Biopolymer production method

Abstract

The present invention provides a method for reducing the amount of DNA remaining in a biopolymer so as to increase the purity of the biopolymer when producing the biopolymer by means of a microorganism. This biopolymer production method includes: an endodeoxyribonuclease treatment step for bringing an endodeoxyribonuclease into contact with biopolymer granules obtained by culturing a microorganism capable of producing a biopolymer; and a pH adjustment step for keeping the biopolymer granules under the condition of a pH of 7.0 or higher at least at one time point after the start of said endodeoxyribonuclease treatment.

Description

Biopolymer manufacturing methods

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

[0002] Biopolymers, manufactured from biomass, 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] PHA, a type of biopolymer, can be produced by microbial fermentation. Industrial production of PHA requires purification of PHA from within densely cultured cells. Cell lysation is performed using heat treatment, mechanical physical treatment, chemical treatment with alkalis and surfactants, and biological treatment with enzymes. It is known that insoluble proteins remain in PHA granules obtained by cell lysation, and methods using surfactants and proteases are known to reduce these residual proteins (Patent Document 1). In addition, PHA granules may contain impurities derived from the producing cells other than proteins, with nucleic acids being cited as an example (Patent Document 2). On the other hand, it is generally believed that nucleic acids derived from PHA-producing cells are released into the processing solution by cell lysation and dissolution, and methods are known to reduce the viscosity of the processing solution by acting on the nucleic acids released into the processing solution with nucleases (Patent Document 3, Non-Patent Document 1). Therefore, it has not been clarified whether nucleic acids actually remain in the PHA granules obtained by cell lysation.

[0004] Since particularly high-purity PHA is required for applications such as medicine, further improvements in PHA purity are desired.

[0005] (Patent Document 1) Chinese Patent Application Publication No. 1190674 (Patent Document 2) International Publication No. 2018 / 186278 (Patent Document 3) International Publication No. 2023 / 027953 (Non-Patent Document 1) Gamero, Jesus E. Rodriguez, et al. Bioresource technology 261 (2018): 176-181.

[0006] The present invention relates to a method for producing a biopolymer, comprising: an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease; and a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher at least one time point after the start of the endodeoxyribonuclease treatment. The present invention also relates to a method for reducing the amount of residual DNA in a biopolymer produced by a microorganism capable of producing biopolymers, comprising an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease.

[0007] A diagram showing the effect of the pH of the treatment solution on the amount of residual nucleic acid in PHA. A diagram showing the effect of the type of nuclease on the amount of residual nucleic acid in PHA. A diagram showing the effect of the presence or absence of nuclease treatment on the powder properties of PHA. A diagram showing the effect of the presence or absence of nuclease treatment on the amount of residual nucleic acid in PHA. A diagram showing the effect of the presence or absence of nuclease treatment on the centrifugal washing efficiency of PHA. A diagram showing the effect of the presence or absence of nuclease treatment on the centrifugal washing efficiency of PHA. A diagram showing the effect of the pH of the treatment solution on the amount of residual nucleic acid in PHA. A diagram showing the effect of the combination of nuclease treatment and surfactant treatment on the amount of residual nucleic acid in PHA. A diagram showing the effect of the combination of nuclease treatment and surfactant (SDS) treatment on the amount of residual nucleic acid in PHA. Detailed description of the invention

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

[0009] In this specification, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12, with the Unit size to compare (ktup) set to 2.

[0010] 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. Also, "at least 90% identity" means identity of 90% or more, preferably preferably 91% or more, 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.

[0011] 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: 5) 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 Crystal W Multiple Alignment Program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Crystal W, such as Crystal W2 or Crystal omega, can be used. Crystal W, Crystal W2, and Crystal omega can be used, for example, on the Crystal website operated by University College Dublin [www.crystal.org], the European Bioinformatics Institute (EBI [www.ebii.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.

[0012] 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 examples 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.

[0013] The inventors of this invention have revealed that DNA unexpectedly adheres to PHA produced by microorganisms and purified by conventional purification methods, becoming insoluble and remaining as an impurity, thus identifying the problem of DNA residue in PHA. Therefore, the present invention relates to providing a method for reducing the amount of DNA remaining in a biopolymer and improving the purity of the biopolymer when producing a biopolymer using microorganisms capable of biopolymer production.

[0014] The inventors have found that the amount of DNA remaining in the biopolymer can be reduced by contacting biopolymer granules obtained by culturing microorganisms capable of producing biopolymers with endodeoxyribonuclease, and that the amount of DNA remaining in the biopolymer can be further reduced by placing the biopolymer granules under conditions of pH 7.0 or higher.

[0015] According to the method of the present invention, in the production of biopolymers using microorganisms capable of producing biopolymers, the amount of DNA remaining in the biopolymer can be reduced and the purity of the biopolymer can be improved.

[0016] 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 an endodeoxyribonuclease treatment step in which biopolymer granules obtained by culturing microorganisms capable of producing biopolymers are contacted with endodeoxyribonuclease, and a pH adjustment step in which the biopolymer granules are placed under conditions of pH 7.0 or higher at least one time point after the start of the endodeoxyribonuclease treatment. Hereinafter, the method for producing biopolymers of the present invention will be simply referred to as the method of the present invention.

[0017] 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.

[0018] 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.

[0019] 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. The hydroxyalkanoic acid is not particularly limited, but examples include (R)-3-hydroxybutanoic acid ((R)-3-hydroxybutyric acid: 3HB), 4-hydroxybutanoic acid (4-hydroxybutyric acid: 4HB), 3-hydroxypropionic acid (3-hydroxypropionic acid: 3HP), (R)-3-hydroxypentanoic acid ((R)-3-hydroxyvaleric acid, (R)-3-hydroxyvaleric acid: 3HV), (R)-3-hydroxyhexanoic acid ((R)-3-hydroxyoctanoic acid: 3HHx), (R)-3-hydroxyoctanoic acid ((R)-3-hydroxyoctanoic acid Examples include (R)-3-hydroxydecanoic acid ((R)-3-hydroxydecanoic acid: 3HD), (R)-3-hydroxydodecanoic acid ((R)-3-hydroxydodecanoic acid: 3HDD), and (R)-3-hydroxytetradecanoic acid ((R)-3-hydroxytetradecanoic acid: 3HTD). PHA may be a homopolymer composed of one monomer unit or a copolymer composed of two or more monomer units.As PHA, there are poly[(R)-3-hydroxybutanoic acid]{poly[(R)-3-hydroxybutyric acid]:P(3HB)}, poly[(R)-3-hydroxybutanoic acid-co-(R)-3-hydroxypentanoic acid]{poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid]:P(3HB-co-3HV)}, and poly[(R)-3-hydroxybutanoic acid-co-(R)-4-hydroxybutanoic acid]{poly[(R)-3-hydroxybutyric acid-co-(R)-4-hydroxybutyric Examples include {(R)-3-hydroxybutanoic acid-co-(R)-3-hydroxyhexanoic acid}:P(3HB-co-3HHx)}, and {(R)-3-hydroxybutanoic 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.

[0020] 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, microorganisms belonging to the genera Aeromonas, Cupriavidus, and Escherichia are preferred in terms of PHA productivity, 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 the preferred choice.

[0021] By culturing microorganisms capable of producing biopolymers, biopolymers are produced within the cells of these microorganisms. The produced biopolymers typically exist in the form of biopolymer granules.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 may 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.

[0026] Culturing is preferably carried out under aerobic conditions, and general 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.

[0027] In the endodeoxyribonuclease treatment process, residual DNA derived from bacterial cells attached to the biopolymer in the biopolymer granules can be reduced in molecular weight and solubilized by the action of endodeoxyribonuclease. The solubilized low-molecular-weight DNA is removed when the biopolymer is recovered. Therefore, the endodeoxyribonuclease treatment process can reduce the amount of DNA attached to and remaining on the biopolymer. Thus, the endodeoxyribonuclease treatment process is a process for purifying the biopolymer and increasing its purity.

[0028] Here, "endodeoxyribonuclease" refers to a protein that possesses endodeoxyribonuclease activity, which hydrolyzes phosphodiester bonds within DNA strands.

[0029] The endodeoxyribonuclease used in the present invention is not particularly limited, but is preferably a bacterial endodeoxyribonuclease, more preferably an endodeoxyribonuclease derived from bacteria of the genus Bacillus, an endodeoxyribonuclease derived from bacteria of the genus Staphylococcus, and an endodeoxyribonuclease derived from bacteria of the genus Serratia. A preferred example of an endodeoxyribonuclease is Mn derived from Bacillus subtilis. ２＋ / Mg ２＋ Dependent endodeoxyribonuclease (e.g., BsNucB in the examples), Mn derived from Bacillus licheniformis. ２＋ / Mg２＋ Dependent endodeoxyribonuclease (e.g., BlNucB in the example), Ca derived from Staphylococcus aureus ２＋ Examples of endodeoxyribonucleases that hydrolyze DNA and RNA in an endo-type manner depending on the enzyme (e.g., SNase in the examples), and endonucleases derived from Serratia marcescens are also mentioned. Another preferred example of an endodeoxyribonuclease is an endodeoxyribonuclease containing a Pfam domain such as the DNase_NucA_NucB domain (PF14040), DUF1524 domain (PF07510), Endonuclease_NS domain (PF01223), or SNase domain (PF00565). Preferred examples of endodeoxyribonucleases containing the DNase_NucA_NucB domain (PF14040) include BsNucB from the example, BlNucB from the example, and endodeoxyribonucleases derived from Aspergillus oryzae (SEQ ID NOs. 12, 13) described in SEQ ID NOs. 14 and 14 of JP 2022-550112. Preferred examples of endodeoxyribonucleases containing the DUF1524 domain (PF07510) include endodeoxyribonucleases derived from the Bacillus genus (SEQ ID NOs. 14-18) described in SEQ ID NOs. 5-9 of JP 2022-550112. A preferred example of an endodeoxyribonuclease containing the Endonuclease_NS domain (PF01233) is the endonuclease derived from Serratia marcescens in the example. A preferred example of an endodeoxyribonuclease containing the SNase domain (PF00565) is SNase in the example. Sequence information of polypeptides containing each Pfam domain can be obtained from the InterPro database (ebi.ac.uk / interpro / ).

[0030] A preferred example of BsNucB is a polypeptide (mature enzyme) consisting of the amino acid sequence shown in SEQ ID NO: 5. Another example of BsNucB is a polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 5, and possessing endodeoxyribonuclease activity. A preferred example of BlNucB is a polypeptide (mature enzyme) consisting of the amino acid sequence shown in SEQ ID NO: 6. Another example of BlNucB is a polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 6, and possessing endodeoxyribonuclease activity. A preferred example of SNase is a polypeptide (mature enzyme) consisting of the amino acid sequence shown in SEQ ID NO: 10. Another example of SNase is a polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 10, and possessing endodeoxyribonuclease activity. A preferred example of an endonuclease derived from Serratia marcescens is a polypeptide (mature enzyme) consisting of the amino acid sequence shown in SEQ ID NO: 11. Another example of an endonuclease derived from Serratia marcescens is a polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 11, and having endodeoxyribonuclease activity. Here, endodeoxyribonuclease activity can be measured by methods known in the art.

[0031] The endodeoxyribonucleases used in the present invention can be extracted or prepared from microorganisms containing them or from their cultures. For example, BsNucB can be extracted or prepared from Bacillus subtilis strain 168 (NBRC 111470), etc., or from its culture; BlNucB can be extracted or prepared from Bacillus licheniformis NBRC 12200, etc., or from its culture; SNase can be extracted or prepared from Staphylococcus aureus NCTC 8325, etc., or from its culture; and Serratia marcescens-derived endonucleases can be extracted or prepared from Serratia marcescens NBRC 102204, etc., or from its culture. The above-mentioned microorganisms can be purchased from public microorganism storage institutions. The microorganisms containing the endodeoxyribonuclease can be cultured under appropriate conditions using a culture medium containing assimilated carbon sources, nitrogen sources, metal salts, vitamins, etc. From the microorganisms or culture medium thus obtained, the enzyme 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 the enzyme from the culture can be carried out using conventional methods such as separation of microorganisms 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.

[0032] Alternatively, the endodeoxyribonuclease used in the present invention can be produced by chemical synthesis or microbiological methods. In the microbiological production of endodeoxyribonuclease, it is preferable to express the endodeoxyribonuclease as an endodeoxyribonuclease precursor containing a signal sequence and a mature enzyme region.

[0033] The signal sequence is involved in the extracellular secretion of endodeoxyribonuclease. In an endodeoxyribonuclease precursor containing a signal sequence and a maturation enzyme region, the signal sequence is preferably located at the N-terminus of the maturation enzyme region. The signal sequence is cleaved by the signal peptidase when the endodeoxyribonuclease precursor passes through the cell membrane. Examples of signal sequences include sequences consisting of amino acids that are at least 90% identical to the amino acid sequence at positions 1-26 of SEQ ID NO: 2, and sequences consisting of amino acids that are at least 90% identical to the amino acid sequence at positions 1-32 of SEQ ID NO: 4, for example, the signal sequences of BsNucB and BlNucB mentioned above. Another example of a signal sequence is a sequence consisting of amino acids that are at least 90% identical to the amino acid sequence of SEQ ID NO: 8, for example, the signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0034] Therefore, the endodeoxyribonuclease precursor may be a polypeptide comprising a signal sequence and a mature enzyme region, and having an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2. Alternatively, the endodeoxyribonuclease precursor may be a polypeptide comprising a signal sequence and a mature enzyme region, and having an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 4. Alternatively, the endodeoxyribonuclease precursor may be a polypeptide comprising a signal sequence and a mature enzyme region, and having an amino acid sequence that is at least 60% identical to the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10.

[0035] Endodeoxyribonuclease precursors can be produced by expressing a polypeptide from a polynucleotide encoding the endodeoxyribonuclease precursor. This polynucleotide can be prepared by extracting genomic DNA from a microorganism producing the target endodeoxyribonuclease precursor using conventional methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence may be chemically synthesized based on the amino acid sequence of the target endodeoxyribonuclease precursor and used as the polynucleotide encoding the target endodeoxyribonuclease precursor. This polynucleotide may include a nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). Furthermore, this polynucleotide may be codon-optimized to suit the species of transformant for endodeoxyribonuclease precursor production.

[0036] The polynucleotide encoding the obtained endodeoxyribonuclease precursor can be incorporated into a vector. The vector can be prepared by inserting the polynucleotide into any vector using a conventional method. 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, but is not limited, preferably a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or its mutants), and more preferably an expression vector that can induce the expression of the transgene in Bacillus bacteria. Among these, a shuttle vector, which is a vector that can replicate in either Bacillus bacteria or other organisms, can be suitably used for recombinant production of the mutants of the present invention. 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 (Nuclide Acids Res, 1988, 16:8732); and plasmid vectors usable for transforming Bacillus bacteria such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0037] When recombinantly producing an endodeoxyribonuclease precursor, the vector is preferably an expression vector. The expression vector may contain various elements essential for expression in a host, such as a transcription promoter, a terminator, a ribosome binding site; cis elements such as a polylinker, an enhancer; a polyA addition signal; a ribosome binding sequence (SD sequence); a selection marker gene such as a drug (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloramphenicol, etc.) resistance gene, and other useful sequences as required. Alternatively, the polynucleotide encoding the protease precursor may contain the above useful sequences.

[0038] By introducing a polynucleotide encoding an endodeoxyribonuclease precursor or a vector containing the same into a host, a transformant containing the polynucleotide encoding the endodeoxyribonuclease precursor or the vector containing the same can be obtained.

[0039] Examples of the host for the transformant include bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Clostridium, yeast, etc. Among them, bacteria of the genus Bacillus are preferred, and Bacillus subtilis or its mutant strain is more preferred. Therefore, the transformant is preferably a recombinant bacterium of the genus Bacillus, and more preferably a recombinant of Bacillus subtilis or its mutant strain. Examples of the Bacillus subtilis mutant strain include a strain lacking aprX and a gene selected from aprE, nprB, nprE, bpr, vpr, mpr, epr, and wprA (Japanese Patent Laid-Open No. 2006-174707).

[0040] For the introduction of polynucleotides and vectors into host cells, well-known transformation techniques such as the protoplast method, calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method, etc. can be applied. For example, methods applicable to Bacillus subtilis or its mutants include the competent cell transformation method (J Bacteriol, 1967, 93: 1925 - 1937), the electroporation method (FEMS Microbiol Lett, 1990, 55: 135 - 138), the protoplast transformation method (Mol Gen Genet, 1979, 168: 111 - 115), the Tris-PEG method (J Bacteriol, 1983, 156: 1130 - 1134), etc.

[0041] If the transformant is cultured in an appropriate medium, the endodeoxyribonuclease precursor can be expressed. The expressed endodeoxyribonuclease precursor becomes the mature enzyme of endodeoxyribonuclease through the cleavage of the signal sequence. Furthermore, when the endodeoxyribonuclease precursor has a signal sequence, the mature enzyme is secreted and produced extracellularly.

[0042] The transformant may be cultured under appropriate conditions using a medium containing an assimilable carbon source, nitrogen source, metal salt, vitamin, etc. From the thus obtained microorganism or culture, the mature enzyme of endodeoxyribonuclease can be collected and prepared by a general method, and further, the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization, etc. For example, the recovery and preparation of the enzyme from the culture can be carried out by separating the microorganism by centrifugation or filtration, precipitation by adding a salt such as ammonium sulfate or precipitation by adding an organic solvent such as ethanol to the enzyme in the supernatant or filtrate, concentration and desalting using an ultrafiltration membrane, etc., and purification using various chromatographies such as ion exchange or gel filtration, etc., by ordinary methods.

[0043] Alternatively, the endodeoxyribonuclease used in the present invention can be produced by enhancing the expression of endodeoxyribonuclease in microorganisms capable of biopolymer production through transformation. For example, in the microbiological production of the endodeoxyribonuclease described above, by using microorganisms capable of biopolymer production as hosts for the transformants, the expression of endodeoxyribonuclease can be enhanced in the microorganisms capable of biopolymer production. In this case, since endodeoxyribonuclease is also expressed during the biopolymer production culture by the microorganisms capable of biopolymer production, the action of the endodeoxyribonuclease can be utilized in the endodeoxyribonuclease treatment step.

[0044] Alternatively, the endodeoxyribonuclease used in the present invention may be a commercially available endodeoxyribonuclease. Examples of commercially available endodeoxyribonucleases include endonuclease derived from Serratia marcescens (e.g., SIGMA, E1014), deoxyribonuclease I derived from bovine pancreas (e.g., SIGMA, D4513), high-salt-tolerant nuclease (SAN) (e.g., SIGMA, SRE0015), micrococcal nuclease derived from Staphylococcus aureus (e.g., TaKaRa, 2910A), and DENARASE (c-LEcta).

[0045] The above endodeoxyribonucleases may be used individually or in combination of two or more.

[0046] The means for contacting biopolymer granules with endodeoxyribonuclease is not particularly limited, as long as it allows endodeoxyribonuclease to directly contact the biopolymer granules. For example, such means include lysing cultured cells of a microorganism capable of producing biopolymers and adding endodeoxyribonuclease to a suspension containing the resulting lysate; adding endodeoxyribonuclease when lysing cultured cells of a microorganism capable of producing biopolymers; adding endodeoxyribonuclease to the culture medium of a microorganism capable of producing biopolymers and then lysing the cultured cells of the microorganism; or lysing cultured cells of a microorganism capable of producing biopolymers in which endodeoxyribonuclease expression is enhanced. By lysing cultured cells of a microorganism capable of producing biopolymers, biopolymer granules are separated from the cultured cells, facilitating contact with endodeoxyribonuclease. Therefore, the method of the present invention may further include a lysing step of lysing cultured cells of a microorganism capable of producing biopolymers. From the viewpoint of improving the purity of the biopolymer, the crushing process should preferably be performed before or simultaneously with the endodeoxyribonuclease treatment process.

[0047] Methods for disrupting the cells of microorganisms capable of producing biopolymers include physical disruption using mechanical forces such as heat treatment, ultrasound, high-pressure homogenation, shearing force using in-line mixers, and crushing; chemical treatment with alkalis and surfactants; and biological treatment with lytic enzymes. Autolysis, which utilizes the action of enzymes such as proteases and esterases produced by the microorganisms themselves, is also a method of disruption.

[0048] The heat treatment can be carried out by maintaining the cells of microorganisms capable of producing biopolymers at a temperature of preferably 40 to 80°C, more preferably 50 to 80°C, and even more preferably 60 to 80°C. The treatment time is preferably 0.2 to 20 hours, more preferably 0.5 to 10 hours.

[0049] Lysozymes are enzymes that break down bacterial peptidoglycans. Examples of lysozymes include muramidase, amidase, glucosaminidase, and endopeptidase, with muramidase (defined as lysozyme in this specification) being preferred, and more preferably lysozymes classified as GH22, GH23, GH24, or GH25 in the glycoside hydrolase (GH) family (CAZy, www.cazy.org). A preferred example of GH22 lysozyme is chicken egg white lysozyme. Furthermore, preferred examples of GH25 lysozyme include lysozyme acm (UniProt id: P25310, SEQ ID NO: 36), Ssp12Lys (SEQ ID NO: 32), Ssp14Lys (SEQ ID NO: 33), Asp3Lys (SEQ ID NO: 34), and KaLys (SEQ ID NO: 35) derived from Streptomyces globisporus. Another preferred example of lysozyme is the LYS polypeptide described in Japanese Patent No. 7275048 (for example, the LYS polypeptides of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, or 45 of Japanese Patent No. 7275048). Other preferred examples of GH25 lysozyme include GH25 muramidase derived from Acremonium alcalophilum (SEQ ID NOs. 23, 24) as described in SEQ ID NOs. 1 or 3 in WO2023 / 110957A1, and GH25 muramidase derived from Aspergillus fumigatus (UniProt id: A4DA29, SEQ ID NOs. 25). A preferred example of GH24 lysozyme is GH24 muramidase derived from Trichophaea saccate (SEQ ID NOs. 26) as described in SEQ ID NOs. 2 in WO2023 / 110957A1. Lysolytic enzymes can be produced, as with endodeoxyribonucleases described above, by preparing them from microorganisms containing lysolytic enzymes or their cultures, by chemical synthesis or microbiological methods, or by enhancing the expression of lysolytic enzymes in microorganisms capable of biopolymer production through transformation.The lytic enzyme can be a commercially available lytic enzyme, such as chicken egg white lysozyme (Fujifilm Wako Pure Chemical Industries, 127-06724), mutanolicin (SIGMA, M9901), a lytic enzyme mainly composed of acm, achromopeptidase (Fujifilm Wako Pure Chemical Industries, 014-09661), a lytic enzyme derived from Lysobacter enzymogenes, and Raviace (Cosmo Bio, OZ-30EX), a lytic enzyme derived from Streptomyces fulvissimus.

[0050] The above-described crushing methods can be used individually or in combination of two or more, but it is preferable to use at least heat treatment of the microbial cells, lysozyme treatment in which lysozyme is brought into contact with the microbial cells, or heat treatment and lysozyme treatment of the microbial cells. Appropriate microbial cell crushing conditions can be appropriately determined by those skilled in the art in accordance with the type, shape, and amount of microorganisms capable of producing biopolymers, the type, shape, and amount of biopolymers, etc.

[0051] The reaction conditions between biopolymer granules and endodeoxyribonuclease are not particularly limited, as long as the endodeoxyribonuclease is not inactivated. Appropriate reaction conditions can be determined by those skilled in the art in accordance with the type, shape, and amount of microorganisms capable of producing biopolymers, the type, shape, and amount of biopolymer, and the type and amount of endodeoxyribonuclease. Examples of reaction conditions are described below.

[0052] The amount of endodeoxyribonuclease used in the reaction is appropriately determined depending on the type, shape, and quantity of the microorganism having biopolymer production ability. For example, the amount of endodeoxyribonuclease 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 100% of the mass of the microorganism having biopolymer production ability or its cell lysate, in terms of dry mass.

[0053] The pH conditions (25°C) for this reaction are preferably pH 6.0 to 13.0, more preferably pH 7.0 to 12.0, and even more preferably pH 8.0 to 11.5, from the viewpoint of maintaining enzyme activity and improving the purity of the biopolymer.

[0054] The temperature conditions for this reaction are preferably 30 to 80°C, more preferably 40 to 70°C, and even more preferably 40 to 60°C, from the viewpoint of improving the purity of the biopolymer.

[0055] 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.

[0056] In the pH adjustment step, by placing the biopolymer granules under conditions of pH 7.0 or higher at least one time point after the start of endodeoxyribonuclease treatment, the amount of DNA adhering to and remaining on the biopolymer can be further reduced. Thus, the pH adjustment step is a process for purifying the biopolymer and increasing its purity.

[0057] The pH conditions (at 25°C) in which the biopolymer granules are placed should be pH 7.0 or higher, 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 greater than 9.0, 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, and even more preferably pH 12.0 or lower. Furthermore, the pH conditions should preferably be pH 7.0 to 13.0, more preferably pH 7.5 to 13.0, even more preferably pH 8.0 to 12.5, even more preferably pH 8.5 to 12.5, even more preferably pH 9.0 to 12.5, even more preferably pH greater than 9.0 and pH 12.5 or lower, even more preferably pH 9.5 to 12.0, and even more preferably pH 10.0 to 12.0. The pH is measured by the glass electrode method.

[0058] For pH adjustment, an alkaline agent can be used, for example. 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.

[0059] The order in which the endodeoxyribonuclease treatment and pH adjustment steps are performed is not particularly limited, as long as the order ensures that the biopolymer granules are under conditions of pH 7.0 or higher at least at one point after the start of the endodeoxyribonuclease treatment, from the viewpoint of improving the purity of the biopolymer. It is preferable to perform the pH adjustment step after the endodeoxyribonuclease treatment step or to perform the endodeoxyribonuclease treatment step and pH adjustment step simultaneously. It is more preferable to perform the endodeoxyribonuclease treatment step and pH adjustment step consecutively in this order, to perform the endodeoxyribonuclease treatment step and pH adjustment step independently in this order, or to perform the endodeoxyribonuclease treatment step and pH adjustment step simultaneously. Performing the endodeoxyribonuclease treatment step and pH adjustment step consecutively preferably means treating the biopolymer granules with endodeoxyribonuclease and then adjusting the pH of the treatment solution to 7.0 or higher after the treatment. Independent implementation of the endodeoxyribonuclease treatment step and the pH adjustment step preferably means treating the biopolymer granules with endodeoxyribonuclease, removing the supernatant after treatment, and adding an aqueous solution with a pH of 7.0 or higher to the biopolymer granules. Here, the aqueous solution with a pH of 7.0 or higher can function as a washing solution for the biopolymer granules. Simultaneous implementation of the endodeoxyribonuclease treatment step and the pH adjustment step preferably means treating the biopolymer granules with endodeoxyribonuclease under conditions of a pH of 7.0 or higher. In this case, the pH condition should be set appropriately within the range of pH 7.0 or higher, taking into consideration the maintenance of the enzyme activity of endodeoxyribonuclease. When the endodeoxyribonuclease treatment process and the pH adjustment process are carried out simultaneously, the pH adjustment process may be performed again after the simultaneous treatment. Preferably, this involves treating the biopolymer granules with endodeoxyribonuclease under conditions of pH 7.0 or higher, removing the supernatant of the treatment solution after treatment, and adding an aqueous solution with a pH of 7.0 or higher to the biopolymer granules. The time for which the biopolymer granules are kept under conditions of pH 7.0 or higher is not particularly limited and can be set appropriately considering the embodiment of the pH adjustment process.

[0060] The method of the present invention may further include a protease treatment step in which a protease is brought into contact with biopolymer granules as a step for purifying the biopolymer, in order to improve the purity of the biopolymer.

[0061] Here, "protease" refers to a protein that possesses protease activity, which hydrolyzes the peptide bonds of protein molecules to produce peptides and amino acids.

[0062] The protease used in the present invention is not particularly limited, but preferably it is a protease that has protease activity in an alkaline environment, more preferably it is a serine protease (EC number 3.4.21), even more preferably it is a protease of the S8 peptidase family in the MEROPS classification system, and even more preferably it is a protease derived from bacteria of the genus Bacillus. Preferred examples of proteases include alkaline protease KP43 (SEQ ID NO: 19) derived from Bacillus sp. KSM-KP43 as described in Japanese Patent Application No. 2024-071822 and its variants (e.g., variant 12, SEQ ID NO: 4 (SEQ ID NO: 20) of Japanese Patent Application No. 2024-071822), and Bacillus sp. KSM-K16 (FERM) as described in Japanese Patent Application No. 2024-071822. Examples include alkaline protease K16 (SEQ ID NO: 21) derived from BP-3376 and its variants (e.g., quadruple variant, SEQ ID NO: 8 (SEQ ID NO: 22) of Japanese Patent Application No. 2024-071822), subtilisin derived from Bacillus lentus and its variants, HH844 (SEQ ID NO: 27) and its variants (SEQ ID NOs: 28, 29), and variants of LL147 (SEQ ID NOs: 30, 31). The protease used in the method of the present invention can be prepared from a microorganism containing a protease or a culture thereof, manufactured by chemical synthesis or microbiological methods, or produced by enhancing the protease expression of a microorganism capable of biopolymer production through transformation, similar to the case of the endodeoxyribonuclease described above. 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, Everase, Savinase, Kannase, Progress Uno (registered trademark; Novonesys Corporation), Preferenz (registered trademark; IFF Corporation) series, and Lavergy (registered trademark; BASF Corporation).

[0063] The above proteases may be used individually or in combination of two or more.

[0064] The means for contacting the biopolymer granules with the protease, and the reaction conditions between the biopolymer granules and the protease, are the same as those for the endodeoxyribonuclease described above.

[0065] The order in which the protease treatment steps are performed is not particularly limited, and the order in which the endodeoxyribonuclease treatment step and the pH adjustment step, or both steps, are performed with respect to the protease treatment step can be any. From the viewpoint of improving the purity of the biopolymer and increasing the efficiency of production, it is preferable to perform the protease treatment step at least simultaneously with the endodeoxyribonuclease treatment step.

[0066] When the endodeoxyribonuclease treatment step and the protease treatment step are performed simultaneously, it is preferable to use an endodeoxyribonuclease that is protease-resistant to the extent that it can maintain its activity in degrading DNA remaining in the biopolymer in the presence of a protease, in order to maintain the activity of the endodeoxyribonuclease. Examples of such endodeoxyribonucleases include BsNucB, BlNucB, SNase, and endonucleases derived from Serratia marcescens. Alternatively, protease-resistant endodeoxyribonuclease mutants may be used. Examples of such endodeoxyribonucleases include the endodeoxyribonuclease mutants described in WO2022 / 194668A1.

[0067] The method of the present invention may further include a surfactant treatment step in which a surfactant is brought into contact with biopolymer granules as a step for purifying the biopolymer, in order to improve the purity of the biopolymer.

[0068] The surfactants used in the present invention include one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, but anionic surfactants are preferred from the viewpoint of reducing residual DNA in biopolymers. Examples of anionic surfactants include sulfate ester salts of alcohols having 10 to 18 carbon atoms, sulfate ester 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, at least one anionic surfactant selected from sulfated alcohols having 10 to 14 carbon atoms in the alkyl chain, sulfated ethoxylated alcohols having 10 to 14 carbon atoms in the alkyl chain, and linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain is preferred, and at least one anionic surfactant selected from sulfated alcohols having 10 to 14 carbon atoms in the alkyl chain and sulfated ethoxylated alcohols having 10 to 14 carbon atoms in the alkyl chain is more preferred. Alternatively, among at least one anionic surfactant selected from sulfated alcohols having 10 to 14 carbon atoms in the alkyl chain, sulfated ethoxylated alcohols having 10 to 14 carbon atoms in the alkyl chain, and linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain, at least one anionic surfactant selected from sulfated alcohols having 12 carbon atoms in the alkyl chain, sulfated ethoxylated alcohols having 12 carbon atoms in the alkyl chain, and linear alkylbenzene sulfonates having 12 carbon atoms in the alkyl chain is more preferred, at least one anionic surfactant selected from sulfated alcohols having 12 carbon atoms in the alkyl chain and sulfated ethoxylated alcohols having 12 carbon atoms in the alkyl chain is even more preferred, and at least one anionic surfactant selected from dodecyl sulfate and polyoxyethylene lauryl ether sulfate is even more preferred.As counterions, alkali metal salts, alkanolamine salts, and ammonium salts are preferred, sodium and / or potassium, monoethanolamine, diethanolamine, triethanolamine, and ammonium ions are more preferred, and sodium is even more preferred. Preferred specific examples of anionic surfactants include at least one selected from sodium dodecyl sulfate, sodium polyoxyethylene lauryl ether sulfate, sodium dodecylbenzenesulfonate, triethanolamine dodecyl sulfate, and ammonium dodecyl sulfate; more preferably at least one selected from sodium dodecyl sulfate, sodium polyoxyethylene lauryl ether sulfate, and sodium dodecylbenzenesulfonate; and even more preferably at least one selected from sodium dodecyl sulfate and sodium polyoxyethylene lauryl ether sulfate.

[0069] The amount of surfactant used is appropriately determined depending on the type, shape, and quantity of microorganisms capable of producing biopolymers. For example, the amount of surfactant used is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, and even more preferably 0.1 to 3% by mass, based on 100% of the mass of the microorganisms capable of producing biopolymers or their cell lysates, in terms of dry mass.

[0070] The method for bringing the biopolymer granules into contact with the surfactant is the same as in the case of the endodeoxyribonuclease described above.

[0071] The order in which the surfactant treatment steps are performed is not particularly limited, and the order in which the endodeoxyribonuclease treatment step and the pH adjustment step, or both steps, are performed with respect to the surfactant treatment step can be any. From the viewpoint of improving the purity of the biopolymer and increasing the efficiency of production, it is preferable to perform the surfactant treatment step simultaneously with at least one of the endodeoxyribonuclease treatment step and the pH adjustment step.

[0072] In a preferred embodiment, the method of the present invention includes an endodeoxyribonuclease treatment step, a lysozyme treatment step as a crushing step, a pH adjustment step, a protease treatment step, and a surfactant treatment step. The order in which each step is performed is not particularly limited, as long as the order ensures that the biopolymer granules are under conditions of pH 7.0 or higher at least at one point in time after the start of the endodeoxyribonuclease treatment. From the viewpoint of improving the purity of the biopolymer and increasing the efficiency of production, it is preferable to perform two or more steps simultaneously, more preferably three or more steps simultaneously, and even more preferably four or more steps simultaneously. For example, methods include performing the endodeoxyribonuclease treatment step and the lysozyme treatment step simultaneously, followed by the pH adjustment step, the protease treatment step, and the surfactant treatment step in that order; performing the endodeoxyribonuclease treatment step, the lysozyme treatment step, and the protease treatment step simultaneously, followed by the pH adjustment step and the surfactant treatment step simultaneously; and performing the endodeoxyribonuclease treatment step, the lysozyme treatment step, the pH adjustment step, the protease treatment step, and the surfactant treatment step all simultaneously.

[0073] The method of the present invention may further include, from the viewpoint of improving the purity of the biopolymer, at least one step selected from an enzyme 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] In the enzyme treatment process, the purity of the biopolymer can be improved by acting on the cells of a microorganism capable of producing biopolymers or biopolymer granules with enzymes other than endodeoxyribonuclease and protease. The enzymes other than endodeoxyribonuclease and protease can be any enzyme capable of hydrolyzing components derived from the cells, such as lysozyme, muramidase, glycosidase, cellulase, lipase, amylase, cutinase, laccase, nuclease, and nucleases other than endodeoxyribonuclease. 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 producing biopolymers, depending on the type of enzyme used.

[0075] In the oxidizing agent treatment step, contact with the cells of microorganisms capable of producing biopolymers or biopolymer granules with an oxidizing agent reduces the molecular weight of impurities derived from the cells, improving the purity of the biopolymer, reducing the membrane filtration load in the membrane filtration step, and suppressing membrane clogging. Furthermore, by combining the endodeoxyribonuclease treatment step and the oxidizing agent treatment step, the molecular weight reduction of impurities such as nucleic acids in the treatment solution is further promoted, reducing the viscosity of the treatment solution and improving the stirring efficiency in each step, the centrifugal efficiency in the centrifugation step, and the efficiency of the membrane filtration step. In addition, the decolorization of the biopolymer can improve 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. 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 demolecularization, 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, 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. To improve 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, preferably 0.02 to 0.08 g, and more preferably 0.02 to 0.07 g.

[0076] In the centrifugation process, the purity of the biopolymer can be improved by removing unwanted supernatant from the processing liquid containing the biopolymer granules. 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.

[0077] 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.

[0078] In the washing process, impurities derived from bacterial cells can be removed by washing the biopolymer granules 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.

[0079] The order in which at least one of the above processes is performed is not particularly limited, and multiple processes can be performed simultaneously.

[0080] The solid component of the processing liquid obtained after the purification process described above contains a biopolymer, and the biopolymer can be recovered by recovering the solid component of the processing liquid. Therefore, the method of the present invention may further include a recovery step for recovering the biopolymer from the processing liquid. 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 liquid into solid and liquid components by centrifugation or the like to obtain a solid component, washing the solid component as necessary, and then drying the solid component by spray drying, evaporation to dryness, freeze-drying, or the like. By this step, a biopolymer powder can be obtained.

[0081] According to the method of the present invention, the amount of DNA adhering to and remaining in the final biopolymer can be effectively reduced, making it possible to produce a biopolymer with high purity. The amount of DNA remaining in the biopolymer produced by the method of the present invention can be reduced to preferably 70% or less, more preferably 50% or less, and even more preferably 40% or less per unit amount of biopolymer powder, compared to the amount of DNA remaining in the biopolymer produced by the same method as the method of the present invention, except that the endodeoxyribonuclease treatment step is not included. Furthermore, the method of the present invention can also contribute to improving the efficiency of biopolymer recovery, reducing the adhesion of the final biopolymer, and improving its flowability.

[0082] The above-described endodeoxyribonuclease treatment step is a step to reduce the amount of DNA adhering to and remaining on the biopolymer and to improve the purity of the biopolymer. Therefore, the present invention also provides a method for improving the purity of a biopolymer produced by a microorganism capable of producing biopolymers, or a method for reducing the amount of DNA remaining on a biopolymer produced by a microorganism capable of producing biopolymers. The method includes an endodeoxyribonuclease treatment step in which biopolymer granules obtained by culturing a microorganism capable of producing biopolymers are contacted with endodeoxyribonuclease. The method may include at least one step selected from the above-described pH adjustment step, protease treatment step, and surfactant treatment step, and may further include at least one step selected from the above-described heat treatment step, enzyme 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 present invention's method for producing biopolymers using a microorganism capable of producing biopolymers. The amount of residual DNA reduced by the method for reducing the amount of residual DNA in the biopolymer of the present invention may be preferably 30% or more, more preferably 50% or more, and even more preferably 60% or more per unit amount of biopolymer powder.

[0083] 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.

[0084] [1] A method for producing a biopolymer, comprising: an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease; and a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher at least one time point after the start of the endodeoxyribonuclease treatment. [2] The method according to [1], wherein the endodeoxyribonuclease treatment step is a step of reducing the molecular weight of DNA attached to the biopolymer by endodeoxyribonuclease and solubilizing it. [3] The method according to [1] or [2], wherein the pH adjustment step is performed after the endodeoxyribonuclease treatment step, or the endodeoxyribonuclease treatment step and the pH adjustment step are performed simultaneously. [4] The method according to any one of [1] to [3], further comprising a surfactant treatment step of contacting the biopolymer granules with a surfactant. [5] The method according to [4], wherein the surfactant is an anionic surfactant. [6] The method according to [4] or [5], wherein at least one of the endodeoxyribonuclease treatment step and the pH adjustment step and the surfactant treatment step are performed simultaneously. [7] The method according to any one of [1] to [6], further comprising a protease treatment step of contacting the biopolymer granules with a protease. [8] The method according to [7], wherein the endodeoxyribonuclease treatment step and the protease treatment step are performed simultaneously. [9] The method according to any one of [1] to [8], further comprising a lysozyme treatment step of contacting the cells of a microorganism having biopolymer production ability with lysozyme.

[10] The method according to [9], wherein the endodeoxyribonuclease treatment step, the protease treatment step and the lysozyme treatment step are performed simultaneously.

[11] The method according to [9] or

[10] , wherein the endodeoxyribonuclease treatment step, the pH adjustment step, the protease treatment step, the lysozyme treatment step and the surfactant treatment step are carried out simultaneously.

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

[11] , further comprising a culture step of culturing the microorganism having biopolymer production ability before the endodeoxyribonuclease treatment step.

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

[12] , further comprising a crushing step of crushing cultured cells of a microorganism having biopolymer production ability.

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

[13] , further comprising a recovery step of recovering a biopolymer.

[0085]

[15] A method for improving the purity of a biopolymer produced by a microorganism capable of producing biopolymers, comprising an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease.

[16] A method for reducing the amount of DNA remaining in a biopolymer produced by a microorganism capable of producing biopolymers, comprising an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease.

[17] The method according to

[15] or

[16] , wherein the endodeoxyribonuclease treatment step is a step of reducing the molecular weight of DNA attached to the biopolymer with endodeoxyribonuclease and solubilizing it.

[18] The method according to any one of

[15] to

[17] , further comprising a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher at least one point in time after the start of the endodeoxyribonuclease treatment.

[19] The method according to

[18] , wherein the pH adjustment step is performed after the endodeoxyribonuclease treatment step, or the endodeoxyribonuclease treatment step and the pH adjustment step are performed simultaneously.

[20] The method according to any one of

[15] to

[19] , further comprising a surfactant treatment step of contacting the biopolymer granules with a surfactant.

[21] The method according to

[20] , wherein the surfactant is an anionic surfactant.

[22] The method according to

[20] or

[21] , wherein the endodeoxyribonuclease treatment step, at least one of the pH adjustment step and the surfactant treatment step are performed simultaneously.

[23] The method according to any one of

[15] to

[22] , further comprising a protease treatment step of contacting the biopolymer granules with a protease.

[24] The method according to

[23] , wherein the endodeoxyribonuclease treatment step and the protease treatment step are carried out simultaneously.

[0086]

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

[14] and

[18] to

[24] , wherein the condition of pH 7.0 or higher 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 greater than 9.0, 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, and also preferably pH 7.0 to 13.0, more preferably pH 7.5 to 13.0, even more preferably pH 8.0 to 12.5, even more preferably pH 8.5 to 12.5, even more preferably pH 9.0 to 12.5, even more preferably pH greater than 9.0 and pH 12.5 or lower, even more preferably pH 9.5 to 12.0, and even more preferably pH 10.0 to 12.0.

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

[14] and

[18] to

[24] , wherein the pH condition of the pH adjustment step is greater than pH 9.0.

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

[14] and

[18] to

[24] , wherein the pH condition of the pH adjustment step is pH 9.5 or higher.

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

[14] and

[18] to

[24] , wherein the pH condition of the pH adjustment step is pH 10.0 or higher.

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

[14] and

[18] to

[28] , wherein the pH condition of the pH adjustment step is pH 13.0 or lower.

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

[14] and

[18] to

[28] , wherein the pH condition of the pH adjustment step is pH 12.5 or lower.

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

[14] and

[18] to

[28] , wherein the pH condition of the pH adjustment step is pH 12.0 or less.

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

[31] , wherein the endodeoxyribonuclease is at least one selected from endodeoxyribonuclease derived from bacteria of the genus Bacillus, endodeoxyribonuclease derived from bacteria of the genus Staphylococcus, and endodeoxyribonuclease derived from bacteria of the genus Serratia.

[33] The endodeoxyribonuclease is Mn derived from Bacillus subtilis.２＋ / Mg ２＋ Manganese-dependent endodeoxyribonuclease from Bacillus licheniformis ２＋ / Mg ２＋ Calcium-dependent endodeoxyribonuclease from Staphylococcus aureus ２＋The method according to any one of [1] to

[31] , wherein the endodeoxyribonuclease is at least one selected from endodeoxyribonucleases that hydrolyze DNA and RNA in an endo-type manner and endonucleases derived from Serratia marcescens.

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

[31] , wherein the endodeoxyribonuclease is an endodeoxyribonuclease comprising a DNase_NucA_NucB domain (PF14040), a DUF1524 domain (PF07510), an Endonuclease_NS domain (PF01223), or an SNase domain (PF00565).

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

[34] , wherein the endodeoxyribonuclease is at least one selected from the group consisting of (A) and (B) below: (A) a polypeptide comprising an amino acid sequence represented by any of SEQ ID NOs. 5, 6, 10 and 11; and (B) a polypeptide comprising an amino acid sequence having at least 60% identity with the amino acid sequence represented by any of SEQ ID NOs. 5, 6, 10 and 11, and having endodeoxyribonuclease activity.

[36] The method according to

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

[37] The method according to

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

[38] The method according to

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

[39] The method according to any one of [4] to

[14] and

[20] to

[38] , wherein the surfactant is an anionic surfactant, preferably at least one selected from sulfate salts of alcohols having 10 to 18 carbon atoms, sulfate 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 esters, and fatty acid salts; more preferably at least one selected from sulfate salts of alcohols having 10 to 14 carbon atoms in the alkyl chain, sulfate salts of ethoxylated alcohols having 10 to 14 carbon atoms in the alkyl chain, and linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain; and even more preferably at least one selected from sulfate salts of alcohols having 10 to 14 carbon atoms in the alkyl chain and sulfate salts of ethoxylated alcohols having 10 to 14 carbon atoms in the alkyl chain.

[40] The method according to any one of [4] to

[14] and

[20] to

[38] , wherein the surfactant is an anionic surfactant, preferably at least one selected from a sulfated alcohol of an alkyl chain having 12 carbon atoms, a sulfated ethoxylated alcohol of an alkyl chain having 12 carbon atoms, and a linear alkylbenzene sulfonate of an alkyl chain having 12 carbon atoms; more preferably at least one selected from a sulfated alcohol of an alkyl chain having 12 carbon atoms and a sulfated ethoxylated alcohol of an alkyl chain having 12 carbon atoms; and even more preferably at least one selected from a dodecyl sulfate and a polyoxyethylene lauryl ether sulfate.

[41] The method according to any one of [4] to

[14] and

[20] to

[38] , wherein the surfactant is an anionic surfactant, preferably at least one selected from sodium dodecyl sulfate, sodium polyoxyethylene lauryl ether sulfate, and sodium dodecylbenzenesulfonate, and more preferably at least one selected from sodium dodecyl sulfate and sodium polyoxyethylene lauryl ether sulfate.

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

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

[43] The method according to

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

[44] The method according to

[42] or

[43] , 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%.

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

[41] , wherein the biopolymer is a polyhydroxyalkanoic acid (PHA).

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

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

[0087] 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.

[0088] Example 1 (1) Construction of an enzyme expression plasmid Plasmid pHY-BsNucB was obtained by substituting the entire ORF of plasmid pHY-BLP2 described in WO2019 / 142773, which consists of the S237 secretion signal sequence and the BLP proprotein, with the BsnucB gene containing the native secretion signal (encoding the polynucleotide of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2). Plasmid pHY-BlNucB was obtained by similarly substituting the entire ORF with the BlnucB gene containing the native secretion signal (encoding the polynucleotide of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4). The amino acid sequences of the mature BsNucB and BlNucB are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Plasmid pHY-SNase was obtained by substituting the full-length ORF containing the VHH gene of the VHH expression plasmid of SEQ ID NO: 26, which contains the Bacillus subtilis spoVG gene-derived promoter described in WO2021 / 153129, with the SNase gene (which encodes the polynucleotide of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10) to which the S237 secretion signal sequence (which encodes the polynucleotide of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8) is ligated to the N-terminus. For the expression of the protease KP43 mutant, the KP43 12-duplex expression plasmid (SEQ ID NO: 4 of Japanese Patent Application No. 2024-071822) was used.

[0089] (2) Preparation of nuclease solution: The enzyme expression plasmid was introduced into Bacillus subtilis strain by protoplast, and 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. The culture supernatant containing the enzyme was then collected by centrifugation. 2 mM CaCl was detected using Amicon 10K. ２ The buffer was changed to 20 mM Tris-HCl (pH 7.5) containing the enzyme. 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 the protein amount.

[0090] (3) Preparation of PHA-containing bacterial cells: Strain 102504 of Cupriavidus necator (Ralstonia eutropha) NBRC was inoculated into LB liquid medium and cultured with shaking at 30°C for 24 hours. One mL of this culture medium 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 suspension of PHA-containing bacterial cells.

[0091] (4) PHA purification by nuclease 1 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. 1N sodium hydroxide aqueous solution was added to the PHA-containing bacterial cell suspension obtained in (3) to adjust the pH to 11.0, and the cells were inactivated by heating at 60°C for 1 hour. 2N sulfuric acid was added to adjust the pH to 7.0, and chicken egg white lysozyme (Fujifilm Wako Pure Chemical Industries, 127-06724) was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 9.0, and then 1 mL was dispensed into 2 mL tubes. The enzyme described in Enzyme Treatment 1 in Table 1 was added, and the mixture was incubated at 50°C for 1 hour. The enzyme described in Enzyme Treatment 2 in Table 1 was added, and the mixture was incubated at 50°C for 1 hour. The nuclease used was BlNucB at a final concentration of 4 mg / L, and the protease used was alcalase (SIGMA, 126741) at a final concentration of 60 mg / L. 1 mL of 6 mM sodium hydroxide aqueous solution containing 1.2 (w / v)% SDS was added and mixed by inversion (pH approximately 10.0 after addition). Centrifuge at 12000 rpm for 5 minutes, and 1 mL of 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, after which the supernatant was completely removed. This procedure was repeated twice. 1 mL of deionized water was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes, after which the supernatant was completely removed. The tubes were opened and dried overnight at 60°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE). 100 μL of the suspension and 10 μL of ×100 GelRed™ aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN) (Table 1). High fluorescence was observed without nuclease addition, and the fluorescence intensity decreased significantly with nuclease treatment. This indicates that nucleic acids unexpectedly remain in the recovered PHA, and that nuclease treatment reduces the residual nucleic acids.In short, it was found that, unexpectedly, at least some insoluble DNA remained in the recovered PHA, and that nuclease treatment reduced at least some of the residual insoluble DNA. Furthermore, the combined use of proteases unexpectedly reduced residual nucleic acids even further.

[0092]

[0093] (5) PHA purification by nuclease 2 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (3) was heated at 60°C for 5 hours to inactivate the cells. 2N sulfuric acid was added to adjust the pH to 6.9, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 8.0, followed by the addition of nuclease BlNucB at a final concentration of 1 mg / L and alcalase at a final concentration of 100 mg / L, followed by incubation at 50°C for 2 hours. The enzyme-treated samples were dispensed in 1 mL portions into 2 mL tubes. 1 mL of 0.4 (w / v)% SDS aqueous solution was added to the dispensed treatment solution, and 1N sodium hydroxide aqueous solution was added to adjust the pH to 9.1, 10.0, and 10.5, respectively. The pH of the sample without alkali was 8.0. The sample was centrifuged at 12000 rpm for 5 minutes, and 1 mL of supernatant was removed. 1 mL of deionized water was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes, after which the entire supernatant was removed. This procedure was repeated twice. The tubes were opened and dried overnight at 50°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes. After cooling at room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The pH of the treatment solution was plotted on the x-axis, and the fluorescence intensity (RFU) on the y-axis (Figure 1). The addition of an alkaline agent after nuclease treatment reduced the fluorescence intensity in a pH-dependent manner, improving the effect of reducing residual nucleic acids.

[0094] (6) PHA purification by nuclease 3 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (3) was heated at 60°C for 5 hours to inactivate the cells. 2N sulfuric acid was added to adjust the pH to 7.0, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 8.0, then alcalase at a final concentration of 100 mg / L was added and dispensed into 2 mL tubes in 1 mL portions. Each nuclease at a final concentration of 1 mg / L was added and incubated at 50°C for 2 hours. 1 mL of 1.2 (w / v)% SDS aqueous solution containing 12 mM sodium hydroxide was added and mixed by inversion. Centrifuge at 12000 rpm for 5 minutes and remove 1 mL of the supernatant. 1 mL of 1 mM sodium hydroxide aqueous solution was added, mixed by inversion, and centrifuged at 12,000 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 12,000 rpm for 5 minutes. The supernatant was then completely removed. The tubes were opened and dried overnight at 60°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE). 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10,000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The ΔRFU value was calculated by subtracting the fluorescence intensity of each sample from the fluorescence intensity of the sample without nuclease (Figure 2). The nucleases used are listed below.endo-DNase 1...BsNucB endo-DNase 2...BlNucB endo-DNase 3...SNase endo-DNase 4...Endonucleases derived from Serratia marcescens (SIGMA, E1014) (SEQ ID NO: 11) RNase...RNaseA (NIPPON GENE, 313-01461) exo-DNase...Exonuclease III (TaKaRa, 2170A) The effect of reducing residual nucleic acids in PHA was particularly pronounced in enzymes with endodeoxyribonuclease activity among nucleases.

[0095] (7) PHA purification by nuclease 4 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (3) was dispensed into 50 mL Falcon tubes and centrifuged at 8000 rpm for 10 minutes. The supernatant was removed so that the volume was reduced to one-fifth, and the bacterial cells were resuspended. The bacterial cells were inactivated by heating at 70°C for 1 hour. 2N sulfuric acid was added to adjust the pH to 7.0, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 8.5, then alcalase at a final concentration of 30 mg / L was added, and the mixture was dispensed into two 50 mL tubes, each containing 20 mL. One tube was left as is, and the other tube was treated with nuclease BlNucB at a final concentration of 30 mg / L and incubated at 50°C for 1 hour. 0.4 mL of 30 (w / w)% SDS aqueous solution was added one by one, and the pH was adjusted to 10 by adding 1 M sodium hydroxide aqueous solution. A final concentration of 30 mg / L KP43 mutant protease was added and incubated at 50°C for 1 hour. 20 mL of 1 mM sodium hydroxide aqueous solution was added one by one and centrifuged at 3000 × g for 20 minutes. 20 mL of the supernatant was removed, and the procedure of adding 20 mL of 1 mM sodium hydroxide aqueous solution one by one and centrifuging at 3000 × g for 20 minutes was repeated twice. Furthermore, the entire supernatant was removed, and the procedure of adding 30 mL of 1 mM sodium hydroxide aqueous solution one by one and centrifuging at 3000 × g for 20 minutes was repeated twice. The entire supernatant was removed, and 30 mL of deionized water was added one by one and centrifuged at 3000 × g for 20 minutes. The entire supernatant was removed, the tube cap was opened and the tube was dried overnight at 60°C. PHA powder was placed in glass bottles in 200 mg portions. After tapping the bottles to collect the PHA at the bottom, the bottles were gently inverted, and the appearance of the PHA adhering to the bottom was photographed. This procedure was repeated four times (Figure 3). The amount of PHA powder adhering to the bottom of the container was reduced in the PHA powder treated with nuclease compared to the PHA powder that was not treated with nuclease, suggesting that nuclease treatment tends to reduce the adhesion of PHA powder and improve its fluidity. 10 mg portions of PHA powder were weighed out and suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE).50 μL of the suspension and 50 μL of ×50 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 200-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). A blank was used in which deionized water was used instead of the GelRed aqueous solution. Instead of the PHA suspension, a calibration curve was created using salmon semen-derived deoxyribonucleic acid (FUJIFILM, 043-31381) dissolved in Tris-EDTA buffer (pH 8.0), and the amount of residual nucleic acid in the PHA powder was determined (Figure 4). Nucleic acid remained in the recovered PHA, and the residual nucleic acid was reduced by nuclease treatment.

[0096] (8) PHA purification by nuclease 5 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (3) was heated at 60°C for 5 hours to inactivate the cells. 1N sodium hydroxide was added to adjust the pH to 7.0, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1 mL was dispensed into two 2 mL tubes, and alcalase at a final concentration of 30 mg / L was added to one tube, and alcalase at a final concentration of 30 mg / L and nuclease (BlNucB) at a final concentration of 100 mg / L were added to the other tube. After incubation at 50°C for 2 hours, 1 mL of 0.4 (w / v)% SDS aqueous solution was added and mixed by inversion. After adding a 1N sodium hydroxide aqueous solution to adjust the pH to 9.0, the sample was centrifuged at 12,000 rpm for 5 minutes. The appearance after centrifugation was observed (Figure 5). Compared to the sample without nuclease addition, the supernatant of the nuclease-added sample was clearer, indicating that nuclease addition improved the centrifugation sedimentation efficiency. This suggests that the use of nuclease not only reduces the amount of DNA remaining in the PHA, but also reduces the water and energy load during centrifugation washing.

[0097] (9) 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.

[0098] (10) PHA purification by nuclease 6 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. 1N HCl was added to the PHA-containing bacterial cell suspension obtained in (9) to adjust the pH to 7.0. 1 mL was dispensed into two 2 mL tubes, and nuclease (BlNucB) at a final concentration of 30 mg / L was added to one of the tubes. After heating each tube at 70°C for 10 minutes, lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L was added and incubated at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 9.0, then 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 samples were centrifuged at 12,000 rpm for 5 minutes. At the end of centrifugation, in the sample without nuclease, a viscous white suspended substance was observed around the solid pellet (Figure 6). In the sample with nuclease, this suspended substance was not observed, and only the solid pellet was seen. 1 mL of the supernatant was removed. 1 mL of 1 mM sodium hydroxide aqueous solution was added, mixed by inversion, and centrifuged at 12,000 rpm for 5 minutes, followed by the removal of the entire supernatant. This procedure was repeated twice. The tube caps were opened and the tubes were dried overnight at 60°C. Each pellet was suspended in 500 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE). 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes in the dark. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of the sample without nuclease treatment were calculated (Table 2). The decrease in fluorescence intensity after nuclease treatment indicates that nucleic acids remain in the recovered PHA, and that nuclease treatment reduced the amount of residual nucleic acids.

[0099]

[0100] (11) PHA purification by nuclease 7 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (9) 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. 1N sodium hydroxide aqueous solution was added to adjust the pH to 9.0, then 30 mg / L alcalase was added, and incubated 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. Centrifuge at 12000 rpm for 5 minutes and remove 1 mL of supernatant. The procedure of adding 1 mL of 1 mM sodium hydroxide aqueous solution, mixing by inversion, centrifugation at 12000 rpm for 5 minutes, and then removing the entire supernatant was repeated twice. The caps of the tubes were opened and dried overnight at 60°C. Each pellet was suspended in 500 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated in the dark at 60°C for 30 minutes. After cooling at room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of the sample without nuclease treatment were calculated (Table 3). The decrease in fluorescence intensity after nuclease treatment indicates that nucleic acids remained in the recovered PHA, and that nuclease treatment reduced the amount of residual nucleic acids.

[0101]

[0102] (12) PHA purification by nuclease 8 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (9) 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 added with lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and alcalase at a final concentration of 30 mg / L. The other tube was added 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. The tubes were incubated at 50°C for 1 hour. After adding a 1N sodium hydroxide aqueous solution to adjust the pH to 9.0, the mixture was incubated 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. At this time, the pH was 9.4. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. The procedure of adding 1 mL of a 1 mM sodium hydroxide aqueous solution, mixing by inversion, centrifuging at 12000 rpm for 5 minutes, and then removing the entire supernatant was repeated twice. The tube cap was opened and the mixture was dried at 60°C overnight. Each pellet was suspended in 500 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE). 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes in the dark. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of the sample without nuclease treatment were calculated (Table 4). The decrease in fluorescence intensity after nuclease treatment indicates that nucleic acids remain in the recovered PHA, and that nuclease treatment reduced the amount of residual nucleic acids.

[0103]

[0104] (13) PHA purification by nuclease 9 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (9) 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. 1 mL each was dispensed into two 2 mL tubes. One tube was added with lysozyme (mutanolysin, SIGMA M9901) at a final concentration of 20 mg / L and alcalase at a final concentration of 30 mg / L, and the other tube was added 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. The tubes were incubated 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 9.6. Centrifuge at 12000 rpm for 5 minutes and remove 1 mL of supernatant. The procedure of adding 1 mL of 1 mM sodium hydroxide aqueous solution, mixing by inversion, centrifugation at 12000 rpm for 5 minutes, and then removing the entire supernatant was repeated twice. The caps of the tubes were opened and dried overnight at 60°C. Each pellet was suspended in 500 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated in the dark at 60°C for 30 minutes. After cooling at room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of the sample without nuclease treatment were calculated (Table 5). The decrease in fluorescence intensity after nuclease treatment indicates that nucleic acids remained in the recovered PHA, and that nuclease treatment reduced the amount of residual nucleic acids.

[0105]

[0106] (14) PHA purification by nuclease 10 A pH meter LAQUAact D-72 (HORIBA) was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (3) 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. 1 mL each 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, and 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, and incubated 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 11.3. Centrifuge at 12000 rpm for 5 minutes and remove 1 mL of supernatant. The procedure of adding 1 mL of 1 mM sodium hydroxide aqueous solution, mixing by inversion, centrifugation at 12000 rpm for 5 minutes, and then removing the entire supernatant was repeated twice. The caps of the tubes were opened and dried overnight at 60°C. Each pellet was suspended in 500 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 100 μL of the suspension and 10 μL of ×100 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 100-fold with deionized water) were mixed in a black 96-well plate and incubated in the dark at 60°C for 30 minutes. After cooling at room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of the sample without nuclease treatment were calculated (Table 6). The decrease in fluorescence intensity after nuclease treatment indicates that nucleic acids remained in the recovered PHA, and that nuclease treatment reduced the amount of residual nucleic acids.

[0107]

[0108] (15) Preparation of PHA-containing bacterial cells Cupriavidus necator (Ralstonia eutropha) strain H16 was inoculated into BD Diffco® Nutrient Broth medium and cultured with shaking at 30°C for 24 hours. One mL of this culture medium 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 suspension of PHA-containing bacterial cells.

[0109] (16) PHA purification by nuclease 11 A pH meter LAQUAact D-72 (HORIBA) was used to measure pH. The PHA-containing bacterial cell suspension obtained in (15) was heated at 70°C for 1 hour to inactivate the cells. 1N sodium hydroxide aqueous solution was added to adjust the pH to 6.5, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. The pH after incubation was 6.5. Alcalase at a final concentration of 100 mg / L was added and 1 mL was dispensed into two 2 mL tubes. Nuclease (BlNucB) at a final concentration of 1 mg / L was added and incubated at 50°C for 2 hours. 1 mL of 0.4 (w / v)% SDS aqueous solution was added to the enzyme-treated samples, and 1N sodium hydroxide aqueous solution was added to adjust the pH to 7.6, 10.6, and 11.8, respectively. The pH of the sample without alkali was 6.7. The sample was centrifuged at 12000 rpm for 5 minutes, and 1 mL of supernatant was removed. 1 mL of deionized water was added, mixed by inversion, and centrifuged at 12000 rpm for 5 minutes, after which the entire supernatant was removed. This procedure was repeated twice. The tubes were opened and dried overnight at 50°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 50 μL of the suspension and 50 μL of ×50 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 200-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes. After cooling to room temperature, the fluorescence intensity (RFU) at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). ΔRFU was calculated by subtracting the fluorescence intensity of each sample from the fluorescence intensity of a sample without nuclease addition and without pH adjustment after SDS addition. A larger ΔRFU indicates a greater nucleic acid reduction effect. The pH adjusted after SDS addition was plotted on the x-axis, and ΔRFU on the y-axis (Figure 7). The pH reduction effect of residual nucleic acids improved in a pH-dependent manner with the addition of an alkaline agent after nuclease treatment.

[0110] (17) Purification of PHA by nuclease 12. Purification of PHA is performed in the same manner as in (12), except that KP43 (SEQ ID NO: 19), KP43 mutant (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Esperase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (SEQ ID NO: 28, 29), or LL147 mutant (SEQ ID NO: 30, 31) is used as the protease instead of alcalase.

[0111] (18) Purification of PHA by nuclease 13 Purification of PHA is carried out in the same manner as in (12), except that egg white lysozyme, Ssp12Lys (SEQ ID NO: 32), Ssp14Lys (SEQ ID NO: 33), Asp3Lys (SEQ ID NO: 34), or KaLys (SEQ ID NO: 35) is used instead of mutanolisin as lysozyme, and KP43 (SEQ ID NO: 19), KP43 mutant (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Esperase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (SEQ ID NO: 28, 29), or LL147 mutant (SEQ ID NO: 30, 31) is used instead of alcalase as protease.

[0112] (19) Purification of PHA by nuclease 14. Purification of PHA is performed in the same manner as in (13), except that KP43 (SEQ ID NO: 19), KP43 mutant (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Esperase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (SEQ ID NO: 28, 29), or LL147 mutant (SEQ ID NO: 30, 31) are used as the protease instead of alcalase.

[0113] (20) Purification of PHA by nuclease 15 The PHA is purified in the same manner as in (13), except that a nuclease consisting of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 is used as the nuclease instead of BlNucB.

[0114] (21) Purification of PHA by nuclease 16 The PHA is purified in the same manner as in (12), except that egg white lysozyme is used instead of mutanolisin as lysozyme, and a nuclease consisting of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 is used instead of BlNucB as the nuclease.

[0115] (22) Purification of PHA by nuclease 17 The PHA is purified in the same manner as in (12), except that a lysozyme consisting of the amino acid sequence shown in SEQ ID NO: 23 is used as the lysozyme instead of mutanolisin, and a nuclease consisting of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 is used as the nuclease instead of BlNucB.

[0116] (23) Purification of PHA by nuclease 18 The PHA is purified in the same manner as in (12), except that a lysozyme consisting of the amino acid sequence shown in SEQ ID NO: 26 is used as the lysozyme instead of mutanolisin, and a nuclease consisting of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 is used as the nuclease instead of BlNucB.

[0117] (24) Purification of PHA by nuclease 19 The PHA is purified in the same manner as in (12), except that a lysozyme consisting of the amino acid sequence shown in SEQ ID NO: 24 is used instead of mutanolisin as the lysozyme, a nuclease consisting of the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 is used instead of BlNucB as the nuclease, and Esperase is used as the protease.

[0118] (25) Purification of PHA by nuclease 20 The PHA is purified in the same manner as in (13), except that Ssp12Lys (SEQ ID NO: 32), Ssp14Lys (SEQ ID NO: 33), Asp3Lys (SEQ ID NO: 34), or KaLys (SEQ ID NO: 35) is used as lysozyme instead of mutanolisin.

[0119] (26) Purification of PHA by nuclease 21 The PHA is purified in the same manner as in (13), except that Ssp12Lys (SEQ ID NO: 32), Ssp14Lys (SEQ ID NO: 33), Asp3Lys (SEQ ID NO: 34), or KaLys (SEQ ID NO: 35) is used as lysozyme instead of mutanolisin, and KP43 (SEQ ID NO: 19), KP43 mutant (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Esperase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (SEQ ID NO: 28, 29), or LL147 mutant (SEQ ID NO: 30, 31) is used as the protease instead of alcalase.

[0120] (27) PHA purification by nuclease 22 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 60°C for 5 hours to inactivate the cells. 1N sodium hydroxide was added to adjust the pH to 6.9, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 8.5, followed by the addition of alcalase at a final concentration of 100 mg / L and nuclease (BlNucB) at a final concentration of 1 mg / L, followed by incubation at 50°C for 2 hours. 1 mL was dispensed into six 2 mL tubes, and 1 mL of 0.2 (w / v)% surfactant aqueous solution was added to two of each tube and mixed by inversion. After adding 1N sodium hydroxide aqueous solution to adjust the pH to 10.0, the mixture was centrifuged at 12000 rpm for 5 minutes. After centrifugation, 1 mL of the supernatant was removed. 1 mL of deionized water 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 caps were opened and the mixture was dried overnight at 50°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE), and 50 μL of the suspension was mixed with 50 μL of ×50 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 200-fold with deionized water) in a black 96-well plate. The mixture was then incubated in the dark at 60°C for 30 minutes. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The average fluorescence intensity of two samples with each surfactant added was calculated and shown as a relative value with the SDS-added sample set to 100 (Figure 8). The surfactants used are listed below. Anionic surfactants SDS...Sodium dodecyl sulfate (Kao Corporation) ES...Sodium polyoxyethylene lauryl ether sulfate (Emal 20C, Kao Corporation) Nonionic surfactant E320L...Polyoxyethylene stearyl ether, average number of moles added of EO 20 (Emulgen 320L, Kao Corporation) The effect of reducing residual nucleic acids (PHA) was particularly pronounced with anionic surfactants among the surfactants.

[0121] (28) PHA purification by nuclease 23 A pH meter LAQUAact D-72 (HORIBA) was used to measure pH. The PHA-containing bacterial cell suspension obtained in (15) was heated at 60°C for 5 hours to inactivate the cells. 1N sodium hydroxide was added to adjust the pH to 6.9, and chicken egg white lysozyme was added at a final concentration of 20 mg / L, followed by incubation at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added to adjust the pH to 8.0, then alcalase at a final concentration of 100 mg / L was dispensed and 1 mL was dispensed into three 2 mL tubes. Nuclease (BlNucB) at a final concentration of 1 mg / L was added to one tube, and the remaining two tubes were incubated at 50°C for 2 hours without adding nuclease. 1 mL of 0.4 (w / v)% SDS aqueous solution was added to the sample with added nuclease and mixed by inversion. Of the samples without added nuclease, 1 mL of deionized water was added to one tube, and 1 mL of 0.4 (w / v)% SDS aqueous solution was added to the other tube, and the mixture was inverted and mixed. After adjusting the pH to 10.0 by adding 1 N sodium hydroxide aqueous solution, the samples were centrifuged at 12,000 rpm for 5 minutes. After centrifugation, 1 mL of the supernatant was removed. The procedure of adding 1 mL of deionized water, inverting and mixing, centrifuging at 12,000 rpm for 5 minutes, and then removing the entire supernatant was repeated twice. The caps of the tubes were opened and dried overnight at 50°C. Each pellet was suspended in 400 μL of Tris-EDTA buffer (pH 8.0) (NIPPON GENE). 50 μL of the suspension and 50 μL of ×50 GelRed aqueous solution (Biotium) (×10000 stock solution diluted 200-fold with deionized water) were mixed in a black 96-well plate and incubated at 60°C for 30 minutes in the dark. After cooling to room temperature, the fluorescence intensity at an excitation wavelength of 280 nm and an emission wavelength of 600 nm was measured using a plate reader Infinite 200 PRO (TECAN). The relative values ​​to the fluorescence intensity of samples without nuclease and SDS were calculated (Figure 9). The combined use of nuclease and SDS improved the reduction effect of residual nucleic acids.

Claims

1. A method for producing a biopolymer, comprising: an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with endodeoxyribonuclease; and a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher at least one time point after the start of the endodeoxyribonuclease treatment.

2. The method according to claim 1, further comprising a surfactant treatment step of contacting the biopolymer granules with a surfactant.

3. The method according to claim 2, wherein the surfactant is an anionic surfactant.

4. The method according to any one of claims 1 to 3, further comprising a protease treatment step of contacting the biopolymer granules with a protease.

5. The method according to claim 4, wherein the endodeoxyribonuclease treatment step and the protease treatment step are performed simultaneously.

6. The method according to claim 3, further comprising a lysozyme treatment step of contacting the cellular cells of the biopolymer-producing microorganism with lysozyme.

7. The method according to claim 4, further comprising a lysozyme treatment step of contacting the cellular cells of the biopolymer-producing microorganism with lysozyme.

8. The method according to claim 7, wherein the endodeoxyribonuclease treatment step, the protease treatment step, and the lysozyme treatment step are performed simultaneously.

9. A method for reducing the amount of residual DNA in a biopolymer produced by a microorganism capable of producing biopolymers, comprising an endodeoxyribonuclease treatment step of contacting biopolymer granules obtained by culturing a microorganism capable of producing biopolymers with an endodeoxyribonuclease.

10. The method according to claim 9, further comprising a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher at least one time point after the start of the endodeoxyribonuclease treatment.

11. The method according to claim 10, further comprising a surfactant treatment step of contacting the biopolymer granules with a surfactant.

12. The method according to claim 11, wherein the surfactant is an anionic surfactant.

13. The method according to any one of claims 9 to 12, further comprising a protease treatment step of contacting the biopolymer granules with a protease.

14. The method according to claim 13, wherein the endodeoxyribonuclease treatment step and the protease treatment step are performed simultaneously.

15. The method according to any one of claims 1 to 14, wherein the biopolymer is a hydroxyalkanoic acid-containing polymer.

16. The method according to any one of claims 1 to 14, wherein the biopolymer is polyhydroxyalkanoic acid (PHA).

17. The method according to any one of claims 1 to 16, wherein the microorganism having biopolymer production ability is a microorganism belonging to the genus Cupriavidus.

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