Biopolymer manufacturing method

Abstract

When producing biopolymers using microorganisms, residual substances remain in the biopolymer. Providing a method to reduce the amount of DNA and increase the purity of biopolymers. [Solution] A method for producing biopolymers, comprising a microorganism having biopolymer production ability. Endodeoxyribonuclease was brought into contact with biopolymer granules obtained by culturing. Endodeoxyribonuclease treatment process and the endodeoxyribonuclease treatment At least one point in time after the start, the biopolymer granules were subjected to conditions of pH 7.0 or higher. A method comprising a pH adjustment step in which the body is placed.

Description

[Technical Field]

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

[0002] Biopolymers, manufactured using biomass as a raw material, are a substitute for petrochemical plastics. They are used in a variety of applications. Many biopolymers have high biodegradability. In recent years, demand for biopolymers has been expanding from an environmental protection perspective. Typical biopolymers include... Examples include pulp, polylactic acid (PLA), and polyhydroxyalkanoic acid (PHA).

[0003] PHA, a type of biopolymer, can be produced by microbial fermentation. In the industrial production of A, it is necessary to purify PHA from within densely cultured cells. Cell disruption can be achieved through heat treatment, mechanical physical treatment, or chemical treatment using alkalis or surfactants. Biological treatments such as enzymes are used. Insoluble PHA granules are obtained by disrupting cells. It is known that residual proteins remain, and to reduce these residual proteins... Therefore, methods using surfactants or proteases are known (Patent Document 1). Also, pH Granule A may contain impurities derived from the production cells in addition to proteins, for example, Nucleic acids have been cited as examples (Patent Document 2). On the other hand, generally, those derived from PHA-producing cells are also cited. Nucleic acids are thought to be released into the treatment solution by cell disruption and lysis, and in the treatment solution A method to reduce the viscosity of the treatment solution by acting a nuclease on the released nucleic acids. This is known (Patent Document 3, Non-Patent Document 1). Therefore, PHA granules obtained by disrupting cells. It has not been clarified whether nucleic acids actually remain in the granules.

[0004] Because particularly high purity PHA is required for medical and other applications, further high purity PHA is needed. Purification is desired. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Chinese Patent Application Publication No. 1190674 Specification [Patent Document 2] International Public Gazette No. 2018 / 186278 [Patent Document 3] International Public Gazette No. 2023 / 027953 [Non-patent literature]

[0006] [Non-Patent Document 1] Gamero, Jesus E. Rodriguez, et al. Bioresource technology 261 (2018): 176-181. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors have discovered that PHA produced by microorganisms and purified by conventional purification methods unexpectedly contains It was revealed that DNA attaches as an impurity, becomes insoluble, and remains in PHA. We found a problem in that residue remains. Therefore, the present invention has a micropolymer production capability. When manufacturing biopolymers using biological materials, the amount of DNA remaining in the biopolymer This invention relates to providing a method for reducing and increasing the purity of biopolymers. [Means for solving the problem]

[0008] The present inventors have found that by contacting endodeoxyribonuclease with biopolymer granules obtained by culturing a microorganism capable of producing a biopolymer, the amount of DNA remaining in the biopolymer can be reduced, and that subjecting the biopolymer granules to a condition of pH 7.0 or higher can further reduce the amount of DNA remaining in the biopolymer. The present invention relates to a method for producing a biopolymer, comprising : an endodeoxyribonuclease treatment step of contacting endodeoxyribonuclease with biopolymer granules obtained by culturing a microorganism capable of producing a biopolymer; and a pH adjustment step of subjecting the biopolymer granules to a condition of pH 7.0 or higher at at least one time point after the start of the endodeoxyribonuclease treatment.

[0009] The present invention also relates to a method for reducing the amount of DNA remaining in a biopolymer produced by a microorganism capable of producing a biopolymer, comprising : an endodeoxyribonuclease treatment step of contacting endodeoxyribonuclease with biopolymer granules obtained by culturing a microorganism capable of producing a biopolymer. Effects of the Invention

[0010] According to the method of the present invention, in the production of a biopolymer using a microorganism capable of producing a biopolymer, the amount of DNA remaining in the biopolymer can be reduced, and the purity of the biopolymer can be improved. Brief Description of Drawings

[0011] [Figure 1]A diagram showing the effect of the pH of the treatment solution on the amount of residual PHA nucleic acid. [Figure 2] A diagram showing the effect of nuclease type on the amount of residual PHA nucleic acid. [Figure 3] A diagram showing the effect of nuclease treatment on the powder properties of PHA. [Figure 4] A diagram showing the effect of nuclease treatment on the amount of residual PHA nucleic acid. [Figure 5] A diagram showing the effect of nuclease treatment on the centrifugal washing efficiency of PHA. [Figure 6] A diagram showing the effect of nuclease treatment on the centrifugal washing efficiency of PHA. [Figure 7] A diagram showing the effect of the pH of the treatment solution on the amount of residual PHA nucleic acid. [Figure 8] This figure shows the effect of the combination of nuclease treatment and surfactant treatment on the amount of residual PHA nucleic acid. [Figure 9] This figure shows the effect of the combination of nuclease treatment and surfactant (SDS) treatment on the amount of residual PHA nucleic acid. [Modes for carrying out the invention]

[0012] All patent, non-patent, and other publications cited herein are entirely subject to the terms of their respective owners. The Body is incorporated herein by reference.

[0013] In this specification, the identity of an amino acid sequence or nucleotide sequence is defined as Lipman-P The calculation is performed using the Earnson method (Science, 1985, 227:1435-1441). It is calculated. Specifically, homozygous genetic information processing software GENETYX Ver.12 Using a genealogy analysis (search homology) program, Unit size It is calculated by performing the analysis with e to compare(ktup) set to 2.

[0014] In this specification, "at least 60% of the amino acid sequence or nucleotide sequence" "Identity" means 60% or more, preferably 70% or more, more preferably 75% or more, and further Preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, More preferably 91% or more, even more preferably 92% or more, and even more preferably 93% or more. , more preferably 94% or more, more preferably 95% or more, and more preferably 96% More preferably 97% or more, even more preferably 98% or more, even more preferably 9 This refers to a 9% or higher degree of identity. Furthermore, "at least 90% identity" means 90% or more of the preferred identity. More preferably 91% or more, more preferably 92% or more, even more preferably 93% or more, even more preferably Preferably 94% or more, more preferably 95% or more, even more preferably 96% or more, and Preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more It refers to identity.

[0015] In this specification, "corresponding position" on an amino acid sequence or nucleotide sequence is the target The sequence and the reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 5) are given maximum homology. This can be determined by aligning them in a specific way. Alignment of column or nucleotide sequences can be performed using known algorithms. This can be done, and the procedure is known to those skilled in the art. For example, alignment is Clustal W Multiple Alignment Program (Thompson, JDet al, 1 994, Nucleic Acids Res. 22:4673-4680) default This can be done by using the settings. Alternatively, the revised version of Clustal W You can also use Clustal W2 or Clustal Omega. Lustal W, Clustal W2, and Clustal Omega are, for example, Clustal, operated by University College Dublin Clustal [www.clustal.org], European Institute of Bioinformatics ( European Bioinformatics Institute:EBI[ww [w.ebi.ac.uk / index.html]) and the National Institute of Genetics, which operates the Japan This DNA Databank (DDBJ [www.ddbj.nig.ac.jp / search It can be used on the website of hes-j.html. The above alignment The position of the target sequence aligned to any position in the reference sequence by the input is " It is considered to be the "corresponding position".

[0016] Those skilled in the art will optimize the alignment of the amino acid sequence obtained above. Further fine-tuning is possible. Such optimal alignment is based on the similarity of amino acid sequences. It is preferable to determine this by considering factors such as sex and the frequency of insertion into the gap. Here, amino acid composition Column similarity refers to the similarity between two amino acid sequences when they are aligned. Alternatively, the ratio (%) of the number of positions where similar amino acid residues exist relative to the total number of amino acid residues. Similar amino acid residues are those among the 20 amino acids that make up a protein, with differences in polarity and other properties. amino acids that have similar properties in terms of charge and undergo so-called conservative substitution. This refers to acid residues. Such groups of similar amino acid residues are well known to those skilled in the art. For example, arginine and lysine or glutamine; glutamic acid and asparagine Acid or glutamine; serine and threonine or alanine; glutamine and asparagine or a Ruginine; leucine and isoleucine are examples, but the list is not limited to these. .

[0017] The present invention provides a method for producing biopolymers. The method has a biopolymer production capacity. A method for producing biopolymers using microorganisms that possess biopolymer production capabilities. Endodeoxyribonuclease was applied to biopolymer granules obtained by culturing microorganisms. The process of treating the endodeoxyribonuclease in contact with the endodeoxyribonuclease At least one point in time after the start of processing, the biopolymer granules were subjected to conditions with a pH of 7.0 or higher. The method for producing the biopolymer of the present invention is described below as the pH adjustment step. This is called the Ming method.

[0018] The "microorganism having biopolymer production ability" of the present invention produces biopolymers through fermentation, This refers to microorganisms that have the ability to accumulate within their cells. These microorganisms are not particularly limited and include wild-type microorganisms. Mutations such as insertions, substitutions, and deletions of base sequences occur in certain types of microorganisms or through various genetic manipulations. Any of the mutant microorganisms (mutants) may be used, and by applying known artificial modifications... It may also be a genetically modified microorganism that has been given the desired biopolymer production ability.

[0019] The "biopolymer" produced by the microorganisms having biopolymer production capabilities of the present invention and Among polymers produced from biomass as a raw material, it is produced by fermentation by microorganisms, and the microbial cells This refers to polymers that accumulate inside. A preferred example of a biopolymer is hydroxyalkali. Examples include hydroxyalkanoic acid-containing polymers. Hydroxyalkanoic acid-containing polymers are, in other words, hydroxyalkanoic acid-containing polymers. This polymer contains roxyalkanoic acid as a monomer unit. It contains hydroxyalkanoic acid. An example of a polymer is polyester containing only hydroxyalkanoic acid as the monomer unit. Polyhydroxyalkanoate (PH) A) In addition to hydroxyalkanoic acid, it also contains amino groups such as amino acids as monomer units. Polyesteramide further comprising a carboxylic acid and having ester and amide bonds. These include the monomer units that make up the hydroxyalkanoic acid-containing polymer. The proportion of hydroxyalkanoic acid is preferably 50% or more, more preferably 60% or more. It is above, more preferably 70% or more, even more preferably 80% or more, More preferably 90% or more, even more preferably 95% or more, even more preferably The content is 98% or more, and more preferably 100%. Hydroxyalkanoic acid-containing poly Hydroxylalkanoic acid is the proportion of hydroxyalkanoic acid in the monomer units that make up the polymer, which is 100%. PHA is a polymer containing sialic acid.

[0020] PHA is a polyester with hydroxyalkanoic acid as its monomer unit, and is a sugar, Produced by microorganisms capable of PHA production using alcohols, fatty acids, and oils as carbon sources. It is then accumulated within the cells of microorganisms. The hydroxyalkanoic acid is not particularly limited. However, for example, (R)-3-hydroxybutanoic acid ((R)-3-hydroxybutyr ic acid (3HB), 4-hydroxybutanoic acid (4-hydroxybutyri c acid:4HB), 3-hydroxypropionic acid (3-hydroxypropi onic acid:3HP), (R)-3-hydroxypentanoic acid ((R)-3-Hyd Roxyvaleric acid, (R)-3-hydroxyvaleric acid:3HV), (R ((R)-3-hydroxyhexanoic acid d:3HHx), (R)-3-hydroxyoctanoic acid ((R)-3-hydroxyoc tanoic acid:3HO), (R)-3-hydroxydecanoic acid ((R)-3-h Hydroxydecanoic acid (3HD), (R)-3-hydroxydodecane Acid ((R)-3-hydroxydodecanoic acid:3HDD), (R) -3-hydroxytetradecanoic acid ((R)-3-hydroxytetradecanoic acid Examples include ic acid (3HTD). PHA is composed of one type of monomer unit. This may be a homopolymer or a copolymer composed of two or more monomer units. Yes. As for PHA, poly[(R)-3-hydroxybutanoic acid]{poly[(R)-3 [Hydroxybutyric acid]:P(3HB)}, Poly[(R)-3- [poly[(R)-3 hydroxypentanoic acid]{(R)-3 hydroxypentanoic acid-co-(R)-3 -hydroxybutyric acid-co-(R)-3-hydroxyval eric acid]:P(3HB-co-3HV)}, poly[(R)-3-hydroxy Butanoic acid-co-(R)-4-hydroxybutanoic acid]{poly[(R)-3-hydr oxybutyric acid-co-(R)-4-hydroxybutyric [(R)-3-hydroxybutanoic acid- co-(R)-3-hydroxyhexanoic acid]{poly[(R)-3-hydroxyb utyric acid-co-(R)-3-hydroxyhexanoic acid d]:P(3HB-co-3HHx)}, poly[(R)-3-hydroxybutanoic acid-co -(R)-3-hydroxydecanoic acid]{poly[(R)-3-hydroxybuty ric acid-co-(R)-3-hydroxydecanoic acid]: Examples include {P(3HB-co-3HD)}. Composition of monomer units in copolymers. The ratio is not particularly limited and depends on the type of microorganism capable of producing PHA used, the type of carbon source, and the culture. Depending on the method used, it is possible to obtain PHA with various composition ratios.

[0021] Microorganisms capable of producing PHA include wild-type microorganisms that inherently possess PHA-producing ability. However, if the gene encoding the PHA polymerase is modified, the PHA production capacity is altered. Even if it is a mutant microorganism, if a gene encoding PHA polymerase is introduced from an external source... It may also be a genetically modified microorganism to which the ability to produce PHA has been conferred. These include Aeromonas sp., Bacillus sp., Cupriavidus sp., Esc. Examples include microorganisms belonging to the genera Herichia and Pseudomonas. However, in terms of PHA productivity, the genera Aeromonas, Cupriavidus, and Es Microorganisms belonging to the genus Cherichia are preferred, and microorganisms belonging to the genus Cupriavidus are preferred. Biological organisms are preferable. As for microorganisms belonging to the genus Cupriavidus, Cupria vidus necator (formerly Ralstonia eutropha), Cupr Examples include *Iavidus metallidurans*, and among them, Cupriav Idus necator is preferred.

[0022] By culturing microorganisms capable of producing biopolymers, the microorganisms can introduce biopolymers into the cells. Opolymers are produced. The produced biopolymers are usually in the form of biopolymer granules. It exists in a certain state.

[0023] The cultivation of microorganisms capable of producing biopolymers is This can be carried out under the common conditions used in the production of biopolymers by culturing. The culture medium for nourishment should contain a carbon source, a nitrogen source, inorganic salts, and microorganisms capable of producing biopolymers. Whether it's a synthetic or natural culture medium, as long as it contains the nutrients necessary for the production of biopolymers by material. But that's fine.

[0024] As a carbon source, any carbon source that can be utilized by microorganisms capable of producing biopolymers is acceptable. Any difference is fine, including glucose, glycerol, fructose, sucrose, maltose, ma Sugars such as ethanol, galactose, starch hydrolysates, and molasses; alcohols such as ethanol. Acids; dodecanoic acid (lauric acid), tridecanoic acid, tetradecanoic acid (myristic acid), pe Pentadecanoic acid (pentadecyl acid), hexadecanoic acid (palmitic acid), hexadecenoic acid, Heptadecanoic acid (margaric acid), octadecanoic acid (stearic acid), octadecenoic acid, Octadecadienoic acid, octadecandridecaenoic acid, nonadecanoic acid, eicosanoic acid, ei Fatty acids such as cosadienoic acid, eicosatrienoic acid, and eicosatetraenoic acid; coconut oil, parsley Oils such as oat oil, palm kernel oil, olive oil, rapeseed oil, rice bran oil, soybean oil, castor oil, and mahua oil. Examples include oils and fats. These carbon sources can be used individually or in combination of two or more. It can be added to the culture medium by any method, such as all at once, in divided doses, or continuously. .

[0025] Nitrogen sources include ammonia, ammonium sulfate, ammonium chloride, and heptamolybdic acid. Ammonium salts such as hexaammonium, nitrogen compounds such as amines, peptone, soybean water Examples include natural nitrogen sources such as lye.

[0026] Inorganic salts include disodium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate. Iron(III) chloride, calcium chloride, cobalt chloride, copper sulfate, nickel chloride Examples include zinc sulfate, iron sulfate, sodium tetraborate, and manganese sulfate. Furthermore, vitamins and other substances can be added to the culture medium as needed. Examples include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), and pa Examples include antotenic acid, inositol, and nicotinic acid.

[0027] Culture is preferably carried out under aerobic conditions, and general methods such as aerated stirring culture and shaking culture can be applied. It is possible to select any culture format from batch culture, semi-batch culture, continuous culture, etc. It can be selected. The culture temperature is preferably 10-50°C, and more preferably 20-42°C. A temperature of 25-35°C is even more preferable. The initial pH of the culture medium (at 30°C) is preferably 6-9, and 7 ~8 is more preferable. The incubation time is preferably 24 to 200 hours, and 50 to 100 hours is preferable. It is preferable.

[0028] In the endodeoxyribonuclease treatment process, the production of endodeoxyribonuclease As a result, residual DNA derived from bacterial cells attached to the biopolymer in the biopolymer granules It can be broken down into smaller molecules and made solubilized. The solubilized small-molecular-weight DNA is used in biopolymers. - is removed during recovery. Therefore, by the endodeoxyribonuclease treatment process This reduces the amount of DNA that adheres to and remains on the biopolymer. The deoxyribonuclease treatment process increases the purity of the biopolymer. This is a process for purification.

[0029] Here, "endodeoxyribonuclease" refers to phosphodiesters within the DNA strand. This refers to proteins that possess endodeoxyribonuclease activity, which hydrolyzes bonds. ru.

[0030] The endodeoxyribonuclease used in the present invention is not particularly limited, but is preferred. Examples include bacterial endodeoxyribonucleases, and more preferably Bacillus ( Endodeoxyribonuclease derived from bacteria of the genus Bacillus, Staphylococcus Endodeoxyribonuclease and se Endodeoxyribonucleases derived from bacteria of the genus Serratia are examples. A preferred example of an endodeoxyribonuclease is Bacillus subtilis. Mn (derived from subtilis) 2+ / Mg 2+ Dependent endodeoxyribonuclease (For example, BsNucB in the example), Bacillus licheniformis Mn derived from licheniformis 2+ / Mg 2+ Dependent endodeoxyribonu Clease (e.g., BlNucB in the example), Staphylococcus aureus Ca derived from Ccus aureus 2+ Depending on the process, DNA and RNA are hydrolyzed in an endotype. Endodeoxyribonucleases that decompose Serratia (e.g., SNase in the example), Endonucleases derived from marcescens (Serratia marcescens) Some examples include: Another preferred example of an endodeoxyribonuclease is the Pfam domain. DNase_NucA_NucB domain (PF14040), DUF1524 domain (PF07510), Endonuclease_NS domain (PF01223), also Endodeoxyribonucleases containing the SNase domain (PF00565) are examples. Endodeo containing the DNase_NucA_NucB domain (PF14040) Preferred examples of xylibonucleases include BsNucB in the example and BlNu in the example. cB, Aspergillus olii described in Sequence IDs 1 and 14 of JP 2022-550112 Endodeoxyribonuclease derived from Aspergillus oryzae Examples include (Sequence IDs 12 and 13). DUF1524 domain (PF07510) A preferred example of an endodeoxyribonuclease containing is JP 2022-5501 Endodeoxyl from the genus Bacillus, as described in sequence numbers 5-9 of 12. Examples include endonucleases (sequence numbers 14-18). Preferred examples of endodeoxyribonucleases containing the NS domain (PF01233) and Therefore, the example is Serratia marcescens Examples include endonucleases derived from ) and SNase domain (PF00565). Preferred examples of endodeoxyribonucleases containing this include SNase as described in the examples. The sequence information of polypeptides containing each Pfam domain is available from InterProDe. It can be obtained from the database (ebi.Ac.Uk / interpro / ).

[0031] A preferred example of BsNucB is a poly(A) consisting of the amino acid sequence shown in SEQ ID NO: 5. Peptides (mature enzymes) are one example. Another example of BsNucB is shown in SEQ ID NO: 5 It consists of an amino acid sequence that has at least 60% identity with the amino acid sequence, and Polypeptides possessing dodeoxyribonuclease activity are examples. BlNucB is preferred. A good example is a polypeptide consisting of the amino acid sequence shown in Sequence ID No. 6 (mature enzyme ) is one example. Another example of BlNucB is the amino acid sequence shown in SEQ ID NO: 6 and It consists of amino acid sequences with at least 60% identity, and endodeoxyribonucle Examples include polypeptides having rease activity. Preferred examples of SNases include A polypeptide (mature enzyme) consisting of the amino acid sequence shown in column number 10 is an example. Another example of Nase is the amino acid sequence shown in SEQ ID NO: 10 and at least 60% It consists of identical amino acid sequences and possesses endodeoxyribonuclease activity. Examples of polypeptides include those derived from Serratia marcescens. A preferred example of a rease is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11. One example is cido (maturation enzyme). Endonu derived from Serratia marcescens. Another example of crease is the amino acid sequence shown in SEQ ID NO: 11 and at least 60% It consists of an amino acid sequence having the same identity and possesses endodeoxyribonuclease activity. Polypeptides that perform this function are listed. Here, the endodeoxyribonuclease activity is the same as that of the polypeptides. It can be measured in the field using known methods.

[0032] The endodeoxyribonuclease used in this invention is a microorganism containing it or It can be extracted or prepared from the culture of Bacillu. For example, BsNucB is derived from Bacillu Extracts from strain 168 of *S. subtilis* (NBRC 111470), etc., or cultures thereof. It can be produced or prepared, and BlNucB is Bacillus lichenifor It can be extracted or prepared from strain mis NBRC 12200, etc., or its culture. SNases include Staphylococcus aureus NCTC 8325 strain, etc. , or can be extracted or prepared from its culture, Serratia marcesc Endonucleases derived from ens include Serratia marcescens NBRC. It can be extracted or prepared from strain 102204, etc., or its culture. The above microorganism is It can be purchased from a public microbiological preservation institution. It contains the endodeoxyribonuclease. The microorganisms present can be adapted using a culture medium containing carbon sources, nitrogen sources, metal salts, vitamins, etc. that they can utilize. Cultivation should be carried out under appropriate conditions. From the microorganisms or culture medium thus obtained, use a general method. Therefore, enzymes are collected and prepared, and then the necessary enzymes are obtained through freeze-drying, spray-drying, crystallization, etc. The form can be obtained. For example, the recovery and preparation of enzymes from cultures can be done by centrifugation or filtering. Separation of microorganisms by filtration, and addition of salts such as ammonium sulfate to enzymes in the supernatant or filtrate. Precipitation by means of precipitation, or by adding an organic solvent such as ethanol, or by using an ultrafiltration membrane, etc. Purification using various chromatography methods such as concentration, desalting, ion exchange, or gel filtration, etc. It can be done using the usual methods.

[0033] Alternatively, the endodeoxyribonuclease used in the present invention may be chemically synthesized or microorganized. It can be manufactured by physical methods. Microbiological aspects of endodeoxyribonuclease In manufacturing, endodeoxyribonuclease has a signal sequence and a mature enzyme region. It is preferable to express it as an endodeoxyribonuclease precursor containing [the specified compound].

[0034] The signal sequence is involved in the extracellular secretion of endodeoxyribonuclease. In an endodeoxyribonuclease precursor containing a nucleotide sequence and a mature enzyme region, sig The N-terminal sequence is preferably located at the N-terminus of the maturation enzyme region. The signal sequence is at the end Deoxyribonuclease precursors are cleaved by signal peptidases as they pass through the cell membrane. It is blocked. An example of a signal sequence is the amino acid sequence from positions 1 to 26 of sequence number 2, and few others. Both sequences consist of amino acid sequences that have 90% identity, and the amino acids at positions 1-32 of sequence number 4. Examples include sequences consisting of amino acid sequences that have at least 90% identity with the no-acid sequence, for example. For example, the signal sequences of BsNucB and BlNucB mentioned above can be cited. Another example of a signal sequence is the amino acid sequence of SEQ ID NO: 8, which is at least 90% identical. Examples include sequences consisting of a monogeneous amino acid sequence, such as Bacillus spy (Baci The signal sequence of the cellulase gene from the KSM-S237 strain of Illus sp. is an example.

[0035] Therefore, the endodeoxyribonuclease precursor consists of a signal sequence and a mature enzyme region. Including amino acid sequences having at least 60% identity with the amino acid sequence of Sequence ID No. 2 It may be a polypeptide. Alternatively, the endodeoxyribonuclease precursor is It contains a Gnar sequence and a mature enzyme region, and at least 60% of the amino acid sequence is the same as that of SEQ ID NO: 4. It may be a polypeptide consisting of a monogenetic amino acid sequence. Alternatively, it may be an endodeoxy. The ribonuclease precursor contains a signal sequence and a mature enzyme region, and sequence number 8 and sequence number 8. Polypeptide consisting of an amino acid sequence having at least 60% identity with the amino acid sequence of No. 10 It could be Petit Do.

[0036] Endodeoxyribonuclease precursors are endodeoxyribonuclease precursors It can be manufactured by expressing a polypeptide from the encoded polynucleotide. Yes, it is possible. The polynucleotide produces the target endodeoxyribonuclease precursor. Genomic DNA is extracted from the microorganism by conventional methods, or RNA is extracted and reverse transcribed. It can be prepared by synthesizing cDNA. Alternatively, the desired endodeo Based on the amino acid sequence of the xyribonuclease precursor, the corresponding nucleotide sequence is converted A polynucleotide encoding the target endodeoxyribonuclease precursor is synthesized. It may be used as a dot. The polynucleotide is an open reading frame (ORF). In addition to the above, the nucleotide sequence of the untranslated region (UTR) may also be included. Nucleotides are the seeds of transformants for the production of endodeoxyribonuclease precursors. Furthermore, codon optimization may also be necessary.

[0037] The polynucleotide encoding the obtained endodeoxyribonuclease precursor is vector It can be incorporated into a vector. The vector can be made by conventional methods to obtain any polynucleotide. It can be produced by inserting it into a vector. The type of vector is not particularly limited. Plasmid, phage, phagemid, cosmid, virus, YAC vector, sha It may be any vector, such as a Torvector. Furthermore, the vector is not limited to, Preferably, it grows within bacteria, preferably within Bacillus bacteria (e.g., Bacillus subtilis or its mutants). A broad vector, more preferably one that induces the expression of a transgene within Bacillus bacteria. It is a viable expression vector. In particular, it can replicate in both Bacillus bacteria and other organisms. The shuttle vector, which is a vector, is suitably used for recombinant production of the mutant of the present invention. It is possible. Examples of preferred vectors include, but are not limited to, pHA304. 0SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY30 0PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Gene) t,1985,60:235-243), pAC3(Nucleic Acids Re Shuttle vectors such as s,1988,16:8732; pUB110 (J Bact eriol,1978,134:318-329), pTA10607(Plasmid Plasmids usable for the transformation of Bacillus bacteria, such as those mentioned in 1987, 18:8-15. Examples include vectors. Also, plasmid vectors derived from E. coli (e.g., pET22) b(+), pBR322, pBR325, pUC57, pUC118, pUC119, p You can also use UC18, pUC19, pBluescript, etc.

[0038] When recombinantly producing an endodeoxyribonuclease precursor, the vector is an expression vector It is preferable that it be a ter. The expression vector is a transcription promoter, terminator, ribo Various elements essential for expression in the host, such as some-binding sites; polylinkers, enhancers cis elements such as sensors; poly(A) addition signals; ribosome-binding sequences (SD sequences); Drugs (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloram) Useful sequences such as selection marker genes (e.g., phenicol) for resistance genes, as needed. It may contain. Alternatively, a polynucleotide encoding a protease precursor may be the above It may contain useful sequences.

[0039] Polynucleotides encoding endodeoxyribonuclease precursors or containing the same By introducing a vector into the host, the endodeoxyribonuclease precursor can be converted to A transformant containing a polynucleotide or a vector containing the same can be obtained. Cut.

[0040] The host for the transformed organism is Bacillus species such as Bacillus subtilis, and Examples include Clostridium species and yeasts, among which basil A species of *S* is preferred, and *Bacillus subtilis* or its mutant strains are more preferred. Therefore, the transformant is Preferably recombinant Bacillus bacteria, more preferably Bacillus subtilis or a recombinant mutant thereof. It is a bacillus subtilis mutant strain, and includes aprX, aprE, nprB, nprE, and bpr A strain lacking a gene selected from vpr, mpr, epr and wprA (Japanese Patent Publication No. 20 Examples include 06-174707).

[0041] For introducing polynucleotides and vectors into host cells, methods such as the protoplast method and Calcium phosphate method, electroporation method, lipofection method, particle gun Well-known transformation techniques such as the PEG method can be applied. For example, Bacillus subtilis or its Applicable methods for mutant strains include competent cell transformation (J Bacterio l,1967,93:1925-1937), electroporation method (FEMS M Icrobiol Lett, 1990, 55:135-138), protoplast trait Conversion method (Mol Gen Genet, 1979, 168:111-115), Tris - PEG method (J Bacteriol, 1983, 156:1130-1134), etc. It can be listed.

[0042] When the transformed organism is cultured in a suitable medium, it produces an endodeoxyribonuclease precursor. It can be expressed. The expressed endodeoxyribonuclease precursor is signaled After the cleavage of the cells, it becomes the mature enzyme endodeoxyribonuclease. Furthermore, endode If the oxyribonuclease precursor has a signal sequence, the mature enzyme is secreted extracellularly. To be produced.

[0043] The transformants are cultured in a medium containing a carbon source, nitrogen source, metal salt, vitamins, etc., that can be assimilated. They can be cultured under appropriate conditions. From the microorganisms or culture thus obtained, a general method The mature enzyme of endodeoxyribonuclease was collected and prepared, and then freeze-dried. The required enzyme form can be obtained by drying, spray drying, crystallization, etc. For example, from a culture The recovery and preparation of the enzyme involves separating microorganisms by centrifugation or filtration, and extracting the enzyme from the supernatant or filtrate. Precipitation by adding salts such as ammonium sulfate, or by adding organic solvents such as ethanol. Various methods such as precipitation, concentration and desalting using ultrafiltration membranes, ion exchange, or gel filtration. This can be done using conventional methods such as chromatography.

[0044] Alternatively, the endodeoxyribonuclease used in the present invention is transformed by To enhance the expression of endodeoxyribonuclease in microorganisms capable of producing iopolymers. It can be manufactured by the above endodeoxyribonuclease microorganism. In the physical manufacturing process, microorganisms capable of producing biopolymers are used as hosts for transformants. By using it, endodeoxyribonucle is produced in microorganisms capable of biopolymer production. This can enhance ase expression. In this case, microorganisms with biopolymer production capabilities Since endodeoxyribonuclease is also expressed during the biopolymer production culture, In the endodeoxyribonuclease treatment process, the endodeoxyribonuclease is processed It can be used for its intended purpose.

[0045] Alternatively, the endodeoxyribonuclease used in the present invention may be a commercially available endodeoxyribonuclease. Xyribonuclease may also be used. Commercially available endodeoxyribonucleases include , endonucleases derived from Serratia marcescens (e.g., SIGMA E1014), bovine pancreas-derived deoxyribonuclease I (e.g., SIGMA, D4) 513) High-salt concentration tolerant nucleases (SANs) (e.g., SIGMA, SRE0015) , Micrococcal Nucl from Staphylococcus aureus ease (e.g., TaKaRa, 2910A), DENARASE (c-LEcta) These are some examples.

[0046] The above endodeoxyribonucleases may be used individually, You may use two or more types in combination.

[0047] As a means of bringing biopolymer granules into contact with endodeoxyribonuclease, Any means by which endodeoxyribonuclease can directly contact the biopolymer granules is acceptable. The means are not particularly limited. For example, the means may include microorganisms capable of producing biopolymers. The cultured bacterial cells were lysed, and the resulting suspension containing the lysates was treated with endodeoxyribonuclear. Adding enzymes, and when disrupting cultured cells of microorganisms capable of producing biopolymers, enzymes Adding dodeoxyribonuclease allows for the cultivation of microorganisms capable of producing biopolymers. The process involves adding endodeoxyribonuclease to the nutrient solution and then disrupting the cultured microbial cells. , or biopolymer production capacity with enhanced expression of endodeoxyribonuclease This includes disrupting the cultured cells of microorganisms that possess biopolymer production capabilities. By disrupting the cultured microbial cells of the organism, biopolymer granules are separated from the cultured microbial cells, and Contact with dodeoxyribonuclease becomes easier. Therefore, the method of the present invention facilitates bioporation. The process may further include a crushing step for crushing cultured cells of microorganisms capable of producing rimers. The order of the crushing process is as follows, from the standpoint of improving the purity of the biopolymer: endodeoxyribonucle It is preferable to perform the ase treatment step before or simultaneously with the endodeoxyribonuclease treatment step.

[0048] As a means of disrupting the cells of microorganisms capable of producing biopolymers, heating of the cells is used. Processing, ultrasonic, high-pressure homogenate, shear force using in-line mixers, crushing and crushing, etc. Physical crushing by mechanical force, chemical treatment with alkalis and surfactants, lytic enzymes, etc. Examples include biological treatment by microorganisms that possess the ability to produce biopolymers. Autolysis, which utilizes the action of enzymes such as proteases and esterases produced by the organism, is also a form of crushing. It can be cited as one of the means to achieve something.

[0049] The heat treatment involves heating the cells of microorganisms capable of producing biopolymers at a temperature of preferably 40-80°C. This is more preferably carried out by maintaining a temperature of 50 to 80°C, and even more preferably 60 to 80°C. This is possible. The processing time is preferably 0.2 to 20 hours, more preferably 0.5 to 10 hours. It is in between.

[0050] Lysolytic enzymes are enzymes that break down peptidoglycans in bacteria. Examples include lamidase, amidase, glucosaminidase, and endopeptidase. More preferably, muramidase (defined herein as lysozyme), more preferably glyco In the side hydrolase (GH) family (CAZy, www.cazy.org) Examples include lysozymes classified as GH22, GH23, GH24, or GH25. A preferred example of 22-lysozyme is chicken egg white lysozyme. Also, G A preferred example of H25 lysozyme is Streptomyces globispo Lysozyme ACM derived from RUS (UniProt ID: P25310, SEQ ID NO: 36) ), Ssp12Lys (sequence number 32), Ssp14Lys (sequence number 33), Asp3 Lys (sequence number 34) and KaLys (sequence number 35) are examples. A preferred example is the LYS polypeptide described in Japanese Patent No. 7275048 (for example, License No. 7275048 Sequence numbers 3, 6, 9, 12, 15, 18, 21, 24, 27, 3 Examples include LYS polypeptides 0, 33, 36, 39, 42, or 45. GH25 Another preferred example of zozyme is the sequence number listed in WO2023 / 110957A1. GH25 muramidases from Acremonium alcalophilum, 1 or 3. (Sequence IDs 23, 24), GH25 derived from Aspergillus fumigatus Examples include muramidase (UniProt id: A4DA29, SEQ ID NO: 25), A preferred example of GH24 rizozyme is the distribution described in WO2023 / 110957A1. GH24 muramidase from Trichophaea saccate in column number 2 (sequence number) Examples include No. 26). In the case of lytic enzymes, such as the endodeoxyribonuclease mentioned above, Similarly, preparation may be carried out from microorganisms containing lytic enzymes or their cultures, or through chemical synthesis or micro-synthesis. Micropolymers that are manufactured by biological methods or transformed to have biopolymer production capabilities. It can be produced by enhancing the expression of lytic enzymes in organisms. The lytic enzyme is a commercially available lytic enzyme. It can also be a bacterial enzyme, such as chicken egg white lysozyme (Fujifilm Wako Pure Chemical Industries, 127-06) 724), Mutanoricin (SIGMA, M9901), a lytic enzyme whose main component is acm ), achromopeptide, a lytic enzyme derived from Lysobacter enzymogenes Tidase (Fujifilm Wako Pure Chemical Industries, 014-09661), Streptomyces Labiace (Cosmo Bio, OZ-30), a lytic enzyme derived from *Fulvissimus* Examples include:

[0051] The crushing means described above can be used individually or in combination of two or more types. However, at the very least, heat treatment of the bacterial cells, lysozyme treatment in which lysozyme is brought into contact with the bacterial cells, or It is preferable to use heat treatment and lysozyme treatment of the cells. Appropriate cell disruption conditions are The types, shapes, and quantities of microorganisms capable of producing opolymers, the types, shapes, and quantities of biopolymers. The amount can be determined appropriately by those skilled in the art, depending on the quantity and other factors.

[0052] The reaction conditions for biopolymer granules and endodeoxyribonuclease are as follows: The conditions are not particularly limited as long as the siribonuclease is not inactivated. Appropriate reaction conditions are, The types, shapes, and quantities of microorganisms capable of producing biopolymers, the types, shapes, and quantities of biopolymers. The amount, type and amount of endodeoxyribonuclease, etc., will be appropriately determined by a person skilled in the art. This is possible. Examples of reaction conditions are listed below.

[0053] The amount of endodeoxyribonuclease used in this reaction determines the biopolymer production capacity. The appropriate amount is determined based on the type, shape, and quantity of microorganisms present. For example, endodeoxyri The amount of bonuclease used is measured by dry weight and is derived from a microorganism or its biopolymer-producing ability. Preferably 0.00001 to 1% by mass, more preferably, relative to 100% of the mass of the lysated microbial material. The amount is 0.0001 to 0.5% by mass, more preferably 0.001 to 0.1% by mass.

[0054] The pH conditions (25°C) for this reaction are important for maintaining enzyme activity and improving the purity of the biopolymer. Preferably, the pH is 6.0 to 13.0, more preferably 7.0 to 12.0, and even more preferably The pH is typically between 8.0 and 11.5.

[0055] The temperature conditions for this reaction are preferably 30 to 80°C, from the viewpoint of improving the purity of the biopolymer. More preferably, the temperature is 40 to 70°C, and even more preferably, 40 to 60°C.

[0056] The reaction time is preferably 30 minutes to 24 hours, from the viewpoint of improving the purity of the biopolymer. More preferably 1 to 12 hours, and even more preferably 1 to 5 hours.

[0057] In the pH adjustment process, at least one time point is observed after the start of endodeoxyribonuclease treatment. By placing the biopolymer granules under conditions of pH 7.0 or higher, the biopolymer The amount of attached and residual DNA can be further reduced. Thus, the pH adjustment process is This is a process for purifying biopolymers, specifically for increasing the purity of iopolymers.

[0058] The pH conditions (25°C) under which the biopolymer granules are placed are pH 7.0 or higher. It is desirable to have a pH of 7.5 or higher, more preferably 8.0 or higher, even more preferably The pH is 8.5 or higher, more preferably 9.0 or higher, and even more preferably above 9.0. More preferably, pH 9.5 or higher, even more preferably, pH 10.0 or higher, and The pH should be 13.0 or lower, more preferably 12.5 or lower, and even more preferably 12. The condition is 0 or less. Furthermore, the pH condition is preferably pH 7.0 to 13.0. Preferably pH 7.5 to 13.0, more preferably pH 8.0 to 12.5, even more preferably The pH is preferably 8.5 to 12.5, more preferably 9.0 to 12.5, even more preferably The pH is greater than 9.0 and less than or equal to 12.5, more preferably between 9.5 and 12.0, and further Preferably, the pH is between 10.0 and 12.0. Note that the pH is measured using the glass electrode method. It can be done.

[0059] For pH adjustment, for example, an alkaline agent can be used. Examples of alkaline agents include: Examples include alkali metal hydroxides and alkaline earth metal hydroxides. Among them, Potassium metal hydroxides are preferred. Alkali metal hydroxides include potassium, lithium, and sodium. These are alkali metal hydroxides such as potassium hydroxide, lithium hydroxide, Examples include sodium hydroxide. When adjusting the pH, the alkaline agent can be used as is. It may also be dissolved in water and used in the form of an aqueous solution. The amount of alkaline agent used depends on the desired pH. It is possible to set the value as appropriate.

[0060] The order in which the endodeoxyribonuclease treatment process and the pH adjustment process are performed is as follows: - From the standpoint of improving purity, at least 1 hour after the start of endodeoxyribonuclease treatment As long as the biopolymer granules are placed under conditions of pH 7.0 or higher in the implementation sequence... The process is not particularly limited, and involves performing a pH adjustment step following the endodeoxyribonuclease treatment step. Alternatively, it is preferable to perform the endodeoxyribonuclease treatment step and the pH adjustment step simultaneously. The endodeoxyribonuclease treatment process and the pH adjustment process are carried out sequentially in this order. The deoxyribonuclease treatment process and the pH adjustment process are carried out independently in this order, or It is more preferable to perform the dodeoxyribonuclease treatment step and the pH adjustment step simultaneously. The continuous execution of the deoxyribonuclease treatment step and the pH adjustment step is preferably performed by Opolymer granules were treated with endodeoxyribonuclease, and the pH of the treatment solution was adjusted after treatment. This refers to adjusting the pH to 7.0 or higher. This involves the endodeoxyribonuclease treatment process and pH adjustment. Independent execution of the process preferably involves adding endodeoxyribonuclease to biopolymer granules. After processing, the supernatant of the processed solution is removed, and the biopolymer granules are mixed with water-soluble material with a pH of 7.0 or higher. This refers to adding a liquid. Here, an aqueous solution with a pH of 7.0 or higher is used for washing the biopolymer granules. It can function as a purifying solution. Simultaneous endodeoxyribonuclease treatment and pH adjustment processes. The process preferably involves dissolving endodeoxyri in biopolymer granules under conditions of pH 7.0 or higher. This refers to the process of treating with bonuclease. During this process, the pH condition is within the range of pH 7.0 or higher. The settings should be adjusted appropriately to maintain the enzyme activity of endodeoxyribonuclease. When the deoxyribonuclease treatment process and the pH adjustment process are performed simultaneously, after simultaneous execution, A pH adjustment step may also be performed, preferably under conditions of pH 7.0 or higher, for bio Polymer granules are treated with endodeoxyribonuclease, and the supernatant of the treated solution is removed after treatment. This refers to adding an aqueous solution with a pH of 7.0 or higher to biopolymer granules. - The time for keeping the granules under conditions of pH 7.0 or higher is not particularly limited, and depends on the implementation of the pH adjustment process. You should adjust the settings as appropriate, taking the user's needs into consideration.

[0061] The present invention provides a method for purifying biopolymers, with respect to improving the purity of biopolymers. The process involves a protease treatment step in which biopolymer granules are brought into contact with protease. It may also be included in the others.

[0062] Here, "protease" refers to the enzyme that hydrolyzes the peptide bonds of protein molecules. This refers to proteins that possess protease activity, which produces proteins and amino acids.

[0063] The protease used in the present invention is not particularly limited, but is preferably in an alkaline environment. Examples include proteases having protease activity, and more preferable serine proteases. Ze (EC number 3.4.21) is an example, and more preferably S in the MEROPS classification. Examples include proteases of the 8-peptidase family, and more preferably Bacillus (Ba Examples of proteases derived from bacteria of the genus *Cillus* include... Therefore, Bacillus espi described in Japanese Patent Application No. 2024-071822 Alkaline protease KP43 (SEQ ID NO: 19) derived from sp.)KSM-KP43 and Variants of (for example, 12 variants, Sequence ID No. 4 of Japanese Patent Application No. 2024-071822) 20)), Bacillus sp. KSM-K16 as described in Japanese Patent Application No. 2024-071822 ( Alkaline protease K16 (SEQ ID NO: 21) derived from FERM BP-3376 and Variants of (for example, quadruple mutants, Sequence ID No. 8 of Japanese Patent Application No. 2024-071822) 22)) Subtilisin derived from Bacillus lentus and its variants HH844 (SEQ ID NO: 27) and its variants (SEQ ID NOs: 28, 29), variants of LL147 Examples include variants (SEQ ID NOs: 30, 31). The protease used in the method of the present invention is Similar to the case of endodeoxyribonuclease described above, microorganisms containing proteases Alternatively, preparation from the culture, production by chemical synthesis or microbiological methods, This involves enhancing the expression of proteases in microorganisms capable of biopolymer production through transformation. It can be manufactured by the following. Alternatively, the protease used in this invention may be a commercially available one. A protease may also be used. A commercially available protease is Protin SD-AY10. Protease P "Amano" 3SD (Amano Enzyme Co., Ltd.), Bioplase OP (Nagasevi (HBI Corporation), Orientase 22BF, Aloase XA-10 (Yakult Pharmaceutical Co., Ltd.), Alcalase, Esperase, Everlas e, Savinase, Kannase, Progress Uno (registered trademark; Novone) Sys Corporation, Preferenz (registered trademark; IFF Corporation) series, Lavergy (registered trademark) Trademarks (BASF, Inc.) are some examples.

[0064] The above proteases may be used individually or in combination of two or more. They may be used together.

[0065] A means of contacting biopolymer granules with protease, biopolymer granules and protease The reaction conditions with the ase are the same as those for the endodeoxyribonuclease described above.

[0066] The order of the protease treatment process is not particularly limited, and endodeoxyribonuclear The implementation of each or both of the ze treatment process and the pH adjustment process, and the implementation of the protease treatment process. The order of events does not matter. From the standpoint of improving the purity and manufacturing efficiency of biopolymers, The ase treatment process should be performed at least simultaneously with the endodeoxyribonuclease treatment process. preferable.

[0067] When performing the endodeoxyribonuclease treatment process and the protease treatment process simultaneously: From the standpoint of maintaining the activity of endodeoxyribonuclease, bio-propagation in the presence of proteases is recommended. The enzyme has protease resistance to the extent that it can maintain the activity to degrade the DNA remaining in the remer. It is preferable to use endodeoxyribonuclease. Examples of ases include BsNucB, BlNucB, SNase, and Serratia. Examples include endonucleases derived from Marcescens. Also, proteases. A resistant endodeoxyribonuclease mutant may be used. For example, xylibonuclease is described in WO2022 / 194668A1. Examples include endodeoxyribonuclease mutants.

[0068] The present invention provides a method for purifying biopolymers, with respect to improving the purity of biopolymers. As part of the process, a surfactant treatment step is further carried out in which a surfactant is brought into contact with the biopolymer granules. It may be included.

[0069] The surfactants used in this invention include anionic surfactants, nonionic surfactants, Examples include amphoteric surfactants, cationic surfactants, or a combination thereof, but From the viewpoint of reducing residual DNA in iopolymers, anionic surfactants are preferred. Examples of nionic surfactants include sulfate esters of alcohols with 10 to 18 carbon atoms, and those with 8 carbon atoms. ~20 alcohol alkoxylated sulfate salts, alkylbenzene sulfonic acid Salt, paraffin sulfonate, α-olefin sulfonate, internal olefin sulfonate Examples include salts, α-sulfo fatty acid salts, α-sulfo fatty acid alkyl ester salts, or fatty acid salts. This can be done. In particular, sulfate ester salts of alcohols with an alkyl chain having 10 to 14 carbon atoms, and Sulfate esters and alkyl esters of ethoxylated alcohols with 10 to 14 carbon atoms in the hydrate chain. At least one linear alkylbenzene sulfonate salt selected from linear alkylbenzene sulfonates with 10 to 14 carbon atoms in the chain. A single anionic surfactant is preferred, and an alcohol having 10 to 14 carbon atoms in its alkyl chain is preferred. Sulfuric acid of sulfate ester salts and ethoxylated alcohols with 10 to 14 carbon atoms in the alkyl chain. More preferably, at least one anionic surfactant selected from ester salts. , sulfate ester salts of alcohols with 10 to 14 carbon atoms in the alkyl chain, number of carbon atoms in the alkyl chain The sulfate ester salts of ethoxylated alcohols with 10-14 carbon atoms and alkyl chains with 1 carbon atom At least one anionic kingdom selected from 0 to 14 linear alkylbenzene sulfonates Among surfactants, sulfate ester salts of alcohols with 12 carbon atoms in the alkyl chain, alkyl chain The number of carbon atoms in the alkyl chain of the ethoxylated alcohol ester salt and the number of carbon atoms At least one anionic interface selected from 12 linear alkylbenzene sulfonates The properties agent is more preferably a sulfate ester salt of an alcohol having 12 carbon atoms in the alkyl chain and an alcohol Selected from sulfate ester salts of alcohol ethoxylated alcohols with 12 carbon atoms in the kill chain A single anionic surfactant is more preferably used, along with dodecyl sulfate and polyoxy at least one anionic interface selected from ethylene lauryl ether sulfate salts An activator is even more preferred. As a counterion, alkali metal salts, alkanolamine salts, Ammonium salts are preferred, along with sodium and / or potassium, monoethanolamine, and di Ethanolamine, triethanolamine, and ammonium ions are more preferred, and sodium Sodium is even more preferred. Preferred specific examples of anionic surfactants include sodium dodecyl sulfate. Thorium, polyoxyethylene lauryl ether sodium sulfate, dodecylbenzenesulfate Sodium ethanolate, triethanolamine dodecyl sulfate, and ammonium dodecyl sulfate At least one selected from, more preferably sodium dodecyl sulfate, polyoxyethylene Selected from sodium lauryl ether sulfate and sodium dodecylbenzenesulfonate At least one of the following, more preferably sodium dodecyl sulfate and polyoxyethylene At least one of the following can be selected from sodium lauryl ether sulfate.

[0070] The amount of surfactant used depends on the type, shape, and quantity of microorganisms capable of producing biopolymers. It is determined appropriately by the following. For example, the amount of surfactant used is determined by the dry mass of the biopolymer Preferably 0.01 to 1% of the mass of the productive microorganism or its cell lysate. 0% by mass, more preferably 0.05 to 5% by mass, and even more preferably 0.1 to 3% by mass. ru.

[0071] The means of contacting biopolymer granules with a surfactant is the above-mentioned endodeoxyribonu The situation is similar to that of Crease.

[0072] The order of the surfactant treatment process is not particularly limited, and endodeoxyribonuclease Before and after the execution of each or both of the processing and pH adjustment processes and the execution of the surfactant treatment process. The relationship doesn't matter. From the standpoint of improving the purity and manufacturing efficiency of biopolymers, surfactants The processing steps consist of at least one step: an endodeoxyribonuclease treatment step and a pH adjustment step. It is preferable to do this at the same time.

[0073] In one preferred embodiment, the method of the present invention involves endodeoxyribonuclease treatment. Process, lysozyme treatment process as a crushing process, pH adjustment process, protease treatment process and This includes a surfactant treatment step. The order in which each step is performed is: endodeoxyribonuclease treatment. At least one point in time after the start, the biopolymer granules were under conditions of pH 7.0 or higher. As long as the implementation order is as described, it is not particularly limited, and is intended to improve the purity and manufacturing efficiency of biopolymers. From this perspective, it is preferable to perform two or more processes simultaneously, and even more preferable to perform three or more processes simultaneously. Preferably, four or more steps are carried out simultaneously. For example, endodeoxyri The Bonuclease treatment process and the lysozyme treatment process are carried out simultaneously, followed by the pH adjustment process, and then the pro A method in which the thease treatment step and the surfactant treatment step are carried out in this order, endodeoxyribonu The crease treatment process, the lysozyme treatment process, and the protease treatment process are performed simultaneously, and then... A method for simultaneously performing a pH adjustment step and a surfactant treatment step, endodeoxyribonuclear -ase treatment process, lysozyme treatment process, pH adjustment process, protease treatment process and surfactant One example is a method in which all agent treatment processes are carried out simultaneously.

[0074] The present invention provides a method for purifying biopolymers, with respect to improving the purity of biopolymers. The process consists of an enzyme treatment step, an oxidizing agent treatment step, a centrifugation step, a membrane filtration step, and a washing step. It may further include at least one more step that is selected.

[0075] In the enzyme treatment process, enzymes other than endodeoxyribonuclease and protease are used. By acting on the cells of microorganisms capable of producing biopolymers or on biopolymer granules This can improve the purity of biopolymers. Endodeoxyribonuclease and Other enzymes besides proteases can be any enzyme capable of hydrolyzing bacterial cell-derived components, for example. Lysozyme, muramidase, glycosidase, cellulase, lipase, amylase, Nucleases other than tinase, laccase, nuclease, and endodeoxyribonuclease Examples include yeast, and one or more of these can be used in combination. The initial processing step involves using microorganisms capable of producing biopolymers, depending on the type of enzyme used. This can be done under the common conditions used in polymer manufacturing.

[0076] In the oxidizing agent treatment process, the cells of microorganisms capable of producing biopolymers or biopolymers are used. By contacting the granules with an oxidizing agent, impurities derived from bacterial cells are reduced to low molecular weight, and the purity of the biopolymer is reduced. This improves the degree of efficiency, reduces the membrane filtration load in the membrane filtration process, and suppresses membrane clogging. It is possible. Furthermore, the endodeoxyribonuclease treatment process and the oxidizing agent treatment process can be combined. This further promotes the demilitarization of impurities such as nucleic acids in the treatment solution, and the viscosity of the treatment solution is reduced. This reduces the efficiency of stirring in each process, the centrifugal efficiency in the centrifugal process, and the efficiency of the membrane filtration process. It is expected that this will be possible. Also, by decolorizing the biopolymer, the The chromaticity of the final product can be improved. For example, the CIE (Colour International Equalization) can be used as an indicator of the chromaticity of the final product. The b* value of the LAB color coordinates is mentioned, and preferably the b* value is less than 15. As an oxidizing agent, , but not limited to, hydrogen peroxide, ozone; sodium peroxide, sodium perborate Other inorganic peroxides such as sodium chlorite, sodium percarbonate, sodium persulfate; chlorite, chlorine Salts, metachloroperbenzoate perchlorate, perchloric acid, chlorine dioxide, and similar halogen compounds Substances; peracids such as performic acid and peracetic acid; permanganate compounds such as potassium permanganate; perboric acid Sodium; potassium nitrate; sodium bismuthate; cerium ammonium nitrate, cerium sulfate Examples include cerium(IV) compounds such as lium. Preferably sodium chlorite, peroxide. It is hydrogen oxide or ozone. It suppresses the dematuration of biopolymers while removing bacterial-derived impurities. Sodium chlorite is preferred because it can reduce the molecular weight and improve the color. Bacterial cell-derived impurities. Hydrogen peroxide or ozone is preferred because it reduces the molecular weight and thus the load on membrane filtration. The oxidizing agent is One or more types may be used in combination. Concentration of sodium chlorite in the treatment solution. While not particularly limited, from the viewpoint of improving chromaticity while suppressing molecular weight reduction, it is preferable. The amount is 0.01 to 1.3% by weight, more preferably 0.05 to 1.0% by weight. The pH at which thorium is brought into contact with the biopolymer may be acidic, for example, pH 1 The pH is 0.0 to 7.0, preferably 3.0 to 5.0. The degree is not particularly limited, but is preferably 0.01 to 30% by weight, more preferably The concentration is 0.1 to 15% by weight, and more preferably 0.2 to 10% by weight. Hydrogen peroxide When using this, it may be brought into contact with the biopolymer granules under alkaline pH conditions, for example, The pH is 7.0 to 13.0, preferably 8.0 to 10.0. Furthermore, sodium bicarbonate The combined use of thorium can enhance the action of hydrogen peroxide. Additionally, the combined use of a chelating agent may be beneficial. By using it, the hydrogen peroxide solution can be stabilized. The chelating agent is not particularly limited. In addition, sodium silicate, EDTA, and trans-1,2-cyclohexanediamine tetravinegar are used. Examples include acid monohydrates. Sodium chlorate treatment and hydrogen peroxide treatment can be used to improve color. The processes may be carried out in order. The amount of ozone added is not particularly limited, but preferably bio-po The amount is 0.01 to 0.1 g per 1 g of dry mass of cultured microorganisms capable of producing rimers. Preferably, 0.02 to 0.08 g, and more preferably, 0.02 to 0.07 g. be.

[0077] In the centrifugation process, unwanted supernatant is removed from the processing solution containing biopolymer granules. This allows for improving the purity of biopolymers. The method of centrifugation is not particularly limited. Although not always done, a preferred method is to use a decanter-type centrifugal separator. Examples of heart separators include horizontal and vertical types, but the ability to process a large volume of fluid is a key feature. From this perspective, a horizontal configuration is preferable.

[0078] In the membrane filtration process, impurities derived from bacterial cells are removed by filtering the treated liquid through a membrane. Yes, it is possible. The filtration method is not particularly limited, but cross-flow filtration and dead-end filtration are preferred. This is excessive, and more preferably, cross-flow filtration.

[0079] In the washing process, the biopolymer granules are washed with an aqueous solution to remove impurities derived from bacterial cells. This can remove impurities and improve the purity of the biopolymer. The cleaning method is not particularly limited. However, in the centrifugation process, the solution is added to the biopolymer after removing the unnecessary supernatant of the processing liquid. and the step of performing centrifugation and removing the supernatant may be repeated. The solution to be added is an alka line solution. It is preferably alkaline.

[0080] The order of carrying out at least one step selected from the above steps is not particularly limited, and a plur ality of steps may also be carried out simultaneously.

[0081] The solid content of the treated liquid obtained after the above purification step contains a biopolymer, and the solid content of the treated liquid content is recovered, the biopolymer can be recovered. Therefore, the method of the present invention further comprises recovering the biopolymer from the treated liquid and may further include a recovery step of recovering the biopolymer. This step is applicable to the production of biopolymers by microorganisms having biopolymer-producing ability, and can be carried out under general conditions employed for the production of biopolymers by microorganisms having biopolymer-producing ability. For example, in this step, the treated liquid is subjected to solid-liquid separation such as centrifugation to obtain a solid content, after washing the solid content as necessary, the solid content can be dried by spray drying, evaporation to dryness, freeze drying, or the like and drying to obtain biopolymer powder. Through this step, a biopolymer powder can be obtained .

[0082] According to the method of the present invention, the amount of DNA attached to and remaining in the finally obtained 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 is determined by endodeoxy ribonuclease treatment step, except that it does not include the endodeoxyribonuclease treatment step, the biopolymer is produced by the same method as the method of the present invention compared with the amount of DNA remaining in the obtained biopolymer, per unit mass of biopolymer powder, it is preferably 70% or less, more preferably 50% or less, still more preferably 40% or less, can be reduced to can be reduced. In addition, the method of the present invention improves the efficiency during biopolymer recovery and the finally obtained This could also contribute to reducing the adhesion and improving the fluidity of biopolymers.

[0083] The above endodeoxyribonuclease treatment process involves the adhesion and residue of the biopolymer. This is a process to reduce the amount of DNA and improve the purity of the biopolymer. Therefore, the present invention is Furthermore, improving the purity of biopolymers produced by microorganisms capable of biopolymer production. Methods, or residues in biopolymers produced by microorganisms capable of biopolymer production. The present invention provides a method for reducing the amount of DNA involved. The method involves a micro-polymer with biopolymer production capabilities. Endodeoxyribonuclease was contacted with biopolymer granules obtained by culturing organisms. The method includes an endodeoxyribonuclease treatment step. The process involves at least one step selected from the protease treatment step and the surfactant treatment step. It may also include the above heat treatment process, enzyme treatment process, oxidizing agent treatment process, and centrifugation. The process may include at least one step selected from the process, membrane filtration process, and washing process. Details of each step are described above using the biopolymer production ability of the microorganisms of the present invention. The method is the same as in the case of the method for producing the rimer. The amount of DNA remaining in the biopolymer of the present invention The amount of residual DNA reduced by the reduction method is, per unit amount of biopolymer powder, It may be 30% or more, more preferably 50% or more, and even more preferably 60% or more. ru.

[0084] As exemplary embodiments of the present invention, the following compositions, manufacturing methods, uses, or methods may be described. This is disclosed herein. However, the present invention is not limited to these embodiments.

[0085] [1] A method for producing a biopolymer, Biopolymer granules obtained by culturing microorganisms capable of producing biopolymers contain ene The endodeoxyribonuclease treatment process involves contacting the dodeoxyribonuclease with the endodeoxyribonuclease and the preceding process. At least one point in time after the start of endodeoxyribonuclease treatment, the bio A pH adjustment step in which polymer granules are placed under conditions of pH 7.0 or higher, Methods that include... [2] The endodeoxyribonuclease treatment step is performed by endodeoxyribonuclease This is a process that reduces the molecular weight of DNA attached to a biopolymer and makes it solubilized, [1] Method of description. [3] The pH adjustment step is performed after the endodeoxyribonuclease treatment step. This method performs the endodeoxyribonuclease treatment step and the pH adjustment step simultaneously. A method as described in [1] or [2]. [4] Further comprising a surfactant treatment step of contacting the biopolymer granules with a surfactant The method described in any one of items [1] to [3]. [5] The method according to [4], wherein the surfactant is an anionic surfactant. [6] At least one of the endodeoxyribonuclease treatment step and the pH adjustment step The method according to [4] or [5], wherein the step and the surfactant treatment step are performed simultaneously. [7] A protease treatment step is further performed, in which the biopolymer granules are brought into contact with a protease. The method described in any one of items [1] to [6], which is included in [1]. [8] Simultaneous endodeoxyribonuclease treatment step and protease treatment step The method described in [7] is performed in the following manner. [9] The lysozyme is brought into contact with the cellular cells of the microorganism having biopolymer production ability. The method according to any one of [1] to [8], further comprising a -ome treatment step.

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

[11] The method according to [9] or

[10] , wherein said endodeoxyribonuclease treatment step, said pH adjustment step, said prote ase treatment step, said lysozyme treatment step and said surfactant treatment step are performed simultaneously .

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

[11] , further comprising, before said endodeoxyribonuclease treatment step, a culture step of culturing a microorganism having an ability to produce said biopolymer.

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

[12] , further comprising a disruption step of disrupting cultured cells of a microorganism having an ability to produce a biopolymer .

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

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

[0086]

[15] A method for increasing the purity of a biopolymer produced by a microorganism having an ability to produce a biopolymer, comprising: an endodeoxyribonuclease treatment step of bringing endodeoxyribonuclease into contact with biopolymer granules obtained by culturing a microorganism having an ability to produce a biopolymer, the method comprising said step.

[16] A method for reducing the amount of DNA remaining in a biopolymer produced by a microorganism having an ability to produce a biopolymer, comprising: an endodeoxyribonuclease treatment step of bringing endodeoxyribonuclease into contact with biopolymer granules obtained by culturing a microorganism having an ability to produce a biopolymer, the method comprising said step. ​​​​​

[17] The endodeoxyribonuclease treatment step is This is a process in which DNA attached to a biopolymer is broken down into smaller molecules and solubilized by a ze. [1 The method described in (5) or (16).

[18] At least one point in time after the start of the endodeoxyribonuclease treatment The process further includes a pH adjustment step of placing the biopolymer granules under conditions of pH 7.0 or higher, [ The method described in any one of items 15) to 17).

[19] The pH adjustment step is performed following the endodeoxyribonuclease treatment step. Alternatively, the endodeoxyribonuclease treatment step and the pH adjustment step may be performed simultaneously. The method described in

[18] .

[20] A surfactant treatment step is further performed, in which the biopolymer granules are brought into contact with a surfactant. Including the method described in any one of paragraphs

[15] to

[19] .

[21] The method according to

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

[22] At least the endodeoxyribonuclease treatment step and the pH adjustment step The first step and the surfactant treatment step are performed simultaneously, as described in

[20] or

[21] . method.

[23] A protease treatment step is performed in which the biopolymer granules are brought into contact with a protease. The method described in any one of items

[15] to

[22] , including the above.

[24] The endodeoxyribonuclease treatment step and the protease treatment step are performed together. The method described in

[23] , which is performed at times.

[0087]

[25] The condition of pH 7.0 or higher is preferably pH 7.5 or higher, more preferably pH pH 8.0 or higher, more preferably pH 8.5 or higher, even more preferably pH 9.0 or higher, further Preferably, pH above 9.0, more preferably pH 9.5 or higher, and even more preferably pH 1 pH 0.0 or higher, preferably pH 13.0 or lower, more preferably pH 12.5 or lower. Furthermore, the conditions are 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, further Preferably pH 8.5 to 12.5, more preferably pH 9.0 to 12.5, even more preferably More preferably, the pH is greater than 9.0 and less than or equal to 12.5, and more preferably between 9.5 and 12.0. More preferably, the conditions are pH 10.0 to 12.0, [1] to

[14] and

[18] The method described in any one of the items in

[24] .

[26] The pH conditions of the pH adjustment step are greater than pH 9.0, [1] to

[14] and [ The method described in any one of items 18 to 24.

[27] The pH conditions of the pH adjustment step are pH 9.5 or higher, [1] to

[14] and The method described in any one of items

[18] to

[24] .

[28] The pH conditions of the pH adjustment step are pH 10.0 or higher, [1] to

[14] and The method described in any one of items

[18] to

[24] .

[29] The pH conditions of the pH adjustment step are pH 13.0 or lower, [1] to

[14] and The method described in any one of items

[18] to

[28] .

[30] The pH conditions of the pH adjustment step are pH 12.5 or lower, [1] to

[14] and The method described in any one of items

[18] to

[28] .

[31] The pH conditions of the pH adjustment step are pH 12.0 or lower, [1] to

[14] and The method described in any one of items

[18] to

[28] .

[32] The endodeoxyribonuclease is used by bacteria of the genus Bacillus. endodeoxyribonuclease derived from, Staphylococ cus) genus bacteria-derived endodeoxyribonuclease and Serratia ) The method according to any one of [1] to

[31] , which is at least one selected from endodeoxyribonucleases derived from bacteria of the genus .

[33] The endodeoxyribonuclease is Bacillus subt ilis)-derived Mn 2+ / Mg 2+ -dependent endodeoxyribonuclease, Bacill us licheniformis-derived Mn 2 + / Mg 2+ -dependent endodeoxyribonuclease, Staphy lococcus aureus)-derived Ca 2+ -dependent endodeoxyribonuclease that endo hydrolyzes DNA and RNA, and endonuclease derived from Se rratia marcescens), which is at least one selected from the group consisting of , the method according to any one of [1] to

[31] .

[34] The endodeoxyribonuclease has a DNase_NucA_NucB domain in (PF14040), DUF1524 domain (PF07510), Endonucle ase_NS domain (PF01223), or SNase domain (PF0056 5), which is an endodeoxyribonuclease comprising, the method according to any one of [1] to

[31] .

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

[34] , wherein the endodeoxyribonuclease is at least one selected from the group consisting of the following (A) and (B): (A) ​ Polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 5, 6, 10, and 11 ; and (B) the amino acid sequence shown in any of sequence numbers 5, 6, 10 and 11 and at least Both consist of amino acid sequences with 60% identity, and endodeoxyribonuclear A polypeptide possessing xerogenic activity.

[36] The method according to

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

[37] The method according to

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

[38] The method according to

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

[39] The surfactant is an anionic surfactant, preferably having 10 to 18 carbon atoms. Sulfate salts of alcohols, sulfates of alkoxylated alcohols with 8 to 20 carbon atoms Steryl salts, alkylbenzene sulfonates, paraffin sulfonates, α-olefins Sulfonate, internal olefin sulfonate, α-sulfo fatty acid salt, α-sulfo fatty acid At least one selected from chlor ester salts and fatty acid salts, more preferably an ammonium compound. Sulfate esters of alcohols with 10-14 carbon atoms in the kill chain, and alkyl chains with 10 carbon atoms. Sulfate salts of ethoxylated alcohols of ~14 types and alkyl chains with 10 to 1 carbon atoms. At least one selected from 4 linear alkylbenzene sulfonates, more preferably Alternatively, sulfate ester salts of alcohols with an alkyl chain having 10 to 14 carbon atoms and alkyl chains Selected from sulfate ester salts of ethoxylated alcohols having 10 to 14 carbon atoms The method described in any one of the items [4] to

[14] and

[20] to

[38] , which is of type 1.

[40] The surfactant is an anionic surfactant, preferably the number of carbon atoms in the alkyl chain Sulfate salts of 12 alcohols, ethoxy compounds of alcohols with 12 carbon atoms in the alkyl chain. Sulfate salts of compounds and linear alkylbenzene sulfonic acids with 12 carbon atoms in the alkyl chain. At least one selected from salts, more preferably an alkyl chain having 12 carbon atoms. Sulfuric acid salts of kohl and ethoxylated alcohols with 12 carbon atoms in the alkyl chain At least one selected from acid ester salts, more preferably ester dodecyl sulfate. At least one selected from 1 lauryl salt and polyoxyethylene lauryl ether sulfate salt The method described in any one of the following items: [4] to

[14] and

[20] to

[38] .

[41] The surfactant is an anionic surfactant, preferably sodium dodecyl sulfate M, polyoxyethylene lauryl ether sulfate sodium and dodecylbenzene sulfone At least one selected from sodium phosphate, more preferably sodium dodecyl sulfate At least one selected from and polyoxyethylene lauryl ether sodium sulfate The method described in any one of the items [4] to

[14] and

[20] to

[38] .

[42] The biopolymer is a hydroxyalkanoic acid-containing polymer, [1] to [4 The method described in any one of item 1).

[43] The hydroxyalkanoic acid-containing polymer contains only hydroxyalkanoic acid as the monomer. - Polyhydroxyalkanoic acid (PHA), which is a polyester contained as a unit, or monomer - Containing a hydroxyalkanoic acid and a carboxylic acid having an amino group as units, ester bonds A polyester amide having a ion and amide bond, preferably PHA, [42 The method described.

[44] Hydro in the monomer units constituting the hydroxyalkanoic acid-containing polymer The proportion of xyalkanoic acid is preferably 50% or more, and more preferably 60% or more. More preferably 70% or more, even more preferably 80% or more, and even more preferably The percentage is 90% or more, more preferably 95% or more, and even more preferably 98%. The method according to

[42] or

[43] , wherein the amount is % or more, and more preferably 100%.

[45] The biopolymer is polyhydroxyalkanoic acid (PHA), [1] to [ The method described in any one of item 41).

[46] The microorganism having the ability to produce biopolymers belongs to the genus Cupriavidus. A method described in any one of the items [1] to

[45] , wherein the microorganism is used. [Examples]

[0088] The present invention will be described in more detail below using examples, but the technical scope of the present invention is as follows: This is not limited to the examples provided.

[0089] Example 1 (1) Construction of enzyme expression plasmid The S237 secretion of plasmid pHY-BLP2 described in WO2019 / 142773 The full-length ORF, consisting of the Gnar sequence and BLP proprotein, is used as a native secretory signal. The BsnucB gene (polynucleotide of SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2) contains Plasmid pHY-BsNucB was obtained by substituting (which codes for) similarly with ORF The entire length of the BlnucB gene (polynucleotide of SEQ ID NO: 3) containing the native secretion signal. Otide (which encodes the amino acid sequence of SEQ ID NO: 4) can be substituted to plasmid pHY- BlNucB was obtained. The amino acid sequences of the mature BsNucB and BlNucB are as follows: These are sequence numbers 5 and 6. Contains a promoter derived from the Bacillus subtilis spoVG gene as described in WO2021 / 153129. The entire ORF containing the VHH gene of the VHH expression plasmid of SEQ ID NO: 26 is extended to the N-terminus. On the side is the S237 secretion signal sequence (polynucleotide of SEQ ID NO: 7, amino acid of SEQ ID NO: 8). The SNase gene (sequence code 9 polynucleotide, which codes for the sequence) is linked. By substituting (which encodes the amino acid sequence at column number 10), plasmid pHY-SNas We obtained e. The expression of the protease KP43 mutant is described in Japanese Patent Application No. 2024-071822. Using 43 12-duplex expression plasmids (Sequence ID 4 of Japanese Patent Application No. 2024-071822) I used it.

[0090] (2) Preparation of nuclease solution The enzyme expression plasmid was introduced into Bacillus subtilis strains by protoplast, and 2×L-maltose was produced. Culture medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetrasulfate Ikurin; % is (w / v)%, cultured at 30°C for 3 days, then the culture supernatant containing the enzyme is removed by fistula. It was recovered by heart separation. 20 mM T containing 2 mM CaCl2 was detected by Amicon 10K. The buffer was changed to ris-HCl (pH 7.5). The DC Protein Assay Kit (Bio-Rad) was used to measure the concentration of the enzyme solution. The standard solution used for calculating protein content is BSA Standard Solution (W Use AKO.

[0091] (3) Preparation of PHA-containing bacterial cells Cupriavidus necator(Ralstonia eutropha) NBRC strain 102504 was inoculated into LB liquid medium and incubated with shaking at 30°C for 24 hours. Add 1 mL of the culture medium to 100 mL of PHA production medium (1.1% disodium hydrogen phosphate) Dihydrate, 0.19% potassium dihydrogen phosphate, 0.13% ammonium sulfate, 0.1% sulfur Magnesium oxide heptahydrate, 16.2 ppm; iron(III) chloride hexahydrate, 10.3 ppm; salt Calcium chloride dihydrate, 0.218 ppm; Cobalt chloride hexahydrate, 0.156 ppm; Sulfuric acid Copper pentahydrate, 0.118 ppm nickel chloride hexahydrate, 2% coconut oil; % is (w / v) % The bacteria were inoculated into a 500 mL baffled flask with the added ingredients and cultured with shaking at 30°C for 72 hours. A suspension of PHA-containing bacterial cells was obtained.

[0092] (4) PHA purification by nuclease 1 A pH meter, LAQUAact D-72 (HORIBA), was used to measure the pH. (3) Add 1N sodium hydroxide aqueous solution to the PHA-containing bacterial cell suspension obtained in (3) to pH 11 The pH was adjusted to 0.0 and heated at 60°C for 1 hour to inactivate the bacterial cells. 2N sulfuric acid was added to adjust the pH to 7. Adjust to 0.0, chicken egg white lysozyme (Fujifilm Wako Pure Chemical Industries, 127-06724) After adding the solution at a final concentration of 20 mg / L, the mixture was incubated at 50°C for 1 hour. 1N sodium hydroxide After adding thorium aqueous solution to adjust the pH to 9.0, dispense 1 mL into 2 mL tubes. The enzymes listed in Enzyme Treatment 1 in Table 1 were added, and the mixture was incubated at 50°C for 1 hour. Furthermore, the enzymes listed in Enzyme Treatment 2 in Table 1 were added, and the mixture was incubated at 50°C for 1 hour. The nuclease was BlNucB at a final concentration of 4 mg / L, and the protease was alcalase (SI GMA (126741) was used at a final concentration of 60 mg / L. It contained 1.2 (w / v)% SDS. 1 mL of 6 mM sodium hydroxide aqueous solution was added and mixed by inversion (pH after addition was approximately 10.0). The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mM sodium hydroxide Add 1 mL of aqueous solution and mix by inversion, then centrifuge at 12000 rpm for 5 minutes, and then remove the supernatant completely. The volume removal procedure was repeated twice. 1 mL of deionized water was added and mixed by inversion, then 1200 After centrifugation at 0 rpm for 5 minutes, the supernatant was completely removed. The tube cap was opened and the mixture was heated at 60°C. I let it dry overnight. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 400 μL, and mix 100 μL of the suspension with 100 × 100 GelRed (trademark) aqueous solution (B iotium) (10000 concentrate diluted 100 times with deionized water) 10 μL to black 96 Mixed in a perforated plate and incubated at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured (Table 1). High fluorescence was observed without nuclease addition, and the fluorescence intensity was significantly increased with nuclease treatment. It decreased. This suggests that nucleic acids unexpectedly remain in the PHA after recovery. It has been shown that residual nucleic acids are reduced by ase treatment. In other words, the PHA after recovery contains residual nucleic acids. Unexpectedly, at least some insoluble DNA remains, and the amount is reduced by nuclease treatment. It can be seen that the amount of insoluble residual DNA is reduced. Also, by using proteases in combination... Surprisingly, residual nucleic acids were further reduced.

[0093] [Table 1]

[0094] (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 bacterial 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. After addition, it was incubated at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added. After adjusting the pH to 8.0, add 1 mg / L of nuclease BlNucB and the final concentration 100 mg / L of alcalase was added and incubated at 50°C for 2 hours. Enzyme treatment. The subsequent samples were dispensed into 2 mL tubes, 1 mL each. The dispensed processing solution was then treated with 0.4 (w / v Add 1 mL of %) SDS aqueous solution, then add 1N sodium hydroxide aqueous solution to adjust the pH to 9.1 The pH was adjusted to 10.0 and 10.5 respectively. The pH of the sample without added alkali was 8.0. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of deionized water was added. The following steps are performed: Add the substance, mix by inversion, centrifuge at 12000 rpm for 5 minutes, and then remove the entire supernatant. I repeated this process twice. I opened the tube cap and dried it overnight at 50°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 400 μL, and add 100 μL of the suspension to 100 × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate at 60°C for 30 minutes. After cooling at room temperature, plate the mixture. Infinite 200 PRO (TECAN) with excitation wavelength 280nm and fluorescence wavelength The fluorescence intensity at 600 nm was measured, and the pH of the treatment solution was plotted on the x-axis, with the fluorescence intensity RFU on the y-axis. (Figure 1) The fluorescence intensity decreases in a pH-dependent manner upon addition of an alkaline agent after nuclease treatment, and residual nuclei remain. The acid reduction effect has improved.

[0095] (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 bacterial 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. After addition, it was incubated at 50°C for 1 hour. 1N sodium hydroxide aqueous solution was added. After adjusting the pH to 8.0, add 100 mg / L of alcalase and 2 mL of chlorine solution. Each nuclease was dispensed into a tube at a final concentration of 1 mg / L. The mixture was then incubated at 50°C for 2 minutes. Incubated for 1 hour. 1.2(w / v)% SDS water containing 12 mM sodium hydroxide. Add 1 mL of the solution and mix by inversion. Centrifuge at 12000 rpm for 5 minutes, and remove 1 mL of the supernatant. Removed. Add 1 mL of 1 mM sodium hydroxide aqueous solution and mix by inverting, then run at 12000 rpm. After centrifugation for 5 minutes, the supernatant was completely removed, and this process was repeated twice. Deionized water was added in 1 ml. After adding L and mixing by inversion, the mixture was centrifuged at 12000 rpm for 5 minutes, and the supernatant was completely removed. I opened the tube cap and dried it overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 400 μL, and add 100 μL of the suspension to 100 × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate at 60°C for 30 minutes. After cooling at room temperature, plate the mixture. Infinite 200 PRO (TECAN) with excitation wavelength 280nm and fluorescence wavelength The fluorescence intensity at 600 nm was measured. From the fluorescence intensity of the nuclease-free sample, each sample was analyzed. The value ΔRFU was calculated by subtracting the fluorescence intensity of the element (Figure 2). The nucleases used are listed below. endo-DNase 1···BsNucB endo-DNase 2···BlNucB endo-DNase 3···SNase endo-DNase 4···Serratia marcescens-derived endo-DNase Crease (SIGMA, E1014) (Sequence ID 11) RNase...RNaseA(NIPPON GENE,313-01461) exo-DNase...Exonuclease III(TaKaRa,2170A ) The effect of reducing residual nucleic acids from PHA is particularly evident in endodeoxyribonuclease among nucleases. This was particularly pronounced in enzymes possessing -ase activity.

[0096] (7) PHA purification by nuclease 4 A pH meter, LAQUAact D-72 (HORIBA), was used to measure the pH. (3) Dispense the PHA-containing bacterial cell suspension obtained in (3) into 50 mL Falcon tubes and 8000 The mixture was centrifuged at rpm for 10 minutes. The supernatant was removed so that the liquid 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. Then, chicken egg white lysozyme was added at a final concentration of 20 mg / L, and the ink was incubated at 50°C for 1 hour. It was fermented. After adjusting the pH to 8.5 by adding 1N sodium hydroxide solution, the final concentration was added. Add 30 mg / L of alcalase and dispense it into two 50 mL tubes, each containing 20 mL. One sample was left as is, while the other was treated with 30 mg / L of BlNucB nuclease. Then, incubate at 50°C for 1 hour. Add 0.4 mL of 30 (w / w)% SDS aqueous solution. The solution was then added, and the pH was adjusted to 10 by adding a 1M sodium hydroxide solution. Final concentration: 30mg g / L of KP43 mutant protease was added and incubated at 50°C for 1 hour. 1 m 20 mL of M sodium hydroxide solution was added at a time, and the mixture was centrifuged at 3000 × g for 20 minutes. The supernatant was then added. Remove 20 mL of the solution, add 20 mL of 1 mM sodium hydroxide solution at a time, and weigh at 3000 × g. The centrifugation process was repeated twice for 20 minutes. Furthermore, the supernatant was completely removed, and 1 mM sodium hydroxide solution was added. Add 30 mL of the lium solution at a time and centrifuge at 3000 × g for 20 minutes, repeating this process twice. Remove the entire supernatant, add 30 mL of deionized water at a time, and centrifuge at 3000 × g for 20 minutes. The supernatant was completely removed, the tube cap was opened, and it was dried overnight at 60°C. I put 200mg of PHA powder into glass bottles. After tapping the bottles to collect the PHA at the bottom... The bottle was then gently inverted, and the PHA adhering to the bottom was photographed. This procedure was repeated four times. The process was repeated (Figure 3). The PHA powder that underwent nuclease treatment was compared to the PHA powder that did not undergo nuclease treatment. Compared to the PHA powder that was treated, the amount adhering to the bottom of the container was reduced, and the nuclease treatment was performed. This suggests that the adhesion of PHA powder decreases and its fluidity tends to improve. Weigh out 10 mg of PHA powder and add Tris-EDTA buffer (pH 8.0) Suspended in 400 μL of NIPPON GENE. 50 μL of the suspension and ×50 GelRe d Aqueous solution (Biotium) (×10000 concentrate diluted 200 times with deionized water) 50 μm Mix L in a black 96-well plate and incubate at 60°C for 30 minutes. Let cool at room temperature. After that, the excitation wavelength was measured using the plate reader Infinite 200 PRO (TECAN). The fluorescence intensity at 280 nm and 600 nm fluorescence wavelengths was measured. Instead of using GelRed aqueous solution... A blank was prepared using deionized water. Instead of PHA suspension, Tris-ED was used. Deoxyribonucleic acid (FUJIFI) derived from salmon semen dissolved in TA buffer (pH 8.0) A calibration curve was created using LM, 043-31381) to determine the amount of nucleic acid residue in the PHA powder. (Figure 4). Nucleic acids remain in the recovered PHA, and the residual nucleic acids are reduced by nuclease treatment. Ta.

[0097] (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 bacterial cells. 1N Sodium hydroxide was added to adjust the pH to 7.0, and chicken egg white lysozyme was added to a final concentration of 2. After adding at a concentration of 0 mg / L, it was incubated at 50°C for 1 hour. 1 2 mL tube After dispensing in mL portions, one vial contains alcalase at a final concentration of 30 mg / L, and the other vial contains the final concentration... Alcalase at a concentration of 30 mg / L and nuclease at a final concentration of 100 mg / L (BlNucB ) was added. After incubation at 50°C for 2 hours, a 0.4(w / v)% SDS aqueous solution was added. Add 1 mL of [the substance] and mix by inverting the mixture. Add 1N sodium hydroxide aqueous solution to adjust the pH to 9.0. Afterward, it was centrifuged at 12,000 rpm for 5 minutes. The appearance after centrifugation was observed (Figure 5). Compared to the sample without added nuclease, the appearance of the nuclease was observed. In the sample with added nuclease, the supernatant was clear, and the centrifugal sedimentation efficiency was improved by the addition of nuclease. It improved. This suggests that the use of nucleases reduces the amount of DNA remaining in PHA. Furthermore, it is suggested that it also has the effect of reducing the water and energy load in centrifugal washing. It was done.

[0098] (9) Preparation of PHA-containing bacterial cells Cupriavidus necator(Ralstonia eutropha) H16 strain was inoculated into LB liquid medium and cultured with shaking at 30°C for 24 hours. 1 mL of this culture solution was then used. , 100 mL of PHA production medium (0.35% ammonium chloride, 0.175% diphosphate) Potassium hydrogen, 0.12% magnesium sulfate heptahydrate, 0.17% citric acid, 3.5% fu Luctose, 0.0225ppm zinc sulfate heptahydrate, 0.1ppm iron sulfate heptahydrate, 0. 0.02 ppm calcium chloride dihydrate, 0.0023 ppm sodium tetraborate heptahydrate, 0.001 ppm ammonium heptamolybdate tetrahydrate, 0.01 ppm copper sulfate pentahydrate Substance, 0.006 ppm manganese sulfate pentahydrate, 35 (v / v) ppm hydrochloric acid; unless otherwise specified Inoculate the Sakaguchi flask with (w / v) % added and culture with shaking at 30°C for 72 hours. A PHA-containing bacterial cell suspension was obtained.

[0099] (10) PHA purification by nuclease 6 A pH meter, LAQUAact D-72 (HORIBA), was used to measure the pH. (9) The PHA-containing bacterial cell suspension obtained in (9) was adjusted to pH 7.0 by adding 1N HCl. Dispense 1 mL into two 2 mL tubes, then add one tube to a final concentration of 30 mg / L nuclea. -se (BlNucB) was added. After heating each tube at 70°C for 10 minutes, the lysozyme was added. Mutanolicin (SIGMA M9901) was added at a final concentration of 20 mg / L and incubated at 50°C. Incubated for 1 hour. 1N sodium hydroxide solution was added to adjust the pH to 9.0. Afterward, a final concentration of 30 mg / L of alcalase was added and incubated at 50°C for 1 hour. Add 1 mL of a 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide, and turn. The mixture was inverted and mixed. At this time, the pH was 9.4. The mixture was centrifuged at 12000 rpm for 5 minutes. At the end of centrifugation, in samples without added nuclease, a sticky substance formed around the solid pellet. White suspended particles indicating filtration were observed (Figure 6). In the sample to which the nuclease was added, these suspended particles were observed. No playful objects were observed; only solid pellets were found. Remove 1 mL of the supernatant. Add 1 mL of 1 mM sodium hydroxide solution and mix by inversion. The mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was completely removed. This procedure was repeated twice. I opened the lid of the tube and dried it overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 500 μL, and add 100 μL of the suspension to a 100 μL × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate in the dark at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured. The samples were of fireflies without the addition of nuclease. Relative values ​​to light intensity were calculated (Table 2). Fluorescence intensity decreased after nuclease treatment. Therefore, nucleic acids remain in the PHA after recovery, and the residual nucleic acids are removed by nuclease treatment. It was shown that it was reduced.

[0100] [Table 2]

[0101] (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 bacterial cells. NHCl was added to adjust the pH to 7.0. 1 mL was dispensed into two 2 mL tubes. Afterwards, one bottle contains lysozyme (mutanolysine) at a final concentration of 20 mg / L, SIGMA M990 1) and the other bottle contains lysozyme (mutanolysine, SIGMA M) at a final concentration of 20 mg / L. 9901) and a final concentration of 30 mg / L of nuclease (BlNucB) are added, and the mixture is heated at 50°C. Incubated for 1 hour. 1N sodium hydroxide solution was added to adjust the pH to 9.0. Afterward, a final concentration of 30 mg / L of alcalase was added and incubated at 50°C for 1 hour. Add 1 mL of a 1.2 (w / v)% SDS aqueous solution containing 20 mM sodium hydroxide, and turn. Mixed inverted. The pH was 9.4 at this time. Centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken. mL was removed. 1 mL of 1 mM sodium hydroxide aqueous solution was added and mixed by inversion, and 12000 The tube was centrifuged at rpm for 5 minutes, and the supernatant was completely removed. This procedure was repeated twice. I opened the box and dried it overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 500 μL, and add 100 μL of the suspension to a 100 μL × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate in the dark at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured. The samples were of fireflies without the addition of nuclease. Relative values ​​against light intensity were calculated (Table 3). Fluorescence intensity decreased after nuclease treatment. Therefore, nucleic acids remain in the PHA after recovery, and the residual nucleic acids are removed by nuclease treatment. It was shown that it was reduced.

[0102] [Table 3]

[0103] (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 bacterial cells. NHCl was added to adjust the pH to 7.0. 1 mL was dispensed into two 2 mL tubes. Afterwards, one bottle contains lysozyme (mutanolysine) at a final concentration of 20 mg / L, SIGMA M990 1) Alcalase at a final concentration of 30 mg / L, and lysozyme at a final concentration of 20 mg / L in the other bottle. Team (mutanolysin, SIGMA M9901), final concentration 30 mg / L alcalase Then, a nuclease (BlNucB) with a final concentration of 30 mg / L was added and incubated at 50°C for 1 hour. It was incubated. After adding 1N sodium hydroxide solution to adjust the pH to 9.0, it was further incubated. It was incubated at 50°C for 1 hour. 1.2 (w / v) containing 20 mM sodium hydroxide. 1 mL of )% SDS aqueous solution was added and mixed by inversion. The pH at this time was 9.4. 120 Centrifuge at 00 rpm for 5 minutes and remove 1 mL of supernatant. Add 1 mM sodium hydroxide aqueous solution. Add mL and mix by inversion, then centrifuge at 12000 rpm for 5 minutes, and remove the entire supernatant. The procedure was repeated twice. The tube cap was opened and it was dried overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 500 μL, and add 100 μL of the suspension to a 100 μL × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate in the dark at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured. The samples were of fireflies without the addition of nuclease. Relative values ​​against light intensity were calculated (Table 4). Fluorescence intensity decreased after nuclease treatment. Therefore, nucleic acids remain in the PHA after recovery, and the residual nucleic acids are removed by nuclease treatment. It was shown that it was reduced.

[0104] [Table 4]

[0105] (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 bacterial cells. The pH was adjusted to 9.0 by adding an aqueous solution of N sodium hydroxide. 1 ml was added to two 2 mL tubes. After dispensing in liters, one vial contains lysozyme (mutanolysine, SIGM) at a final concentration of 20 mg / L. A M9901) and alcalase at a final concentration of 30 mg / L, and another vial containing alcalase at a final concentration of 20 mg / L. g / L lysozyme (mutanolysine, SIGMA M9901), final concentration 30 mg / L Alcalase and a final concentration of 30 mg / L nuclease (BlNucB) were added, and the temperature was raised at 50°C. Incubated for 1 hour. 1.2(w / v)%SD containing 20 mM sodium hydroxide. 1 mL of aqueous solution S was added and mixed by inversion. The pH at this time was 9.6. 12000 rp Centrifuge at m for 5 minutes and remove 1 mL of supernatant. Add 1 mL of 1 mM sodium hydroxide solution. Then, invert and mix, centrifuge at 12000 rpm for 5 minutes, and then remove the entire supernatant. I repeated the process several times. I opened the tube cap and dried it overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 500 μL, and add 100 μL of the suspension to a 100 μL × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate in the dark at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured. The samples were of fireflies without the addition of nuclease. Relative values ​​against light intensity were calculated (Table 5). Fluorescence intensity decreased after nuclease treatment. Therefore, nucleic acids remain in the PHA after recovery, and the residual nucleic acids are removed by nuclease treatment. It was shown that it was reduced.

[0106] [Table 5]

[0107] (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 bacterial cells. The pH was adjusted to 9.0 by adding an aqueous solution of N sodium hydroxide. 1 ml was added to two 2 mL tubes. After dispensing in liters, one vial contains lysozyme (mutanolysine, SIGM) at a final concentration of 20 mg / L. A M9901) and alcalase at a final concentration of 30 mg / L, and another vial containing alcalase at a final concentration of 20 mg / L. g / L lysozyme (mutanolysine, SIGMA M9901), final concentration 30 mg / L Alcalase and a final concentration of 30 mg / L nuclease (BlNucB) were added, and the temperature was raised at 50°C. Incubated for 1 hour. 1.2(w / v)%SD containing 20 mM sodium hydroxide. 1 mL of aqueous solution S was added and mixed by inversion. At this time, the pH was 11.3. 12000r Centrifuge at 5 minutes with pm and remove 1 mL of the supernatant. Add 1 mL of 1 mM sodium hydroxide solution. After adding the mixture and mixing by inversion, centrifuge at 12000 rpm for 5 minutes, then remove the entire supernatant. I repeated the process twice. I opened the tube cap and dried it overnight at 60°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE Suspend in 500 μL, and add 100 μL of the suspension to a 100 μL × 100 GelRed aqueous solution (Bioti (um) (10000 times the original solution, diluted 100 times with deionized water) 10 μL on a black 96-hole plate Mix in a container and incubate in the dark at 60°C for 30 minutes. After cooling at room temperature, pre- With the Infinite 200 PRO (TECAN) head reader, the excitation wavelength is 280nm. The fluorescence intensity at a fluorescence wavelength of 600 nm was measured. The samples were of fireflies without the addition of nuclease. Relative values ​​against light intensity were calculated (Table 6). Fluorescence intensity decreased after nuclease treatment. Therefore, nucleic acids remain in the PHA after recovery, and the residual nucleic acids are removed by nuclease treatment. It was shown that it was reduced.

[0108] [Table 6]

[0109] (15) Preparation of PHA-containing bacterial cells Cupriavidus necator(Ralstonia eutropha) H16 strain was inoculated into BD Difco® Nutrient Broth medium. The culture was incubated at 30°C for 24 hours with shaking. 1 mL of this culture was added to 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 ferric chloride ( III) Hexahydrate, 10.3 ppm calcium chloride dihydrate, 0.22 ppm cobalt chloride 0.16 ppm copper sulfate pentahydrate, 0.12 ppm nickel chloride hexahydrate, 1 0.5% coconut oil and 2% fructose were added to a Sakaguchi flask, and the cells were inoculated. A PHA-containing bacterial cell suspension was obtained by culturing at 30°C with shaking for 72 hours.

[0110] (16) PHA purification by nuclease 11 A pH meter, LAQUAact D-72 (HORIBA), was used to measure the pH. The PHA-containing bacterial cell suspension obtained in (15) was heated at 70°C for 1 hour to inactivate the bacterial cells. Add sodium hydroxide aqueous solution to adjust the pH to 6.5, and then add chicken egg white lysozyme. The substance was added at a concentration of 20 mg / L and incubated at 50°C for 1 hour. After incubation... The pH was 6.5. Add 100 mg / L of alcalase to a final concentration and add 2 mL of tube. Dispense 1 mL into two vials. Add 1 mg / L nuclease (BlNucB). The sample was incubated at 50°C for 2 hours. 0.4(w / v)%S was added to the enzyme-treated sample. Add 1 mL of DS aqueous solution, then add 1N sodium hydroxide aqueous solution to adjust the pH to 7.6 and 10. The pH was adjusted to 0.6 and 11.8, respectively. The pH of the sample without added alkali was 6.7. The mixture was centrifuged at 12000 rpm for 5 minutes, and 1 mL of the supernatant was removed. 1 mL of deionized water was added. The mixture is then inverted and mixed, centrifuged at 12,000 rpm for 5 minutes, and the supernatant is completely removed. This process is repeated twice. I repeated the process. I opened the tube cap and dried it overnight at 50°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE ) Suspend in 400 μL, add 50 μL of the suspension and ×50 GelRed aqueous solution (Biotium (Dilute the undiluted solution 10,000 times with deionized water 200 times) 50 μL in a black 96-well plate Mixed and incubated at 60°C for 30 minutes. After cooling to room temperature, plate reader Infinite 200 PRO (TECAN) with excitation wavelength 280nm and fluorescence wavelength 60nm The fluorescence intensity (RFU) at 0 nm was measured without the addition of nuclease and after the addition of SDS. ΔR is the value obtained by subtracting the fluorescence intensity of each sample from the fluorescence intensity of the sample that was not pH adjusted. The FU was calculated. A larger ΔRFU indicates a greater nucleic acid reduction effect. After SDS addition. The adjusted pH was plotted on the x-axis and ΔRFU on the y-axis (Figure 7). Adding an alkaline agent after nuclease treatment improves the reduction of residual nucleic acids in a pH-dependent manner. did.

[0111] (17) PHA purification by nuclease 12 As a protease, instead of alcalase, KP43 (SEQ ID NO: 19), KP43 variant Allomorph (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Es perase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (distributed Except for using sequence numbers 28, 29) or the LL147 variant (sequence numbers 30, 31), The PHA is purified using the same method as in (12).

[0112] (18) PHA purification by nuclease 13 As lysozyme, instead of mutanolicin, egg white lysozyme, Ssp12Lys (sequence) Number 32), Ssp14Lys (Sequence ID 33), Asp3Lys (Sequence ID 34) or The use of KaLys (SEQ ID NO: 35), and the use of alcalase as a protease. In contrast, KP43 (sequence number 19), KP43 mutant (sequence number 20), K16 (sequence number 19) 21) K16 mutant (SEQ ID NO: 22), Esperase, Savinase, HH8 44 (SEQ ID NO: 27), HH844 variant (SEQ ID NO: 28, 29), or LL147 variant Except for using (SEQ ID NOs. 30 and 31), the PHA is purified in the same manner as in (12). cormorant.

[0113] (19) PHA purification by nuclease 14 As a protease, instead of alcalase, KP43 (SEQ ID NO: 19), KP43 variant Allomorph (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant (SEQ ID NO: 22), Es perase, Savinase, HH844 (SEQ ID NO: 27), HH844 mutant (distributed Except for using sequence numbers 28, 29) or the LL147 variant (sequence numbers 30, 31), The PHA is purified using the same method as in (13).

[0114] (20) PHA purification by nuclease 15 Instead of BlNucB, use SEQ ID NO: 12, SEQ ID NO: 13, 14, the amino acid shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 Except for using a nuclease consisting of a sequence, the purification of PHA is performed in the same manner as in (13). conduct.

[0115] (21) PHA purification by nuclease 16 As lysozyme, use egg white lysozyme instead of mutanolicin, and nucle As an ase, instead of BlNucB, use SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, From the amino acid sequence shown in sequence number 15, sequence number 16, sequence number 17, or sequence number 18 The PHA is purified in the same manner as in (12), except that a nuclease is used.

[0116] (22) PHA purification by nuclease 17 Lysozyme is derived from the amino acid sequence shown in SEQ ID NO: 23, instead of mutanolysin. By using lysozyme, and by using BlNucB as the nuclease instead of 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 Except for using a nuclease consisting of the amino acid sequence shown in column number 18, (12) The PHA is purified using the same method.

[0117] (23) PHA purification by nuclease 18 Lysozyme is derived from the amino acid sequence shown in SEQ ID NO: 26, instead of mutanolysin. By using lysozyme, and by using BlNucB as the nuclease instead of 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 Except for using a nuclease consisting of the amino acid sequence shown in column number 18, (12) The PHA is purified using the same method.

[0118] (24) PHA purification by nuclease 19 Lysozyme is derived from the amino acid sequence shown in SEQ ID NO: 24, instead of mutanolysin. By using lysozyme, instead of BlNucB as the nuclease, use SEQ ID NO: 12 Alternatively, use a nuclease consisting of the amino acid sequence shown in Sequence ID No. 13, and pro Aside from using Esperase as the thease, the PHA is prepared in the same way as in (12). Purification is performed.

[0119] (25) PHA purification by nuclease 20 As lysozyme, Ssp12Lys (SEQ ID NO: 32) is used instead of mutanolysin. p14Lys (sequence number 33), Asp3Lys (sequence number 34), or KaLys (sequence number 33) The PHA is purified in the same manner as in (13), except that nominal

[0120] (26) PHA purification by nuclease 21 As lysozyme, Ssp12Lys (SEQ ID NO: 32) is used instead of mutanolysin. p14Lys (sequence number 33), Asp3Lys (sequence number 34), or KaLys (sequence number 33) Use number 35), and use KP43 instead of alcalase as the protease. SEQ ID NO: 19), KP43 mutant (SEQ ID NO: 20), K16 (SEQ ID NO: 21), K16 mutant Variant (SEQ ID NO: 22), Esperase, Savinase, HH844 (SEQ ID NO: 22) 7) HH844 variant (SEQ ID NOs. 28, 29) or LL147 variant (SEQ ID NOs. 30, Except for using 31), the PHA is purified in the same manner as in (13).

[0121] (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 bacterial cells. Add sodium hydroxide to adjust the pH to 6.9, and add chicken egg white lysozyme to the final concentration. The solution was added at a concentration of 20 mg / L and incubated at 50°C for 1 hour. (1N sodium hydroxide) After adding an aqueous solution to adjust the pH to 8.5, a final concentration of 100 mg / L of alcalase and Add 1 mg / L nuclease (BlNucB) and incubate at 50°C for 2 hours. The solution was then dispensed into six 2mL tubes, each containing 1mL, and then two tubes were filled with 0.2(w / v)% solution. Add 1 mL of surfactant aqueous solution and mix by inversion. Add 1N sodium hydroxide aqueous solution and p After adjusting to H10.0, the sample was centrifuged at 12000 rpm for 5 minutes. After centrifugation, 1 mL of supernatant was removed. 1 mL of deionized water was added and mixed by inversion. The mixture was centrifuged at 000 rpm for 5 minutes, and the supernatant was completely removed. This process was repeated twice. I opened the lid of the container and dried it overnight at 50°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE ) Suspend in 400 μL, add 50 μL of the suspension and ×50 GelRed aqueous solution (Biotium (Dilute the undiluted solution 10,000 times with deionized water 200 times) 50 μL in a black 96-well plate Mixed and incubated in the dark at 60°C for 30 minutes. After cooling at room temperature, plate Leader Infinite 200 PRO (TECAN) with excitation wavelength 280nm, fluorescence The fluorescence intensity at a wavelength of 600 nm was measured. The fluorescence intensity of two samples with each surfactant added was measured. The average value 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 (manufactured by Kao Corporation) ES...Polyoxyethylene lauryl ether sulfate sodium (Emal 20C, flower (manufactured by Wang Co., Ltd.) Nonionic surfactants E320L...Polyoxyethylene stearyl ether, EO average number of added moles 20 (E Marugen 320L (manufactured by Kao Corporation) The effect of reducing residual nucleic acids from PHA is particularly evident in anionic surfactants among surfactants. He was the author.

[0122] (28) PHA purification by nuclease 23 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 bacterial cells. Add sodium hydroxide to adjust the pH to 6.9, and add chicken egg white lysozyme to the final concentration. The solution was added at a concentration of 20 mg / L and incubated at 50°C for 1 hour. (1N sodium hydroxide) After adding an aqueous solution to adjust the pH to 8.0, a final concentration of 100 mg / L of alcalase was added. The solution was dispensed and 1 mL was aliquoted into three 2 mL tubes. One tube contained a final concentration of 1 mg / L nuclea. Nuclease (BlNucB) was added to one of the tubes, and the remaining two tubes were incubated at 50°C for 2 hours without adding nuclease. The samples were incubated. Samples to which nuclease was added were treated with 0.4(w / v)% SDS water. Add 1 mL of the solution and mix by inversion. Of the samples that did not have nuclease added, one was selected. Add 1 mL of deionized water to one bottle, and 1 mL of 0.4 (w / v)% SDS aqueous solution to the other bottle. L was added and mixed by inversion. pH was adjusted to 10.0 by adding 1N sodium hydroxide solution. Next, it was centrifuged at 12,000 rpm for 5 minutes. After centrifugation, 1 mL of supernatant was removed. 1 mL of deionized water was added and mixed by inversion. The mixture was centrifuged at 000 rpm for 5 minutes, and the supernatant was completely removed. This process was repeated twice. I opened the lid of the container and dried it overnight at 50°C. Each pellet is refrigerated in Tris-EDTA buffer (pH 8.0) (NIPPON GENE ) Suspend in 400 μL, add 50 μL of the suspension and ×50 GelRed aqueous solution (Biotium (Dilute the undiluted solution 10,000 times with deionized water 200 times) 50 μL in a black 96-well plate Mixed and incubated in the dark at 60°C for 30 minutes. After cooling at room temperature, plate Leader Infinite 200 PRO (TECAN) with excitation wavelength 280nm, fluorescence The fluorescence intensity at a wavelength of 600 nm was measured. Sunflower was measured without the addition of nuclease and SDS. The relative values ​​of the pull to the fluorescence intensity were determined (Figure 9). The combined use of nuclease and SDS improved the effectiveness of reducing residual nucleic acids.

Claims

1. A method for producing biopolymers, Biopolymer granules obtained by culturing microorganisms capable of producing biopolymers contain ene The endodeoxyribonuclease treatment process involves contacting the dodeoxyribonuclease with the endodeoxyribonuclease and the preceding process. At least one point in time after the start of endodeoxyribonuclease treatment, the bio A pH adjustment step in which polymer granules are placed under conditions of pH 7.0 or higher, Methods that include...

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

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

4. The process further includes a protease treatment step in which the biopolymer granules are brought into contact with a protease. The method according to any one of claims 1 to 3.

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

6. Lysozyme is brought into contact with the cellular cells of the microorganism having biopolymer production ability. The method according to claim 3, further comprising a processing step.

7. Lysozyme is brought into contact with the cellular cells of the microorganism having biopolymer production ability. The method according to claim 4, further comprising a processing step.

8. The endodeoxyribonuclease treatment step, the protease treatment step and the ri The method according to claim 7, wherein the zozyme treatment process is performed simultaneously.

9. DN remaining in biopolymers produced by microorganisms capable of biopolymer production A method for reducing the amount of A, Biopolymer granules obtained by culturing microorganisms capable of producing biopolymers contain ene Endodeoxyribonuclease treatment process, in which dodeoxyribonuclease is brought into contact with the product. Methods that include...

10. At least one point in time after the start of the endodeoxyribonuclease treatment, the Claim 9 further includes a pH adjustment step of placing the iopolymer granules under conditions of pH 7.0 or higher. Method of description.

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

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

13. The process further includes a protease treatment step in which the biopolymer granules are brought into contact with a protease. The method according to any one of claims 9 to 12.

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

15. The biopolymer is a hydroxyalkanoic acid-containing polymer, according to claim 1 or 9. The method.

16. The biopolymer is polyhydroxyalkanoic acid (PHA), as described in claim 1 or 9. Method of loading.

17. The microorganism having the ability to produce biopolymers belongs to the genus Cupriavidus. The method according to claim 1 or 9.

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