 family lysozyme corresponds to the region from 98th to 105th in the numbering scheme of Sequence ID No. 1, and is Motif DX １ EX ２ NP(X ３ ) ｎ G(X) １ , X ２ , and X ３ The method according to claim 1, wherein each of the following independently represents an arbitrary amino acid residue, and n represents an integer from 1 to 5.

7. where the motif is DX １ EX ２ NP[S / N / Y]G(X １ and X ２ each independently represents any amino acid residue), the method according to claim 6.

8. The method according to claim 1, wherein the GH25 family lysozyme is at least one selected from the group consisting of polypeptides comprising amino acid sequences represented by any of SEQ ID NOs: 1-4 and 59-96, and polypeptides comprising amino acid sequences having at least 60% identity with the amino acid sequences represented by any of SEQ ID NOs: 1-4 and 59-96, and having lysozyme activity.

9. The method according to claim 1, wherein the GH25 family lysozyme is derived from bacteria or fungi.

10. A method for purifying biopolymers, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is reacted with cultured cells of a microorganism capable of producing biopolymers or a processed product thereof under conditions of pH greater than 7.

0.

11. The method according to any one of claims 1 to 10, wherein the biopolymer is a hydroxyalkanoic acid-containing polymer.

12. The method according to any one of claims 1 to 10, wherein the biopolymer is polyhydroxyalkanoic acid (PHA).

13. The method according to any one of claims 1 to 10, wherein the microorganism having biopolymer production ability is a microorganism belonging to the genus Aeromonas, Escherichia, or Cupriavidus.

14. An enzyme composition for purifying biopolymers, containing GH25 family lysozyme.

15. A method for purifying a target substance, comprising a GH25 family lysozyme treatment step in which GH25 family lysozyme is applied to cultured cells of a bacterium that produces the target substance within its cells, or a processed product thereof, under conditions of pH greater than 7.0.

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