WO2022091685A1 - Polyhydroxybutyrate copolymer production method and use therefor - Google Patents
Polyhydroxybutyrate copolymer production method and use therefor Download PDFInfo
- Publication number
- WO2022091685A1 WO2022091685A1 PCT/JP2021/036049 JP2021036049W WO2022091685A1 WO 2022091685 A1 WO2022091685 A1 WO 2022091685A1 JP 2021036049 W JP2021036049 W JP 2021036049W WO 2022091685 A1 WO2022091685 A1 WO 2022091685A1
- Authority
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- WIPO (PCT)
- Prior art keywords
- phb copolymer
- copolymer
- polyhydroxybutyric acid
- aqueous suspension
- acid copolymer
- Prior art date
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005979 thermal decomposition reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
- 239000004711 α-olefin Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/78—Preparation processes
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/12—Powdering or granulating
- C08J3/16—Powdering or granulating by coagulating dispersions
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K5/00—Use of organic ingredients
- C08K5/36—Sulfur-, selenium-, or tellurium-containing compounds
- C08K5/41—Compounds containing sulfur bound to oxygen
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L29/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers
- C08L29/02—Homopolymers or copolymers of unsaturated alcohols
- C08L29/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L67/00—Compositions of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Compositions of derivatives of such polymers
- C08L67/04—Polyesters derived from hydroxycarboxylic acids, e.g. lactones
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
- C08L71/02—Polyalkylene oxides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
Definitions
- the present invention relates to a method for producing a polyhydroxybutyric acid copolymer and its use.
- PHB copolymers Polyhydroxybutyrate copolymers
- the PHB copolymer produced by a microorganism is accumulated in the cells of the microorganism, in order to use the PHB copolymer as a plastic, a step of separating and purifying the PHB copolymer from the inside of the microorganism is required. It becomes.
- the step of separating and purifying the PHB copolymer after solubilizing biological components other than the PHB copolymer, the PHB copolymer is taken out from the obtained aqueous suspension.
- separation operations such as centrifugation, filtration, and drying are performed.
- a spray dryer, a fluidized bed dryer, a drum dryer and the like are used, but since the operation is simple, a spray dryer is preferably used.
- the present inventors have made polyvinyl alcohol before adjusting the pH of the aqueous suspension to 7.0 or less in order to prevent aggregation of the PHB copolymer in the aqueous suspension having a pH of 7.0 or less.
- a technique for adding (PVA) as a dispersant and then spray-drying the obtained aqueous suspension having a pH of 7.0 or less Patent Document 1.
- the present inventor contains the PHB copolymer as a technique for producing a PHB copolymer having a high composition ratio of 3-hydroxyhexanoate (hereinafter, may be referred to as “3HH”) unit.
- 3HH 3-hydroxyhexanoate
- polyhydroxybutyric acid copolymer poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter, may be referred to as "PHBH”) is known.
- an object of the present invention is a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles in the manufacturing process, particularly a specific 3-hydroxybutyrate having a high bulk density (hereinafter referred to as “)”. It may be referred to as "3HB”.) It is an object of the present invention to provide a PHB copolymer having a composition ratio of hydroxyalkanoate units other than units / 3HB units, and a method for producing the same.
- the present inventors have a poly having a high composition ratio of hydroxyalkanoate units other than 3HB units and a specific composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units.
- a step of enzymatically treating a bacterial cell containing a hydroxybutyrate copolymer with a specific enzyme a PHB copolymer having a high bulk density can be obtained while suppressing aggregation of PHB copolymer particles in the manufacturing process. It was the first time to find out that it could be obtained, and the present invention was completed.
- one aspect of the present invention is a method for producing a PHB copolymer, wherein the composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units is 80/20 to 88/12.
- (Mol / mol) a step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing the PHB copolymer, and (b) the step of enzymatically treating the cells.
- An alkaline aqueous solution is added to the culture solution obtained in (a) to adjust the pH to 10.0 to 12.0, and the surfactant is added either before the adjustment, at the same time as the adjustment, or after the adjustment.
- Step of addition (c) From the aqueous suspension obtained in the step (b), the pH is 7.0 or less, and the shear viscosity is 0.005 Pa ⁇ s or more and 0.5 Pa ⁇ s or less.
- a method for producing a PHB copolymer which comprises a step of preparing an aqueous suspension and (d) a step of spray-drying the aqueous suspension prepared in the step (c).
- one aspect of the present invention includes a PHB copolymer, a peptide glycan, and a dispersant, and the PHB copolymer has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20. It is a PHB copolymer powder having a bulk density of 0.45 g / mL or more, a median particle size of 80 to 200 ⁇ m, and a bulk density of about 88/12 (mol / mol).
- one aspect of the present invention comprises a PHB copolymer and a nitrogen compound, and the PHB copolymer has a composition of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit.
- a PHB copolymer powder having a ratio of 80/20 to 88/12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 ⁇ m.
- a PHB copolymer having a high bulk density, particularly a high bulk density, other than a specific 3HB unit / 3HB unit, while suppressing aggregation of PHB copolymer particles in the manufacturing process PHB copolymers having a composition ratio of hydroxyalkanoate units can be provided.
- the composition ratio of the PHB copolymer to a hydroxy alkanoate unit other than 3HB unit / 3HB unit. Is 80/20 to 88/12 (mol / mol)
- an alkaline proteolytic enzyme is added to a culture solution containing the cells containing the PHB copolymer to treat the cells enzymatically.
- Step (b) Add an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and before, at the same time as, or after the adjustment.
- the pH is 7.0 or less and the shear viscosity is 0.005 Pa ⁇ s or more. , 0.5 Pa ⁇ s or less, and (d) spray-drying the aqueous suspension prepared in the step (c).
- Patent Document 1 the present inventor uses a high-concentration slurry (PHB copolymer aqueous suspension), and the composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit.
- PHB copolymer aqueous suspension a high-concentration slurry
- the composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit we have found a new problem that the PHB copolymer particles in the above are aggregated and the viscosity of the aqueous suspension of the PHB copolymer is increased, resulting in difficulty in liquid feeding and spray drying. Further, if the concentration of the aqueous suspension of the PHB copolymer is lowered in order to solve the above problems, a large amount of energy is required for spray drying, which is not preferable in terms of energy efficiency and the bulk of the obtained powder. It was also found that another problem arises: low density and poor powder transportability.
- Patent Documents 2 and 3 have a problem that the process is complicated at the time of scale-up because the bacterial cells are physically crushed by high pressure crushing. Further, the method described in Patent Document 3 has a problem that the manufacturing process itself is long.
- the present inventor has identified a bacterial cell containing a PHB copolymer having a composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit.
- the present inventor states that this is a result of the above-mentioned steps, that the PHB copolymer aqueous suspension contains peptidoglycan derived from cells, and aggregation of PHB copolymer particles can be avoided. Is guessing.
- the present inventor further studied the PHB copolymer (PHB copolymer powder (intermediate)) obtained above from the viewpoint of increasing the purity, and as a result, the PHB copolymer (PHB copolymer weight).
- PHB copolymer powder (intermediate) obtained above from the viewpoint of increasing the purity
- PHB copolymer weight obtained above from the viewpoint of increasing the purity
- PHB copolymer weight obtained above from the viewpoint of increasing the purity
- PHB copolymer (PHB copolymer weight the PHB copolymer weight.
- a high-purity PHB copolymer can be obtained in a state of high bulk specific gravity by washing the combined powder (intermediate) with a specific enzyme (here, high purity means high purity). It indicates that there are few nitrogen compounds that are impurities.).
- a PHB copolymer for example, PHB copolymer powder
- Combined powder can be obtained.
- the PHB copolymer can be obtained without using a physical crushing (for example, high-pressure crushing) step, which is advantageous from the viewpoint of scale-up.
- the amount of plastic waste generated can be reduced, whereby, for example, Goal 12 “Securing a sustainable consumption production form” and Goal 14 “For sustainable development”. It can contribute to the achievement of Sustainable Development Goals (SDGs) such as "Conserving and using marine and marine resources in a sustainable manner.”
- SDGs Sustainable Development Goals
- This manufacturing method is a method including the following steps (a) to (d) as essential steps. Further, the PHB copolymer in this production method has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol).
- Step (c) From the aqueous suspension obtained in the step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa ⁇ s or more and 0.5 Pa ⁇ s or less.
- Step of preparing turbid liquid-Step (d) Step of spray-drying the aqueous suspension prepared in the above-mentioned step (c) In the step (c) in the present production method, the aqueous suspension obtained in the step (b).
- an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa ⁇ s or more and 0.5 Pa ⁇ s or less is prepared.
- the PHB copolymer exists in a dispersed state in the aqueous medium.
- an aqueous suspension containing at least a PHB copolymer may be abbreviated as "PHB copolymer aqueous suspension".
- Step (a) is a step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing a PHB copolymer to enzymatically treat the cells.
- PHB copolymer The PHB copolymer in this production method is a copolymer of 3HB and a hydroxy alkanoate other than 3HB.
- hydroxyalkanoates other than 3HB examples include 3-hydroxyhexanoate (3HH), 3-hydroxyvariate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), and the like. Examples thereof include 3-hydroxyoctadecanoate (3HOD) and 3-hydroxydecanoate (3HD).
- PHBH can be preferably exemplified, but is not limited thereto.
- PHBH will be mainly described as a representative example.
- PHBH can change the melting point and crystallinity by changing the composition ratio of the repeating unit of 3HB and 3HH, and as a result, the physical properties such as Young's modulus and heat resistance can be changed. It is possible to impart physical characteristics between them.
- the PHB copolymer in this production method has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol) and 81/19 to 87/13 (mo1 /). It is preferably mo1), and more preferably 82/18 to 86/14 (mo1 / mo1). Sufficient hardness can be obtained when the composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units is 88/12 (mol / mol) or less, and sufficient when it is 80/20 (mol / mol) or more. Flexibility is obtained.
- the weight average molecular weight of the PHB copolymer (hereinafter, may be referred to as “Mw”) is not particularly limited, but is preferably 150,000 to 800,000, preferably 200,000 to 700,000. More preferably, 250,000 to 600,000 is even more preferable.
- Mw weight average molecular weight
- the weight average molecular weight is 150,000 or more, sufficient mechanical characteristics and the like can be obtained, and when it is 800,000 or less, a sufficient crystallization rate can be obtained and good molding processability can be achieved.
- GPC gel permeation chromatography
- Polystyrene gel Showa Denko's "Shodex K-804"
- the bacterial cells used in the step (a) are not particularly limited as long as they are microorganisms capable of producing a PHB copolymer in the cells.
- microorganisms isolated from nature and microorganisms deposited in a depositary institution of strains (eg, IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used.
- the first bacterial cell that produces P3HB which is an example of a PHB copolymer, is Cupriavidus necator (former classification: Alcaligenes), which was discovered in 1925. Natural microorganisms such as europhos), Ralstonia europha), and Alcaligenes latus can be mentioned. It is known that PHB copolymers are accumulated in the cells of these microorganisms.
- examples of the cells that produce a copolymer of hydroxybutyrate and other hydroxyalkanoates include Aeromonas caviae, which is a P3HB3HV and P3HB3HH-producing bacterium, and P3HB4HB production.
- examples thereof include Alcaligenes europhos, which is a fungus.
- P3HB3HH in order to increase the productivity of P3HB3HH, Alcaligenes utrophas AC32 strain (Alcaligenes europhos AC32, FERM BP-6038) (T. Fukui, Y.
- the bacterial cell may be a recombinant microorganism into which various PHB copolymer synthesis-related genes have been introduced according to the PHB copolymer to be produced.
- the PHB copolymer can also be produced, for example, by the method described in International Publication No. 2010/0134883.
- alkaline proteolytic enzyme As used herein, the term "alkaline proteolytic enzyme” is intended to be a proteolytic enzyme having an activity of degrading a protein in an alkaline environment (for example, in a solution having a pH of 8.5).
- the alkaline proteolytic enzyme is not particularly limited as long as it has an activity of degrading proteins in an alkaline environment, and is, for example, a serine-specific proteolytic enzyme (eg, subtilisin, chymotrypsin), cysteine-specific.
- proteolytic enzymes eg, papaine, bromeline
- serine-specific proteolytic enzymes particularly alcalases containing subtilisin, are preferable.
- One of these types may be used alone, or two or more types may be used in combination.
- alkaline proteolytic enzyme for example, "Alkalase” and “Esperase” manufactured by Novozyme; “Protin SD-AY10” and “Protease P” Amano "3SD” manufactured by Amano Enzyme Co., Ltd .; “Multifect PR6L” and “Optimase PR89L” manufactured by Danisco Japan Co., Ltd .; “Sumiteam MP” manufactured by Shin Nihon Kagaku Kogyo Co., Ltd .; “Delbolase” manufactured by DSM Japan Co., Ltd .; , “Bioplase SP-20FG” and “Bioplase SP-4FG”; “Orientase 22BF” manufactured by HBI Co., Ltd .; “Aloase XA-10” manufactured by Yakult Pharmaceutical Co., Ltd. and the like.
- step (a) when enzymatically treating cells with an alkaline proteolytic enzyme, the pH and temperature of the culture solution should be adjusted according to the optimum pH and temperature of the alkaline proteolytic enzyme to be used. Is preferable. Further, the pH in the step (a) is preferably lower than the pH adjusted by the addition of the alkaline aqueous solution in the step (b).
- the method for adjusting the pH and temperature of the culture solution is not particularly limited, and known methods can be used.
- the optimum pH of the alkaline proteolytic enzyme is not particularly limited as long as the alkaline proteolytic enzyme has activity in an alkaline environment, but is, for example, 8.0 to 12.0, preferably 8.0 to 12.0. It is 8.0 to 11.0, more preferably 8.0 to 10.0, still more preferably 8.0 to 9.0, and most preferably 8.5.
- the optimum temperature of the alkaline proteolytic enzyme is not particularly limited, but it does not require excessive heating and can prevent thermal changes (thermal decomposition) of the PHB copolymer. From the viewpoint, 70 ° C. or lower is preferable, and 60 ° C. or lower is more preferable.
- the lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (for example, 25 ° C.) or higher from the viewpoint of not requiring an excessive cooling operation and being economical.
- the amount of the alkaline proteolytic enzyme added is not particularly limited, but is, for example, 0.05 to 1.0 phr, preferably 0.1 to 0.5 phr, and 0.15 to 0. 3 phr is more preferable. If the amount of the alkaline proteolytic enzyme added is within the above range, the cells can be appropriately decomposed.
- the lytic enzyme is not substantially added at the same time as the alkaline proteolytic enzyme.
- the term "lytic enzyme” is intended to be an enzyme having an activity of degrading (bacteriolytic) the cell wall (for example, peptidoglycan) of a bacterial cell.
- substantially no lytic enzyme is added means that the lytic enzyme is added at 0.0005 phr or less, and may be 0 phr.
- the lytic enzyme is not particularly limited as long as it is included in the above definition, and examples thereof include lysozyme, rabbia, ⁇ -N-acetylglucosaminidase, endolysine, and autolysine.
- the cells containing the PHB copolymer are preferably inactivated.
- the method of inactivation is not particularly limited, but for example, as described in Examples, a method of heating and stirring a culture solution containing cells containing a PHB copolymer at 60 to 70 ° C. for 7 hours is used. Can be mentioned. It is preferable that the culture broth after the heating and stirring treatment is further cooled to a temperature suitable for the step (a).
- Step (b)> an alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and the pH is adjusted before, at the same time as the adjustment, or after the adjustment.
- This is a step of adding a surfactant in any of the above.
- the step (b) includes the following steps (b1) and step (b2).
- -Step (b1) A step of adding an alkaline aqueous solution to the culture solution obtained in the above-mentioned step (a) to adjust the pH to 10.0 to 12.0-
- Step (b2) A step of adding a surfactant.
- Step (b1)) As described above, the step (b1) is a step of adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0.
- a high-purity PHB copolymer can be separated from the cells by dispersing and dissolving impurities (nucleic acid, protein, etc.) derived from the cells.
- the alkaline aqueous solution is an aqueous solution containing a basic compound.
- the basic compound contained in the alkaline aqueous solution is not particularly limited, and is, for example, an alkali metal such as sodium hydroxide or potassium hydroxide or an alkali earth metal hydroxide; a metal carbonate such as sodium carbonate or potassium carbonate; Examples thereof include metal phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate and the like, or metal hydrogen phosphate salts.
- the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and more preferably sodium hydroxide.
- the basic compound one kind may be used alone, or two or more kinds may be used in combination.
- the pH is preferably adjusted to 10.0 to 12.0, more preferably 10.2 to 11.8 by adding an alkaline aqueous solution. It is more preferable to adjust to ⁇ 11.6. Adjusting the pH to 10.0 or higher has the advantage that the bacterial cell components can be decomposed and dissolved. Further, by adjusting the pH to 12.0 or less, unintended damage to the bacterial cells can be prevented.
- the temperature in the step (b1) is preferably less than 100 ° C, more preferably less than 80 ° C.
- the lower limit is not particularly limited, but is preferably 40 ° C. or higher, for example.
- Step (b2) is a step of adding a surfactant to the culture solution obtained in the step (a). According to this step, the cell membrane can be treated particularly efficiently, and more impurities derived from the cells can be removed, so that a higher purity PHB copolymer can be separated from the cells.
- the surfactant is not particularly limited, and examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.
- anionic surface activity is preferable from the viewpoint of high ability to remove cell membranes.
- One of these types may be used alone, or two or more types may be used in combination.
- anionic surfactant examples include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfates, alkenyl sulfates, alkyl ether sulfates, alkenyl ether sulfates, ⁇ -olefin sulfonates, and ⁇ -.
- examples thereof include a sulfo fatty acid salt, an ester of an ⁇ -sulfo fatty acid salt, an alkyl ether carboxylate, an alkenyl ether carboxylate, an amino acid type surfactant, and an N-acyl amino acid type surfactant.
- an alkyl sulfate ester salt is preferable, and sodium dodecyl sulfate (SDS) is particularly preferable from the viewpoint of high ability to remove cell membranes and low cost.
- SDS sodium dodecyl sulfate
- One of these types may be used alone, or two or more types may be used in combination.
- the amount of the surfactant to be added is not particularly limited, and is, for example, 0.1 to 5.0% by weight and 0.3 to 2.5% by weight based on the culture solution. preferable.
- the step (b2) may be performed before the step (b1), at the same time, or after the step (b1). Preferably, step (b2) is performed after step (b1).
- the manufacturing method may further comprise step (b').
- the step (b') is a step of centrifuging the culture solution obtained in the step (b) and removing the supernatant to obtain a PHB copolymer aqueous suspension in which the PHB copolymer is concentrated.
- it is a step of removing impurities from the PHB copolymer separated from the bacterial cells, and concentrating and purifying the mixture.
- the method for centrifuging the culture solution is not particularly limited, and a known method can be used.
- the step (b') it is preferable to repeat the steps of centrifuging the culture solution, removing the supernatant, adding the solution to the sediment, centrifuging again, and removing the supernatant.
- the solution to be added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the culture solution.
- the amount of impurities remaining in the final product is generally determined by the step (b'), it is preferable to reduce these impurities as much as possible.
- impurities may be mixed as long as the physical properties of the final product are not impaired, but if a high-purity PHB copolymer is required for medical applications, etc., the impurities should be reduced as much as possible. It is preferable to let them.
- the amount of protein in the aqueous suspension of the PHB copolymer can be mentioned.
- the amount of protein in the aqueous suspension of PHB copolymer is not particularly limited as long as the amount of residual protein in the PHB copolymer powder described later can be achieved.
- the amount of the protein is preferably 10000 ppm or less, more preferably 5000 ppm or less, still more preferably 3000 ppm or less, based on the weight of the PHB copolymer in the aqueous suspension of the PHB copolymer.
- the solvent constituting the aqueous suspension of the PHB copolymer (the “solvent” is also referred to as “aqueous medium") is not particularly limited, and water or a mixture of water and an organic solvent is not particularly limited. It may be a solvent. Further, in the mixed solvent, the concentration of the organic solvent is not particularly limited as long as it is equal to or less than the solubility of the organic solvent used in water.
- the organic solvent is not particularly limited, and for example, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol; acetone and methyl ethyl ketone.
- Ketones such as; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethylsulfoxide, pyridine, piperidine and the like.
- methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile and the like are preferable because they are easy to remove.
- methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone and the like are more preferable because they are easily available. Further, methanol, ethanol and acetone are particularly preferable.
- the content of water in the aqueous medium constituting the aqueous suspension of the PHB copolymer is preferably 5% by weight or more, more preferably 10% by weight or more, still more preferably 30% by weight or more. Particularly preferably, it is 50% by weight or more.
- the PHB copolymer aqueous suspension in step (b') may contain other solvents, bacterial cell-derived components, compounds generated during purification, etc., as long as the essence of the present invention is not impaired. ..
- Step (c)> In the step (c) of the present production method, the pH is 7.0 or less, the shear viscosity is 0.005 Pa ⁇ s or more, and 0.5 Pa ⁇ s from the aqueous suspension obtained in the step (b). Prepare the following aqueous suspension.
- the PHB copolymer aqueous suspension before being subjected to the step (c) of the present production method usually has a pH exceeding 7.0 by going through the step (b). Therefore, the pH of the aqueous suspension of the PHB copolymer is adjusted to 7.0 or less by the step (c) of the present production method.
- the adjustment method is not particularly limited, and examples thereof include a method of adding an acid.
- the acid is not particularly limited, and may be either an organic acid or an inorganic acid, and may or may not be volatile. More specifically, as the acid, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid and the like can be used.
- the upper limit of the pH of the aqueous suspension of the PHB copolymer adjusted in the above adjustment step from the viewpoint of reducing the coloring when the PHB copolymer is heated and melted, and the stability of the molecular weight during heating and / or drying. From the viewpoint of ensuring the property, it is 7.0 or less, preferably 5.0 or less, and more preferably 4.0 or less.
- the lower limit of pH is preferably 1 or more, more preferably 2.0 or more, still more preferably 3.0 or more, from the viewpoint of acid resistance of the container.
- the concentration of the PHB copolymer in the aqueous suspension adjusted by the step (c) of the present production method is preferably 30% by weight or more, more preferably 40% by weight or more, still more preferably 50% by weight or more.
- the concentration of the PHB copolymer concentration is 30% by weight or more, it is economically advantageous in terms of drying utility, productivity is improved, and the powder bulk density obtained in the next step (d) is increased. As a result, transportability is improved.
- the upper limit of the concentration of the PHB copolymer is preferably close packing, and sufficient fluidity may not be ensured. Therefore, 65% by weight or less is preferable, and 60% by weight or less is more preferable.
- the method for adjusting the concentration of the PHB copolymer is not particularly limited, and an aqueous medium is added and a part of the aqueous medium is removed (for example, by centrifuging and then removing the supernatant).
- the method can be mentioned.
- the adjustment of the concentration of the PHB copolymer may be carried out at the stage of the step (c) or may be carried out at the stage of the step (b).
- the concentration of the PHB copolymer in the aqueous suspension prepared in step (c) is preferably 30 to 65% by weight.
- the shear viscosity of the aqueous suspension of the PHB copolymer is 0.005 to 0.5 Pa ⁇ s and 0.007 to 0. It is preferably 4 Pa ⁇ s, and more preferably 0.009 to 0.3 Pa ⁇ s.
- the shear viscosity of the aqueous suspension of PHB copolymer means the shear viscosity (Pa ⁇ s) at 20 ° C. when a shear rate of 10 (1 / s) is given.
- the shear viscosity of the aqueous suspension of PHB copolymer is measured by the method described in Examples.
- the aqueous suspension in one embodiment of the present invention may contain a dispersant. That is, the step (c) in one embodiment of the present invention may be a step of preparing an aqueous suspension further containing a dispersant in addition to the PHB copolymer.
- the dispersant when used, it is preferable to add the dispersant to the PHB copolymer aqueous suspension before adjusting the pH to 7.0 or less.
- the productivity and thermal stability of the PHB copolymer can be suitably improved.
- the dispersant is not particularly limited, and examples thereof include an alkylene oxide-based dispersant, a cellulose-based dispersant, polyvinyl alcohol (PVA), a sorbitan alkylate-based dispersant, and the like. By using these dispersants, a PHB copolymer having a large particle size can be obtained at a lower hot air temperature.
- the dispersant may be one kind or two or more kinds.
- the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
- the pH of the aqueous suspension of PHB copolymer when the pH of the aqueous suspension of PHB copolymer is adjusted to 7.0 or less, aggregation of the PHB copolymer is suitably prevented, and an extruder for powder processing is used.
- the dispersant is preferably an alkylene oxide-based dispersant from the viewpoint of preferably suppressing the adhesion of the above to the shaft. Further, it is more preferable to use a cellulose-based dispersant in combination with the alkylene oxide-based dispersant because it can be expected to be effective as a binder that further suppresses aggregation of the PHB copolymer and further prevents the powder after drying from breaking.
- the alkylene oxide-based dispersant is not particularly limited as long as it exhibits the above effects, but is composed of a block of poly (ethylene oxide) (PEO) and a block of poly (propylene oxide) (PPO). It is preferably configured and in the form of PEO-PPO-PEO.
- poly (ethylene oxide) (PEO) block means a polymer portion formed by polymerizing ethylene oxide (EO) in the structure of an alkylene oxide-based dispersant.
- poly (propylene oxide) (PPO) block means a polymer portion formed by polymerizing propylene oxide (PO) in the structure of an alkylene oxide-based dispersant.
- the viscosity of the aqueous suspension is kept low and the PHB copolymer is highly productive.
- PHB copolymer powder can be produced.
- the range of PEO molecular weight and PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant is preferably the following combination.
- PEO molecular weight may be referred to as "EO molecular weight”
- PPO molecular weight may be referred to as "PO molecular weight”.
- the PEO molecular weight in the alkylene oxide-based dispersant may be 1500 or more, preferably 1750 or more, and more preferably 2000 or more.
- the upper limit of the PEO molecular weight in the alkylene oxide-based dispersant is, for example, 30,000 or less, preferably 25,000 or less, and more preferably 20,000 or less.
- the PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant is preferably 0.5 or more, more preferably 0.6 or more, and 0.7 or more. Is even more preferable.
- the upper limit of the PEO molecular weight / PPO molecular weight is preferably 5.0 or less, more preferably 4.8 or less, and further preferably 4.5 or less.
- the alkylene oxide-based dispersant has hydrophilicity and the alkylene oxide-based dispersant is dispersed. Since the number of molecules increases with respect to the added weight of the agent, it is easy to maintain the dispersibility of the aqueous suspension.
- the alkylene oxide-based dispersant has a PEO molecular weight of 1500 or more and a PEO molecular weight / PPO molecular weight of 0.5 to 5.0.
- the alkylene oxide-based dispersant used in the step (c) of the present production method is not particularly limited, and for example, a commercially available product can be used.
- commercially available products include Pluronic (registered trademark) 10400 (registered trademark) 10400 (registered trademark) 10500 (BASF), Genapol (registered trademark) PF80 (registered trademark), and Unilube (registered trademark) 70DP-.
- the amount of the dispersant added to the aqueous suspension in the step (c) of the present production method is not particularly limited, but is 0. 1 to 20 parts by weight is preferable, 0.5 to 10 parts by weight is more preferable, and 0.75 to 5 parts by weight is further preferable.
- the addition amount of the dispersant within the above range, the dispersion stability of the PHB copolymer in the aqueous suspension of the PHB copolymer is further improved, and spray drying can be efficiently carried out, and as a result, PHB can be carried out. There is a tendency to more preferably improve the productivity and thermal stability of the copolymer.
- the cellulose-based dispersant is not particularly limited as long as it exhibits the effects of the present invention, and for example, methyl cellulose (MC), ethyl cellulose, propyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose (HEC), and the like.
- MC methyl cellulose
- HEC hydroxyethyl cellulose
- HPMC hydroxypropylmethyl cellulose
- HPMC carboxymethyl cellulose
- CMC carboxyethyl cellulose
- carboxypropyl cellulose carboxymethyl hydroxyethyl cellulose
- acetyl cellulose cyanoethyl cellulose
- sodium cellulose sulfate and the like can be mentioned.
- methyl cellulose and hydroxypropyl methyl cellulose are preferable from the viewpoint of a wide range of degree of substitution that makes them water-soluble. Only one type of cellulosic dispersant may be used, or a plurality of cellulosic dispersants may be used in combination.
- the cellulosic dispersant used in the step (c) of this production method is not particularly limited, and for example, a commercially available product can be used.
- Commercially available products of cellulosic dispersants include, for example, MCE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SFE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.).
- the amount of the cellulosic dispersant added to the aqueous suspension of the PHB copolymer in the step (c) of the present production method is not particularly limited, but is based on 100 parts by weight of the PHB copolymer contained in the aqueous suspension. 0.01 to 10 parts by weight is preferable, 0.05 to 5 parts by weight is more preferable, and 0.08 to 3 parts by weight is further preferable.
- the effect of the present invention can be achieved by setting the addition amount of the cellulosic dispersant within the above range.
- the dispersant is preferably a biodegradable substance from the viewpoint of environmental problems.
- volume median diameter of the PHB copolymer in the aqueous suspension of the PHB copolymer obtained by the step (c) of the present production method (hereinafter, may be simply referred to as "volume median diameter of the PHB copolymer").
- volume median diameter of the primary particles of the PHB copolymer (hereinafter referred to as “primary particle diameter”) is preferably 30 times or less, more preferably 20 times or less, still more preferably 10 times or less. Since the volume median diameter of the PHB copolymer is 30 times or less the primary particle diameter, the PHB copolymer aqueous suspension exhibits better fluidity, and therefore the subsequent step (d) is carried out with high efficiency.
- the volume median diameter of the PHB copolymer is measured, for example, using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA.
- the volume median diameter of the above PHB copolymer can be used as an index of the dispersed state of the PHB copolymer in the aqueous suspension of the PHB copolymer.
- the method for adjusting the volume median diameter of the PHB copolymer is not particularly limited, and known means (stirring or the like) can be applied.
- a PHB copolymer aqueous suspension whose dispersed state has collapsed due to exposure to acidic conditions can be subjected to physical treatment, chemical treatment, biological treatment, etc. that can be considered by those skilled in the art.
- the PHB copolymer in the aqueous suspension of the PHB copolymer can be returned to the dispersed state (for example, the state having the volume median diameter of the above PHB copolymer).
- the PHB copolymer aqueous suspension prepared in the step (c) is spray-dried.
- the spray drying method include a method in which a PHB copolymer aqueous suspension is supplied into a dryer in the form of fine droplets and dried while being in contact with hot air in the dryer.
- the method (atomizer) for supplying the aqueous suspension of the PHB copolymer in the form of fine droplets into the dryer is not particularly limited, and examples thereof include known methods such as a method using a rotating disk and a method using a nozzle. ..
- the contact method between the droplet and the hot air in the dryer is not particularly limited, and examples thereof include a parallel flow type, a countercurrent type, and a method in which these are used in combination.
- the drying temperature at the time of spray drying in the step (d) may be any temperature as long as it can remove most of the aqueous medium from the droplets of the aqueous suspension of the PHB copolymer, and can be dried to the desired moisture content. Moreover, it can be appropriately set under conditions that do not cause deterioration of quality (decrease in molecular weight, decrease in color tone, etc.), melting, etc. as much as possible.
- the temperature of the hot air blown into the spray dryer can be appropriately selected in the range of 100 to 300 ° C.
- the amount of hot air in the dryer can be appropriately set according to, for example, the size of the dryer.
- the present production method may include, after the step (d), a step of further drying the obtained PHB copolymer (PHB copolymer powder or the like) (for example, a step of subjecting to vacuum drying or the like). Further, the present production method may include other steps (for example, a step of adding various additives to the aqueous suspension of PHB copolymer).
- the present manufacturing method may further include the following steps (e) and (f) after the step (d). According to these steps, the nitrogen content derived from the bacterial cell residue contained in the PHB copolymer can be reduced, and a high-purity PHB copolymer can be obtained.
- the step (e) is a step of washing the PHB copolymer powder obtained in the step (d).
- the step (e) preferably includes the following steps (e1) and step (e2).
- Step (e1) The PHB copolymer powder obtained in the step (e) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added to disperse the dispersion.
- Step / Step (e2) of adjusting the slurry pH and stirring with the cleaning liquid A step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), centrifuging and removing the supernatant.
- Step (e1) In the step (e1), as described above, the PHB copolymer powder obtained in the step (d) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added. This is a step of adjusting the pH of the dispersed slurry and stirring it together with the washing liquid. When stirring, the temperature of the liquid may be raised.
- the predetermined concentration of the dispersed slurry in the step (e1) is not particularly limited, but is preferably 20 to 40% from the viewpoint of minimizing the size of the tank to be dispersed and the fluidity of the slurry. , 30-40% is preferable.
- the alkaline aqueous solution is not particularly limited, but the alkaline aqueous solution described in (step (b1)) can be used.
- the pH of the dispersed slurry is preferably adjusted to, for example, 5.0 to 14.0.
- the stirring time in the washing of the step (e1) is not particularly limited, but may be several hours. From the viewpoint of sufficiently washing the PHB copolymer, the stirring time may be 1 to 12 hours or 2 to 10 hours.
- the dispersed slurry in the step (e1) may further contain sodium sulfate.
- the cleaning solution in the step (e1) may contain an alkaline proteolytic enzyme and / or a lytic enzyme.
- the type of the alkaline proteolytic enzyme is not particularly limited, but the one described in (Alkaline Proteolytic Enzyme) of the present specification may be used. Further, the alkaline proteolytic enzyme used in this step may be the same as or different from the alkaline proteolytic enzyme used in step (a).
- the type of lytic enzyme is not particularly limited, but may be the lytic enzyme described in the description of step (a).
- Step (e2) is a step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), separating the PHB copolymer powder, and removing the supernatant.
- the alkaline aqueous solution is not particularly limited, but may be the same as or different from the alkaline aqueous solution used in the step (e1).
- the method for separating the PHB copolymer powder is not particularly limited, and a known method can be used. For example, centrifugation, filter dehydration, and liquid cyclone separation can be used.
- step (e2) it is preferable to repeat the steps of adding an alkaline aqueous solution to the dispersed slurry, centrifuging the dispersion, and removing the supernatant. By this operation, a more concentrated and purified PHB copolymer can be obtained.
- the step (f) is a step of dehydrating and / or drying the polyhydroxyalkanoic acid powder obtained in the step (e).
- a spray dryer for example, a spray dryer, a fluidized bed dryer, a drum dryer, or the like is used, but a spray dryer is preferably used because of its simple operation.
- the spray drying method and drying temperature for example, the method and temperature described in step (d) may be used.
- the PHB copolymer powder according to one embodiment of the present invention contains a PHB copolymer, a peptide glycan, and a dispersant.
- the PHB copolymer has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol), and a bulk density of 0.45 g / mL or more.
- the first PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units, and has a high bulk density, so that it is extremely useful in various fields.
- the above-mentioned substances are used as the "PHB copolymer”, “peptidoglycan”, and “dispersant”.
- the first PHB copolymer powder contains a PHB copolymer.
- the content of the PHB copolymer in the first PHB copolymer powder is not particularly limited, but is, for example, 90 to 99% by weight, preferably 93 to 98% by weight, and 95 to 97% by weight. More preferred. By setting the content of the PHB copolymer in the above range, there is an advantage that the physical properties of PHBH such as fluidity are not lost.
- the first PHB copolymer powder contains peptidoglycan.
- the content of peptidoglycan in the first PHB copolymer powder is not particularly limited, but is 0.1 to 1.5 with respect to 100 parts by weight of the PHB copolymer constituting the PPHB copolymer powder.
- the part by weight (phr) is preferable, and 0.3 to 1.3 parts by weight (phr) is more preferable.
- the content of peptidoglycan in the first PHB copolymer powder is measured by the method described in Examples.
- the first PHB copolymer powder contains a dispersant.
- the dispersant is preferably an alkylene oxide-based dispersant.
- the content of the dispersant in the PHB copolymer powder is not particularly limited, but is preferably 0.1 to 20 parts by weight with respect to 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. 0.5 to 10 parts by weight is more preferable, and 0.75 to 5 parts by weight is further preferable. By setting the addition amount of the dispersant within the above range, the productivity of the PHB copolymer powder tends to be improved.
- the bulk density of the first PHB copolymer powder is 0.45 g / mL or more, preferably 0.48 g / mL or more, preferably 0.50 g, from the viewpoint of increasing powder transportability. More preferably, it is / mL or more.
- the upper limit is not particularly limited, but from the viewpoint of powder transportability, for example, 0.55 g / mL can be mentioned.
- the bulk density of the first PHB copolymer powder is measured by the method described in Examples.
- the median particle size of the first PHB copolymer powder is 80 to 200 ⁇ m, preferably 100 to 180 ⁇ m, and more preferably 105 to 160 ⁇ m from the viewpoint of achieving excellent fluidity.
- the median particle size of the first PHB copolymer powder is measured by the method described in Examples.
- the purity of the first PHB copolymer powder is less than 98%, although the upper limit is not particularly specified.
- the lower limit is 85% or more, preferably 90% or more, and more preferably 92% or more, from the viewpoint of not impairing the physical properties of the PHB copolymer.
- the purity of the PHB copolymer powder means the content (% by weight) of the PHB copolymer in the PHB copolymer powder.
- the purity of the first PHB copolymer powder is measured by the method described in Examples.
- the amount of residual protein in the first PHB copolymer powder is preferably 0.1 to 1.0 phr, preferably 0.12 to 0.5 phr, from the viewpoint of coloring. It is more preferably 0.15 to 0.3 phr, and even more preferably 0.15 to 0.3 phr.
- the amount of residual protein means the amount of protein remaining in the PHB copolymer powder, and is expressed as an amount with respect to 100 parts by weight of the PHB copolymer in the PHB copolymer powder.
- the amount of residual protein in the first PHB copolymer powder is measured by the method described in Examples.
- the first PHB copolymer powder may contain various components generated or not removed in the manufacturing process as long as the effect of the present invention is exhibited.
- the first PHB copolymer powder is an intermediate to the final product described later, and is produced by the above-mentioned production method (for example, steps (a) to (d)). Will be done.
- the PHB copolymer powder according to one embodiment of the present invention contains a PHB copolymer and a nitrogen compound, and the PHB.
- the copolymer has a composition ratio of 80/20 to 88/12 (mol / mol) of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit, and a bulk density of 0.45 g /.
- the second PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate units, and has a high bulk density and high purity. It is extremely useful in the field of.
- nitrogen compound means a compound containing at least nitrogen.
- the nitrogen compound is a decomposition product of the protein remaining in the PHB copolymer powder and peptidoglycan which is a component of the cell wall.
- the above-mentioned ones are used as the "PHB copolymer” and the “nitrogen compound”.
- "bulk density” and “median particle size” those described in the above (intermediate) section are used.
- the second PHB copolymer powder contains a nitrogen compound.
- the total amount of nitrogen in the second PHB copolymer powder is not particularly limited, but is 0.010 to 0.075 weight with respect to 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder.
- the part (phr) is preferable, and 0.01 to 0.06 parts by weight (phr) is more preferable.
- the total amount of nitrogen in the second PHB copolymer powder is measured by the method described in Examples.
- the second PHB copolymer powder may contain various components generated or not removed in the manufacturing process as long as the effect of the present invention is exhibited.
- the second PHB copolymer powder is a final product and is produced by the above-mentioned production method (for example, steps (a) to (f)).
- This PHB copolymer powder can be used for various purposes such as paper, film, sheet, tube, plate, rod, container (for example, bottle container, etc.), bag, parts, and the like.
- one embodiment of the present invention is as follows.
- ⁇ 1> A method for producing a polyhydroxybutyrate copolymer.
- the popopolyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
- B) An alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and either before the adjustment, at the same time as the adjustment, or after the adjustment.
- step (C) From the aqueous suspension obtained in the step (b), an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa ⁇ s or more and 0.5 Pa ⁇ s or less. And (d) the step of spray-drying the aqueous suspension prepared in the above step (c), A method for producing a polyhydroxybutyric acid copolymer.
- ⁇ 4> The method for producing a polyhydroxybutyric acid copolymer according to any one of ⁇ 1> to ⁇ 3>, wherein the surfactant in the step (b) is sodium dodecyl sulfate.
- ⁇ 5> The method for producing a polyhydroxybutyric acid copolymer according to any one of ⁇ 1> to ⁇ 4>, wherein the aqueous suspension further contains a dispersant.
- ⁇ 6> The method for producing a polyhydroxybutyric acid copolymer according to ⁇ 5>, wherein the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
- ⁇ 7> The polyhydroxybutyric acid according to any one of ⁇ 1> to ⁇ 6>, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight.
- ⁇ 8> The method for producing a polyhydroxybutyric acid copolymer according to ⁇ 2>, wherein the cleaning solution of the step (e) contains an alkaline proteolytic enzyme and / or a lytic enzyme.
- ⁇ 9> Containing a polyhydroxybutyrate copolymer, peptidoglycan, and a dispersant
- the polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
- a polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 ⁇ m.
- ⁇ 12> Containing a polyhydroxybutyrate copolymer and a nitrogen compound, The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
- ⁇ 13> The polyhydroxybutyrate copolymer powder according to ⁇ 12>, wherein the total amount of nitrogen in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr.
- volume median diameter The volume median diameter in the aqueous suspension of PHB copolymer was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 manufactured by HORIBA.
- Shear viscosity of aqueous suspension of PHB copolymer The shear viscosity of the aqueous suspension of PHB copolymer was measured by the following method. Specifically, the shear viscosity was measured with a coaxial double cylinder using MCR302 manufactured by Antonio Par. The aqueous suspension of the PHB copolymer was placed in a 20 mL cylinder and cooled under the condition of a shear rate of 10 (1 / s) until the liquid temperature reached 15 ° C. Then, the liquid temperature was raised to 20 ° C., and after reaching the target liquid temperature, the viscosity when the time change of the torque became less than 1% was measured.
- the amount of peptidoglycan remaining in the PHB copolymer powder was measured by the following method. Specifically, first, 50 ⁇ L of peptidoglycan standard product was placed in a microplate. Then, 50 ⁇ L of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the time at which the absorbance became 0.4 times the final absorbance was measured, with the absorbance 3 hours after the start of the measurement as the final absorbance.
- the standard peptidoglycan was diluted 1000 to 10000 times with distilled water, and the same operation as above was carried out to prepare a calibration curve. Then, the aqueous suspension of PHB copolymer prepared in the step (c) was diluted 1000 to 10000 times with distilled water, and 50 ⁇ L of the diluted solution was placed in a microplate. Then, 50 ⁇ L of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate.
- the absorbance was measured every 15 seconds at a wavelength of 650 nm, and the amount of peptide glycan was calculated from the time when the absorbance became 0.4 times the final absorbance, with the absorbance 3 hours after the start of measurement as the final absorbance.
- the amount of remaining protein in the PHB copolymer powder was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was put into a 14 mL falcon tube, 2 mL of the above reagent was added, and the mixture was shaken at 60 ° C. for 30 minutes. After 30 minutes, it was cooled and the absorbance at a wavelength of 562 nm was measured.
- the bulk density of the PHB copolymer powder was measured using a bulk specific gravity measuring instrument (manufactured by Kuramochi Kagaku Kikai) based on JIS K 7365: 1999.
- the median particle size of the PHB copolymer powder was measured by the following method. Specifically, the median particle size was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 (HORIBA). To 20 mL of ion-exchanged water, 0.05 g of sodium dodecyl sulfate was added as a surfactant to obtain an aqueous surfactant solution. Next, 0.2 g of the PHB copolymer powder to be measured was added to the aqueous surfactant solution, and the PHB copolymer powder was dispersed in the aqueous surfactant solution to obtain a dispersion for measurement. rice field. The prepared dispersion was introduced into the laser diffraction / scattering type particle size distribution measuring device and measured.
- the purity of the PHB copolymer powder was measured by the following method. Specifically, in TG-DTA (manufactured by 2000SE NIETZSCHE), about 10 mg of PHBH dry powder (weight is W) is heated at 10 ° C / min from 50 ° C to 500 ° C in a nitrogen atmosphere. Then, a graph of temperature and sample weight was created. Intersection A of a linear line with a weight loss slope of 280-285 ° C. and a linear line with a weight loss slope of 400-450 ° C. A, a linear line with a weight loss slope of 280-285 ° C. and 150 The weight of the intersection B with the linear straight line having a slope of weight reduction of ⁇ 200 ° C. was calculated from the graph. W'was calculated as the amount of PHBH by the weight difference between the intersection A and the intersection B, and W'/ W was defined as the purity of PHBH.
- the total nitrogen content of the PHB copolymer powder was measured using a trace total nitrogen analyzer TN-2100H (Nittoseiko Analytech Co., Ltd.).
- Example 1 (Preparation of cell culture solution)
- Ralstonia eutropha described in International Publication No. WO2019 / 142717 is cultured by the method described in paragraphs [0041] to [0048] of the same document, and a cell culture solution containing cells containing a PHB copolymer is prepared. Obtained. Ralstonia eutropha is now classified as Cupriavidus necator.
- the composition ratio of the repeating unit of the PHB copolymer was 80/20 to 88/12 (mol / mol).
- the PHB copolymer aqueous suspension was concentrated 4-fold to adjust the PHB copolymer concentration to 52% by weight or more.
- the volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 ⁇ m.
- This liquid was stirred, the liquid temperature was raised to 60 ° C., 10% sulfuric acid was added so that the pH became 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. .. Then, the solid content concentration of the mixture was adjusted to 50% by mass by adding water.
- the volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 ⁇ m. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa ⁇ s.
- the obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder before washing was obtained.
- the amount of peptidoglycan contained in the obtained pre-washed PHB copolymer powder was 1.04 phr, the amount of residual protein was 0.18 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g / mL, and the median particles.
- the diameter was 110 ⁇ m, the purity was 97%, and the total amount of nitrogen was 0.099 phr.
- Example 2 Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation).
- the PHB copolymer powder before washing was dispersed in pure water so that the PHB copolymer concentration was 37.5%, and 1 phr of sodium sulfate was added to the dispersed slurry. Then, the pH of the dispersed slurry was adjusted to 10.5 with 30% sodium hydroxide. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours.
- the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.070 phr, and the bulk of the PHB copolymer powder was obtained.
- the density was 0.50 g / mL and the polymer particle size was 108 ⁇ m.
- Example 3 Up to (washing 1), a dispersed slurry of PHB copolymer powder before washing was obtained by the same method as in Example 1. 30% sodium hydroxide was added to the obtained dispersed slurry to adjust the pH of the dispersed slurry to 10.5. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.033 phr, and the bulk of the PHB copolymer powder was obtained. The density was 0.50 g / mL and the polymer particle size was 115 ⁇ m.
- Example 4 Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation). Before washing PHB copolymer powder was dispersed in pure water so that the PHB copolymer concentration was 37.5%, 30% sodium hydroxide was added, and the pH was adjusted to 11 for 2 hours. Stirred. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.098 phr, and the PHB copolymer powder before washing was obtained. The bulk density was 0.51 g / mL, and the polymer particle size was 110 ⁇ m.
- the aqueous suspension of the PHB copolymer was concentrated to a solid content concentration of 50% by filtration through a Büchner funnel, but it was in the form of a wet powder, had no fluidity, and could not be spray-dried. ..
- the volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 ⁇ m. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa ⁇ s.
- the obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary).
- PHB copolymer powder was obtained.
- the amount of peptidoglycan contained in the obtained PHB copolymer powder is 0.01 phr, the amount of residual protein is 0.5 phr, the bulk density of the PHB copolymer powder is 0.20 g / mL, the median particle size is 71 ⁇ m, and the purity. Was 98%.
- the present invention it is possible to produce a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units. Further, since the PHB copolymer obtained by the production method of the present invention has a high bulk density, it can be used in agriculture, fisheries, forestry, horticulture, medicine, sanitary goods, clothing, non-clothing, packaging, automobiles, building materials, and other fields. It can be suitably used.
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Abstract
The purpose of the present invention is to provide a PHB copolymer with which aggregation of PHB copolymer particles during the production process is inhibited and which has high bulk density. The purpose is also to provide a production method for said copolymer. The aforementioned purpose is achieved by providing a PHB copolymer production method comprising: (a) a step for performing an enzymatic treatment using a specific enzyme on a microorganism containing a PHB copolymer having a composition ratio that is a 3HB unit/a hydroxyalkanoate unit other than a 3HB unit and is in a prescribed range; (b) a step for adding a surfactant after the pH is adjusted to within a prescribed range by adding an alkaline aqueous solution; (c) a step for preparing an aqueous suspension having a pH that is at most 7.0 and having a shear viscosity in a prescribed range; and (d) a step for spray drying.
Description
本発明は、ポリヒドロキシ酪酸共重合体の製造方法およびその利用に関する。
The present invention relates to a method for producing a polyhydroxybutyric acid copolymer and its use.
ポリヒドロキシ酪酸共重合体（以下、「ＰＨＢ共重合体」と称する場合がある。）は、生分解性を有することが知られている。
Polyhydroxybutyrate copolymers (hereinafter, may be referred to as "PHB copolymers") are known to have biodegradability.
微生物が生成するＰＨＢ共重合体は、微生物の菌体内に蓄積されるため、ＰＨＢ共重合体をプラスチックとして利用するためには、微生物の菌体内からＰＨＢ共重合体を分離・精製する工程が必要となる。ＰＨＢ共重合体を分離・精製する工程では、ＰＨＢ共重合体以外の生物由来成分を可溶化した後、得られた水性懸濁液からＰＨＢ共重合体を取り出す。このとき、例えば、遠心分離、ろ過、乾燥等の分離操作が行われる。乾燥としては、例えば、噴霧乾燥機、流動層乾燥機、ドラムドライヤー等が用いられるが、操作が簡便であることから、好ましくは、噴霧乾燥機が用いられる。
Since the PHB copolymer produced by a microorganism is accumulated in the cells of the microorganism, in order to use the PHB copolymer as a plastic, a step of separating and purifying the PHB copolymer from the inside of the microorganism is required. It becomes. In the step of separating and purifying the PHB copolymer, after solubilizing biological components other than the PHB copolymer, the PHB copolymer is taken out from the obtained aqueous suspension. At this time, for example, separation operations such as centrifugation, filtration, and drying are performed. As the drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer and the like are used, but since the operation is simple, a spray dryer is preferably used.
これまで、本発明者は、ｐＨ７．０以下の水性懸濁液中でのＰＨＢ共重合体の凝集を防止するために、水性懸濁液のｐＨを７．０以下に調整する前にポリビニルアルコール（ＰＶＡ）を分散剤として添加し、その後、得られたｐＨ７．０以下の水性懸濁液を噴霧乾燥する技術を開発している（特許文献１）。また、本発明者は、３－ヒドロキシヘキサノエート（以下、「３ＨＨ」と称する場合がある。）単位の組成比が高いＰＨＢ共重合体を製造する技術として、前記ＰＨＢ共重合体を含有する菌体を高圧破砕により破砕する工程を含む技術を開発している（特許文献２および３）。
So far, the present inventors have made polyvinyl alcohol before adjusting the pH of the aqueous suspension to 7.0 or less in order to prevent aggregation of the PHB copolymer in the aqueous suspension having a pH of 7.0 or less. We are developing a technique for adding (PVA) as a dispersant and then spray-drying the obtained aqueous suspension having a pH of 7.0 or less (Patent Document 1). Further, the present inventor contains the PHB copolymer as a technique for producing a PHB copolymer having a high composition ratio of 3-hydroxyhexanoate (hereinafter, may be referred to as “3HH”) unit. We are developing a technique including a step of crushing cells by high-pressure crushing (Patent Documents 2 and 3).
ポリヒドロキシ酪酸共重合体としては、ポリ（３－ヒドロキシブチレート－コ－３－ヒドロキシヘキサノエート）（以下、「ＰＨＢＨ」と称する場合もある。）が知られている。
As the polyhydroxybutyric acid copolymer, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter, may be referred to as "PHBH") is known.
上記の技術は優れたものであるが、製造工程のハンドリング容易性の観点から、さらなる改善の余地がある。
The above technology is excellent, but there is room for further improvement from the viewpoint of ease of handling in the manufacturing process.
そこで、本発明の目的は、製造過程でのＰＨＢ共重合体粒子の凝集を抑制しつつ、嵩密度が高いＰＨＢ共重合体、とりわけ、嵩密度が高い、特定の３－ヒドロキシブチレート（以下、「３ＨＢ」と称する場合がある。）単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するＰＨＢ共重合体と、その製造方法を提供することにある。
Therefore, an object of the present invention is a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles in the manufacturing process, particularly a specific 3-hydroxybutyrate having a high bulk density (hereinafter referred to as “)”. It may be referred to as "3HB".) It is an object of the present invention to provide a PHB copolymer having a composition ratio of hydroxyalkanoate units other than units / 3HB units, and a method for producing the same.
本発明者らは、上記課題を解決すべく鋭意検討した結果、３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が高い、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するポリヒドロキシ酪酸共重合体を含有する菌体を、特定の酵素で酵素処理する工程を含むことにより、製造過程でのＰＨＢ共重合体粒子の凝集を抑制しつつ、嵩密度が高いＰＨＢ共重合体が得られることを初めて見出し本発明を完成するに至った。
As a result of diligent studies to solve the above problems, the present inventors have a poly having a high composition ratio of hydroxyalkanoate units other than 3HB units and a specific composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units. By including a step of enzymatically treating a bacterial cell containing a hydroxybutyrate copolymer with a specific enzyme, a PHB copolymer having a high bulk density can be obtained while suppressing aggregation of PHB copolymer particles in the manufacturing process. It was the first time to find out that it could be obtained, and the present invention was completed.
したがって、本発明の一態様は、ＰＨＢ共重合体を製造する方法であって、前記ＰＨＢ共重合体は、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、（ａ）前記ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、を含む、ＰＨＢ共重合体の製造方法である。
Therefore, one aspect of the present invention is a method for producing a PHB copolymer, wherein the composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units is 80/20 to 88/12. (Mol / mol), (a) a step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing the PHB copolymer, and (b) the step of enzymatically treating the cells. An alkaline aqueous solution is added to the culture solution obtained in (a) to adjust the pH to 10.0 to 12.0, and the surfactant is added either before the adjustment, at the same time as the adjustment, or after the adjustment. Step of addition, (c) From the aqueous suspension obtained in the step (b), the pH is 7.0 or less, and the shear viscosity is 0.005 Pa · s or more and 0.5 Pa · s or less. A method for producing a PHB copolymer, which comprises a step of preparing an aqueous suspension and (d) a step of spray-drying the aqueous suspension prepared in the step (c).
また、本発明の一態様は、ＰＨＢ共重合体と、ペプチドグリカンと、分散剤と、を含み、前記ＰＨＢ共重合体は、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。
Further, one aspect of the present invention includes a PHB copolymer, a peptide glycan, and a dispersant, and the PHB copolymer has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20. It is a PHB copolymer powder having a bulk density of 0.45 g / mL or more, a median particle size of 80 to 200 μm, and a bulk density of about 88/12 (mol / mol).
さらに、本発明の一態様は、ＰＨＢ共重合体と、窒素化合物と、を含み、前記ＰＨＢ共重合体が、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。
Further, one aspect of the present invention comprises a PHB copolymer and a nitrogen compound, and the PHB copolymer has a composition of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit. A PHB copolymer powder having a ratio of 80/20 to 88/12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 μm.
本発明の一態様によれば、製造過程でのＰＨＢ共重合体粒子の凝集を抑制しつつ、嵩密度が高いＰＨＢ共重合体、とりわけ、嵩密度が高い、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するＰＨＢ共重合体を提供することができる。
According to one aspect of the present invention, a PHB copolymer having a high bulk density, particularly a high bulk density, other than a specific 3HB unit / 3HB unit, while suppressing aggregation of PHB copolymer particles in the manufacturing process. PHB copolymers having a composition ratio of hydroxyalkanoate units can be provided.
本発明の実施の一形態について、以下に詳細に説明する。なお、本明細書において特記しない限り、数値範囲を表す「Ａ～Ｂ」は、「Ａ以上、Ｂ以下」を意味する。また、本明細書中に記載された文献の全てが、本明細書中において参考文献として援用される。
An embodiment of the present invention will be described in detail below. Unless otherwise specified in the present specification, "A to B" representing a numerical range means "A or more and B or less". In addition, all of the documents described herein are incorporated herein by reference.
〔１．本発明の概要〕
本発明の一実施形態に係るＰＨＢ共重合体の製造方法（以下、「本製造方法」と称する。）は、前記ＰＨＢ共重合体が、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、（ａ）前記ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、を含む、ことを特徴とする。 [1. Outline of the present invention]
In the method for producing a PHB copolymer according to an embodiment of the present invention (hereinafter, referred to as "the present production method"), the composition ratio of the PHB copolymer to a hydroxy alkanoate unit other than 3HB unit / 3HB unit. Is 80/20 to 88/12 (mol / mol), and (a) an alkaline proteolytic enzyme is added to a culture solution containing the cells containing the PHB copolymer to treat the cells enzymatically. Step (b) Add an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and before, at the same time as, or after the adjustment. From the step of adding the surfactant in any of the above steps, (c) the aqueous suspension obtained in the step (b), the pH is 7.0 or less and the shear viscosity is 0.005 Pa · s or more. , 0.5 Pa · s or less, and (d) spray-drying the aqueous suspension prepared in the step (c).
本発明の一実施形態に係るＰＨＢ共重合体の製造方法（以下、「本製造方法」と称する。）は、前記ＰＨＢ共重合体が、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、（ａ）前記ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、を含む、ことを特徴とする。 [1. Outline of the present invention]
In the method for producing a PHB copolymer according to an embodiment of the present invention (hereinafter, referred to as "the present production method"), the composition ratio of the PHB copolymer to a hydroxy alkanoate unit other than 3HB unit / 3HB unit. Is 80/20 to 88/12 (mol / mol), and (a) an alkaline proteolytic enzyme is added to a culture solution containing the cells containing the PHB copolymer to treat the cells enzymatically. Step (b) Add an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and before, at the same time as, or after the adjustment. From the step of adding the surfactant in any of the above steps, (c) the aqueous suspension obtained in the step (b), the pH is 7.0 or less and the shear viscosity is 0.005 Pa · s or more. , 0.5 Pa · s or less, and (d) spray-drying the aqueous suspension prepared in the step (c).
本発明者は、従来の方法（特許文献１）では、高濃度のスラリー（ＰＨＢ共重合体水性懸濁液）を使用するところ、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比のＰＨＢ共重合体粒子は凝集し、ＰＨＢ共重合体水性懸濁液の粘度が高くなる結果、送液困難を生じ、噴霧乾燥が困難となる場合があるとの新規の課題を見出した。また、上記の問題を解決するために、ＰＨＢ共重合体水性懸濁液の濃度を下げると、噴霧乾燥に多くのエネルギーが必要となり、エネルギー効率的に好ましくなく、かつ、得られる粉体の嵩密度が低く、粉体輸送性が悪くなるという別の問題が生じることもわかった。
In the conventional method (Patent Document 1), the present inventor uses a high-concentration slurry (PHB copolymer aqueous suspension), and the composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit. We have found a new problem that the PHB copolymer particles in the above are aggregated and the viscosity of the aqueous suspension of the PHB copolymer is increased, resulting in difficulty in liquid feeding and spray drying. Further, if the concentration of the aqueous suspension of the PHB copolymer is lowered in order to solve the above problems, a large amount of energy is required for spray drying, which is not preferable in terms of energy efficiency and the bulk of the obtained powder. It was also found that another problem arises: low density and poor powder transportability.
一方、特許文献２および３に記載の方法では、菌体を高圧破砕により物理的に破砕するため、スケールアップ時に工程が複雑になるという問題がある。さらに、特許文献３に記載の方法では、製造工程自体が長いという問題もある。
On the other hand, the methods described in Patent Documents 2 and 3 have a problem that the process is complicated at the time of scale-up because the bacterial cells are physically crushed by high pressure crushing. Further, the method described in Patent Document 3 has a problem that the manufacturing process itself is long.
そこで、本発明者は、上記問題を解決するべく鋭意検討を行った結果、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するＰＨＢ共重合体を含有する菌体を、特定の酵素で酵素処理する工程を含むことにより、噴霧乾燥を行う場合であっても製造過程でのＰＨＢ共重合体粒子の凝集を抑制しつつ、嵩密度が高いＰＨＢ共重合体が得られることを初めて見出した。これは、上記工程により、ＰＨＢ共重合体水性懸濁液が菌体由来のペプチドグリカンを含むことになり、ＰＨＢ共重合体粒子同士の凝集が回避できるようになった結果であると、本発明者は推測している。
Therefore, as a result of diligent studies to solve the above problems, the present inventor has identified a bacterial cell containing a PHB copolymer having a composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit. By including the step of enzymatically treating with the above-mentioned enzyme, it is possible to obtain a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles in the manufacturing process even when spray-drying. I found it for the first time. The present inventor states that this is a result of the above-mentioned steps, that the PHB copolymer aqueous suspension contains peptidoglycan derived from cells, and aggregation of PHB copolymer particles can be avoided. Is guessing.
また、本発明者は、上記で得られたＰＨＢ共重合体（ＰＨＢ共重合体粉体（中間体））について、純度を高める観点からさらに検討を進めた結果、ＰＨＢ共重合体（ＰＨＢ共重合体粉体（中間体））を特定の酵素で洗浄することで、嵩比重が高い状態で、純度の高いＰＨＢ共重合体が得られることを初めて見出した（ここで、純度が高いとは、不純物である窒素化合物が少ないことを示す。）。
Further, the present inventor further studied the PHB copolymer (PHB copolymer powder (intermediate)) obtained above from the viewpoint of increasing the purity, and as a result, the PHB copolymer (PHB copolymer weight). For the first time, we have found that a high-purity PHB copolymer can be obtained in a state of high bulk specific gravity by washing the combined powder (intermediate) with a specific enzyme (here, high purity means high purity). It indicates that there are few nitrogen compounds that are impurities.).
したがって、本製造方法によれば、嵩密度が高い、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するＰＨＢ共重合体（例えば、ＰＨＢ共重合体粉体）を得ることができる。また、本製造方法によれば、不純物である窒素化合物が少なく、嵩密度が高い、特定の３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比を有するＰＨＢ共重合体（例えば、ＰＨＢ共重合体粉体）を得ることができる。さらに、本製造方法によれば、物理的破砕（例えば、高圧破砕）工程を用いることなく上記ＰＨＢ共重合体を得ることができ、スケールアップの観点からも有利である。
Therefore, according to this production method, it is possible to obtain a PHB copolymer (for example, PHB copolymer powder) having a high bulk density and a composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit. can. Further, according to this production method, a PHB copolymer having a composition ratio of hydroxyalkanoate units other than a specific 3HB unit / 3HB unit, which has a small amount of nitrogen compound as an impurity and a high bulk density (for example, PHB copolymer weight). Combined powder) can be obtained. Further, according to this production method, the PHB copolymer can be obtained without using a physical crushing (for example, high-pressure crushing) step, which is advantageous from the viewpoint of scale-up.
また、上述したような構成によれば、プラスチックゴミの発生量を低減でき、これにより、例えば、目標１２「持続可能な消費生産形態を確保する」や目標１４「持続可能な開発のために、海・海洋資源を保全し、持続可能な形で利用する」等の持続可能な開発目標（ＳＤＧｓ）の達成に貢献できる。以下、本製造方法の構成について詳説する。
In addition, according to the configuration as described above, the amount of plastic waste generated can be reduced, whereby, for example, Goal 12 “Securing a sustainable consumption production form” and Goal 14 “For sustainable development”. It can contribute to the achievement of Sustainable Development Goals (SDGs) such as "Conserving and using marine and marine resources in a sustainable manner." Hereinafter, the configuration of this manufacturing method will be described in detail.
〔２．ＰＨＢ共重合体の製造方法〕
本製造方法は、下記の工程（ａ）～工程（ｄ）を必須の工程として含む方法である。また、本製造方法におけるＰＨＢ共重合体は、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）である。
・工程（ａ）：ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程
・工程（ｂ）：前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程
・工程（ｃ）：前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程
・工程（ｄ）：前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程
本製造方法における工程（ｃ）では、工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する。当該水性懸濁液において、ＰＨＢ共重合体は水性媒体中に分散した状態で存在している。本明細書では、少なくともＰＨＢ共重合体を含む水性懸濁液を、「ＰＨＢ共重合体水性懸濁液」と略して表記する場合がある。 [2. Method for producing PHB copolymer]
This manufacturing method is a method including the following steps (a) to (d) as essential steps. Further, the PHB copolymer in this production method has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol).
-Step (a): A step of adding an alkaline proteolytic enzyme to a culture solution containing a bacterial cell containing a PHB copolymer to enzymatically treat the bacterial cell-Step (b): In the above-mentioned step (a). A step of adding an alkaline aqueous solution to the obtained culture solution to adjust the pH to 10.0 to 12.0, and adding a surfactant either before the adjustment, at the same time as the adjustment, or after the adjustment. Step (c): From the aqueous suspension obtained in the step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less. Step of preparing turbid liquid-Step (d): Step of spray-drying the aqueous suspension prepared in the above-mentioned step (c) In the step (c) in the present production method, the aqueous suspension obtained in the step (b). From the liquid, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less is prepared. In the aqueous suspension, the PHB copolymer exists in a dispersed state in the aqueous medium. In the present specification, an aqueous suspension containing at least a PHB copolymer may be abbreviated as "PHB copolymer aqueous suspension".
本製造方法は、下記の工程（ａ）～工程（ｄ）を必須の工程として含む方法である。また、本製造方法におけるＰＨＢ共重合体は、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）である。
・工程（ａ）：ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程
・工程（ｂ）：前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程
・工程（ｃ）：前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程
・工程（ｄ）：前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程
本製造方法における工程（ｃ）では、工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する。当該水性懸濁液において、ＰＨＢ共重合体は水性媒体中に分散した状態で存在している。本明細書では、少なくともＰＨＢ共重合体を含む水性懸濁液を、「ＰＨＢ共重合体水性懸濁液」と略して表記する場合がある。 [2. Method for producing PHB copolymer]
This manufacturing method is a method including the following steps (a) to (d) as essential steps. Further, the PHB copolymer in this production method has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol).
-Step (a): A step of adding an alkaline proteolytic enzyme to a culture solution containing a bacterial cell containing a PHB copolymer to enzymatically treat the bacterial cell-Step (b): In the above-mentioned step (a). A step of adding an alkaline aqueous solution to the obtained culture solution to adjust the pH to 10.0 to 12.0, and adding a surfactant either before the adjustment, at the same time as the adjustment, or after the adjustment. Step (c): From the aqueous suspension obtained in the step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less. Step of preparing turbid liquid-Step (d): Step of spray-drying the aqueous suspension prepared in the above-mentioned step (c) In the step (c) in the present production method, the aqueous suspension obtained in the step (b). From the liquid, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less is prepared. In the aqueous suspension, the PHB copolymer exists in a dispersed state in the aqueous medium. In the present specification, an aqueous suspension containing at least a PHB copolymer may be abbreviated as "PHB copolymer aqueous suspension".
＜工程（ａ）＞
工程（ａ）は、ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程である。 <Step (a)>
Step (a) is a step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing a PHB copolymer to enzymatically treat the cells.
工程（ａ）は、ＰＨＢ共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程である。 <Step (a)>
Step (a) is a step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing a PHB copolymer to enzymatically treat the cells.
（ＰＨＢ共重合体）
本製造方法におけるＰＨＢ共重合体は、３ＨＢと３ＨＢ以外のヒドロキシアルカノエートとの共重合体である。 (PHB copolymer)
The PHB copolymer in this production method is a copolymer of 3HB and a hydroxy alkanoate other than 3HB.
本製造方法におけるＰＨＢ共重合体は、３ＨＢと３ＨＢ以外のヒドロキシアルカノエートとの共重合体である。 (PHB copolymer)
The PHB copolymer in this production method is a copolymer of 3HB and a hydroxy alkanoate other than 3HB.
３ＨＢ以外のヒドロキシアルカノエートとしては、例えば、３－ヒドロキシヘキサノエート）（３ＨＨ）、３－ヒドロキシバリレート（３ＨＶ）、４－ヒドロキシブチレート（４ＨＢ）、３－ヒドロキシオクタノエート（３ＨＯ）、３－ヒドロキシオクタデカノエート（３ＨＯＤ）、３－ヒドロキシデカノエート（３ＨＤ）等が挙げられる。
Examples of hydroxyalkanoates other than 3HB include 3-hydroxyhexanoate (3HH), 3-hydroxyvariate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), and the like. Examples thereof include 3-hydroxyoctadecanoate (3HOD) and 3-hydroxydecanoate (3HD).
ＰＨＢ共重合体として、例えば、ＰＨＢＨを好ましく例示し得るが、これに限定されない。以下では、説明の便宜上、主として、ＰＨＢＨを代表例として説明する。
As the PHB copolymer, for example, PHBH can be preferably exemplified, but is not limited thereto. Hereinafter, for convenience of explanation, PHBH will be mainly described as a representative example.
ＰＨＢＨは、３ＨＢと３ＨＨとの繰り返し単位の組成比を変えることで、融点、結晶化度を変化させ、結果として、ヤング率、耐熱性等の物性を変化させることができ、ポリプロピレンとポリエチレンとの間の物性を付与することが可能である。
PHBH can change the melting point and crystallinity by changing the composition ratio of the repeating unit of 3HB and 3HH, and as a result, the physical properties such as Young's modulus and heat resistance can be changed. It is possible to impart physical characteristics between them.
本製造方法におけるＰＨＢ共重合体は、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、８１／１９～８７／１３（ｍｏ１／ｍｏ１）であることが好ましく、８２／１８～８６／１４（ｍｏ１／ｍｏ１）であることがより好ましい。３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が、８８／１２（ｍｏｌ／ｍｏｌ）以下であると、十分な硬度が得られ、８０／２０（ｍｏｌ／ｍｏｌ）以上であると、十分な柔軟性が得られる。
The PHB copolymer in this production method has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol) and 81/19 to 87/13 (mo1 /). It is preferably mo1), and more preferably 82/18 to 86/14 (mo1 / mo1). Sufficient hardness can be obtained when the composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units is 88/12 (mol / mol) or less, and sufficient when it is 80/20 (mol / mol) or more. Flexibility is obtained.
本発明の一実施形態において、ＰＨＢ共重合体の重量平均分子量（以下、「Ｍｗ」と称する場合がある。）は、特に限定されないが、１５万～８０万が好ましく、２０万～７０万がより好ましく、２５万～６０万がさらに好ましい。重量平均分子量が１５万以上であると、十分な機械物性等が得られ、８０万以下であると、十分な結晶化速度が得られ、良好な成形加工性が達成される。Ｐ３ＨＢ系樹脂の重量平均分子量は、ゲル浸透クロマトグラフィー（ＧＰＣ）（昭和電工製「Ｓｈｏｄｅｘ ＧＰＣ－１０１」）によって、カラムにポリスチレンゲル（昭和電工製「Ｓｈｏｄｅｘ Ｋ－８０４」）を用い、クロロホルムを移動相とし、ポリスチレン換算した場合の分子量として求めることができる。
In one embodiment of the present invention, the weight average molecular weight of the PHB copolymer (hereinafter, may be referred to as “Mw”) is not particularly limited, but is preferably 150,000 to 800,000, preferably 200,000 to 700,000. More preferably, 250,000 to 600,000 is even more preferable. When the weight average molecular weight is 150,000 or more, sufficient mechanical characteristics and the like can be obtained, and when it is 800,000 or less, a sufficient crystallization rate can be obtained and good molding processability can be achieved. For the weight average molecular weight of the P3HB resin, gel permeation chromatography (GPC) (Showa Denko's "Shodex GPC-101") is used, and polystyrene gel (Showa Denko's "Shodex K-804") is used for the column to transfer chloroform. It can be obtained as a phase and as a molecular weight when converted to polystyrene.
（菌体）
工程（ａ）で用いられる菌体は、細胞内にＰＨＢ共重合体を生成し得る微生物である限り、特に限定されない。例えば、天然から単離された微生物および菌株の寄託機関（例えば、ＩＦＯ、ＡＴＣＣ等）に寄託されている微生物、またはそれらから調製し得る変異体および形質転換体等を使用できる。例えば、ＰＨＢ共重合体の一例であるＰ３ＨＢを生成する菌体としては、１９２５年に発見されたＢａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍが最初で、他にもカプリアビダス・ネカトール（Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ）（旧分類：アルカリゲネス・ユートロファス（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）、ラルストニア・ユートロフア（Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ））、アルカリゲネス・ラタス（Ａｌｃａｌｉｇｅｎｅｓ ｌａｔｕｓ）等の天然微生物が挙げられる。これらの微生物ではＰＨＢ共重合体が菌体内に蓄積されることが知られている。 (Bacterial cells)
The bacterial cells used in the step (a) are not particularly limited as long as they are microorganisms capable of producing a PHB copolymer in the cells. For example, microorganisms isolated from nature and microorganisms deposited in a depositary institution of strains (eg, IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used. For example, the first bacterial cell that produces P3HB, which is an example of a PHB copolymer, is Cupriavidus necator (former classification: Alcaligenes), which was discovered in 1925. Natural microorganisms such as europhos), Ralstonia europha), and Alcaligenes latus can be mentioned. It is known that PHB copolymers are accumulated in the cells of these microorganisms.
工程（ａ）で用いられる菌体は、細胞内にＰＨＢ共重合体を生成し得る微生物である限り、特に限定されない。例えば、天然から単離された微生物および菌株の寄託機関（例えば、ＩＦＯ、ＡＴＣＣ等）に寄託されている微生物、またはそれらから調製し得る変異体および形質転換体等を使用できる。例えば、ＰＨＢ共重合体の一例であるＰ３ＨＢを生成する菌体としては、１９２５年に発見されたＢａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍが最初で、他にもカプリアビダス・ネカトール（Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ）（旧分類：アルカリゲネス・ユートロファス（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）、ラルストニア・ユートロフア（Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ））、アルカリゲネス・ラタス（Ａｌｃａｌｉｇｅｎｅｓ ｌａｔｕｓ）等の天然微生物が挙げられる。これらの微生物ではＰＨＢ共重合体が菌体内に蓄積されることが知られている。 (Bacterial cells)
The bacterial cells used in the step (a) are not particularly limited as long as they are microorganisms capable of producing a PHB copolymer in the cells. For example, microorganisms isolated from nature and microorganisms deposited in a depositary institution of strains (eg, IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used. For example, the first bacterial cell that produces P3HB, which is an example of a PHB copolymer, is Cupriavidus necator (former classification: Alcaligenes), which was discovered in 1925. Natural microorganisms such as europhos), Ralstonia europha), and Alcaligenes latus can be mentioned. It is known that PHB copolymers are accumulated in the cells of these microorganisms.
また、ＰＨＢ共重合体の一例である、ヒドロキシブチレートとその他のヒドロキシアルカノエートとの共重合体を生成する菌体としては、Ｐ３ＨＢ３ＨＶおよびＰ３ＨＢ３ＨＨ生産菌であるアエロモナス・キヤビエ（Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ）、Ｐ３ＨＢ４ＨＢ生産菌であるアルカリゲネス・ユートロファス（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）等が挙げられる。特に、Ｐ３ＨＢ３ＨＨに関し、Ｐ３ＨＢ３ＨＨの生産性を上げるために、ＰＨＢ共重合体合成酵素群の遺伝子を導入したアルカリゲネス・ユートロファス ＡＣ３２株（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ ＡＣ３２， ＦＥＲＭ ＢＰ－６０３８）（Ｔ．Ｆｕｋｕｉ，Ｙ．Ｄｏｉ，Ｊ．Ｂａｔｅｒｉｏｌ．，１７９，ｐ４８２１－４８３０（１９９７））等がより好ましい。また、菌体は、上記以外にも、生産したいＰＨＢ共重合体に合わせて、各種ＰＨＢ共重合体合成関連遺伝子を導入した遺伝子組換え微生物であっても良い。
In addition, examples of the cells that produce a copolymer of hydroxybutyrate and other hydroxyalkanoates, which are examples of PHB copolymers, include Aeromonas caviae, which is a P3HB3HV and P3HB3HH-producing bacterium, and P3HB4HB production. Examples thereof include Alcaligenes europhos, which is a fungus. In particular, regarding P3HB3HH, in order to increase the productivity of P3HB3HH, Alcaligenes utrophas AC32 strain (Alcaligenes europhos AC32, FERM BP-6038) (T. Fukui, Y. Doi,) into which a gene of the PHB copolymer synthase group was introduced was introduced. J. Polymer., 179, p4821-4830 (1997)) and the like are more preferable. In addition to the above, the bacterial cell may be a recombinant microorganism into which various PHB copolymer synthesis-related genes have been introduced according to the PHB copolymer to be produced.
また、ＰＨＢ共重合体は、例えば、国際公開第２０１０／０１３４８３号公報に記載された方法によっても製造され得る。
The PHB copolymer can also be produced, for example, by the method described in International Publication No. 2010/0134883.
（アルカリ性タンパク質分解酵素）
本明細書において、「アルカリ性タンパク質分解酵素」とは、アルカリ環境下（例えばｐＨ８．５の溶液中）でタンパク質を分解する活性を有するタンパク質分解酵素を意図する。 (Alkaline proteolytic enzyme)
As used herein, the term "alkaline proteolytic enzyme" is intended to be a proteolytic enzyme having an activity of degrading a protein in an alkaline environment (for example, in a solution having a pH of 8.5).
本明細書において、「アルカリ性タンパク質分解酵素」とは、アルカリ環境下（例えばｐＨ８．５の溶液中）でタンパク質を分解する活性を有するタンパク質分解酵素を意図する。 (Alkaline proteolytic enzyme)
As used herein, the term "alkaline proteolytic enzyme" is intended to be a proteolytic enzyme having an activity of degrading a protein in an alkaline environment (for example, in a solution having a pH of 8.5).
本発明の一実施形態において、アルカリ性タンパク質分解酵素は、アルカリ環境下でタンパク質を分解する活性を有する限り特に限定されず、例えば、セリン特異的タンパク質分解酵素（例えば、サブチリシン、キモトリプシン）、システイン特異的タンパク質分解酵素（例えば、パパイン、ブロメライン）等が挙げられる。汎用性・経済性の観点から、セリン特異的タンパク質分解酵素、とりわけ、サブチリシンを含むアルカラーゼが好ましい。これらの１種類を単独で使用してもよく、２種類以上を組み合わせて使用してもよい。
In one embodiment of the invention, the alkaline proteolytic enzyme is not particularly limited as long as it has an activity of degrading proteins in an alkaline environment, and is, for example, a serine-specific proteolytic enzyme (eg, subtilisin, chymotrypsin), cysteine-specific. Proteolytic enzymes (eg, papaine, bromeline) and the like can be mentioned. From the viewpoint of versatility and economy, serine-specific proteolytic enzymes, particularly alcalases containing subtilisin, are preferable. One of these types may be used alone, or two or more types may be used in combination.
アルカリ性タンパク質分解酵素としては、市販品を用いることもでき、例えば、Ｎｏｖｏｚｙｍｅ社製「アルカラーゼ」および「エスペラーゼ」；天野エンザイム株式会社社製「プロチンＳＤ－ＡＹ１０」および「プロテアーゼＰ「アマノ」３ＳＤ」；ダニスコジャパン株式会社製「マルチフェクトＰＲ６Ｌ」および「オプチマーゼＰＲ８９Ｌ」；新日本化学工業株式会社製「スミチームＭＰ」；ディー・エス・エムジャパン株式会社製「デルボラーゼ」；ナガセケムテックス株式会社製「ビオプラーゼＯＰ」、「ビオプラーゼＳＰ－２０ＦＧ」および「ビオプラーゼＳＰ－４ＦＧ」；ＨＢＩ株式会社製「オリエンターゼ２２ＢＦ」；ヤクルト薬品工業株式会社製「アロアーゼＸＡ－１０」等が挙げられる。
Commercially available products can also be used as the alkaline proteolytic enzyme, for example, "Alkalase" and "Esperase" manufactured by Novozyme; "Protin SD-AY10" and "Protease P" Amano "3SD" manufactured by Amano Enzyme Co., Ltd .; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd .; "Sumiteam MP" manufactured by Shin Nihon Kagaku Kogyo Co., Ltd .; "Delbolase" manufactured by DSM Japan Co., Ltd .; , "Bioplase SP-20FG" and "Bioplase SP-4FG"; "Orientase 22BF" manufactured by HBI Co., Ltd .; "Aloase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd. and the like.
工程（ａ）において、アルカリ性タンパク質分解酵素による菌体の酵素処理を行う際には、使用するアルカリ性タンパク質分解酵素の至適ｐＨおよび至適温度に合わせて前記培養液のｐＨおよび温度を調整することが好ましい。また、工程（ａ）におけるｐＨは、工程（ｂ）でアルカリ水溶液の添加により調整されるｐＨよりも低いｐＨであることが好ましい。前記培養液のｐＨおよび温度の調整方法は特に限定されず、公知の方法を用いることができる。
In step (a), when enzymatically treating cells with an alkaline proteolytic enzyme, the pH and temperature of the culture solution should be adjusted according to the optimum pH and temperature of the alkaline proteolytic enzyme to be used. Is preferable. Further, the pH in the step (a) is preferably lower than the pH adjusted by the addition of the alkaline aqueous solution in the step (b). The method for adjusting the pH and temperature of the culture solution is not particularly limited, and known methods can be used.
本発明の一実施形態において、アルカリ性タンパク質分解酵素の至適ｐＨは、当該アルカリ性タンパク質分解酵素がアルカリ環境下で活性を有する限り特に限定されないが、例えば８．０～１２．０であり、好ましくは８．０～１１．０であり、より好ましくは８．０～１０．０であり、さらに好ましくは８．０～９．０であり、最も好ましくは８．５である。
In one embodiment of the present invention, the optimum pH of the alkaline proteolytic enzyme is not particularly limited as long as the alkaline proteolytic enzyme has activity in an alkaline environment, but is, for example, 8.0 to 12.0, preferably 8.0 to 12.0. It is 8.0 to 11.0, more preferably 8.0 to 10.0, still more preferably 8.0 to 9.0, and most preferably 8.5.
本発明の一実施形態において、アルカリ性タンパク質分解酵素の至適温度は、特に限定されないが、過度の加温を必要とせず、ＰＨＢ共重合体の熱変化（熱分解）を防ぐことができるとの観点から、７０℃以下が好ましく、６０℃以下がさらに好ましい。至適温度の下限は、特に限定されないが、過度の冷却操作が必要なく、経済的であるとの観点から、室温（例えば、２５℃）以上であることが好ましい。
In one embodiment of the present invention, the optimum temperature of the alkaline proteolytic enzyme is not particularly limited, but it does not require excessive heating and can prevent thermal changes (thermal decomposition) of the PHB copolymer. From the viewpoint, 70 ° C. or lower is preferable, and 60 ° C. or lower is more preferable. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (for example, 25 ° C.) or higher from the viewpoint of not requiring an excessive cooling operation and being economical.
本発明の一実施形態において、アルカリ性タンパク質分解酵素の添加量は、特に限定されないが、例えば、０．０５～１．０ｐｈｒであり、０．１～０．５ｐｈｒが好ましく、０．１５～０．３ｐｈｒがより好ましい。アルカリ性タンパク質分解酵素の添加量が、上記の範囲であれば、適度に菌体を分解することができる。
In one embodiment of the present invention, the amount of the alkaline proteolytic enzyme added is not particularly limited, but is, for example, 0.05 to 1.0 phr, preferably 0.1 to 0.5 phr, and 0.15 to 0. 3 phr is more preferable. If the amount of the alkaline proteolytic enzyme added is within the above range, the cells can be appropriately decomposed.
工程（ａ）は、アルカリ性タンパク質分解酵素と同時に、溶菌酵素を実質的に添加しないことが好ましい。本明細書において、「溶菌酵素」とは、菌体の細胞壁（例えば、ペプチドグリカン）を分解する（溶菌する）活性を有する酵素を意図する。また、本明細書において、「溶菌酵素を実質的に添加しない」とは、溶菌酵素を０．０００５ｐｈｒ以下で添加することを意図し、０ｐｈｒであってもよい。溶菌酵素としては、上記の定義の範囲に含まれるものであれば特に限定されないが、例えば、リゾチーム、ラビアー、β－Ｎ－アセチルグルコサミニダーゼ、エンドリシン、オートリシン等が挙げられる。工程（ａ）において、溶菌酵素を実質的に添加しないことにより、ＰＨＢ共重合体水性懸濁液中にペプチドグリカンが残存することとなり、本発明の効果を奏することができる。
In step (a), it is preferable that the lytic enzyme is not substantially added at the same time as the alkaline proteolytic enzyme. As used herein, the term "lytic enzyme" is intended to be an enzyme having an activity of degrading (bacteriolytic) the cell wall (for example, peptidoglycan) of a bacterial cell. Further, in the present specification, "substantially no lytic enzyme is added" means that the lytic enzyme is added at 0.0005 phr or less, and may be 0 phr. The lytic enzyme is not particularly limited as long as it is included in the above definition, and examples thereof include lysozyme, rabbia, β-N-acetylglucosaminidase, endolysine, and autolysine. By substantially not adding the lytic enzyme in the step (a), peptidoglycan remains in the aqueous suspension of the PHB copolymer, and the effect of the present invention can be exhibited.
工程（ａ）において、ＰＨＢ共重合体を含有する菌体は、不活化されていることが好ましい。不活化の方法は特に限定されないが、例えば、実施例に記載するように、ＰＨＢ共重合体を含有する菌体を含む培養液を、６０～７０℃で７時間、加熱および攪拌処理する方法が挙げられる。加熱および攪拌処理後の培養液は、さらに、工程（ａ）に適した温度まで、冷却されることが好ましい。
In step (a), the cells containing the PHB copolymer are preferably inactivated. The method of inactivation is not particularly limited, but for example, as described in Examples, a method of heating and stirring a culture solution containing cells containing a PHB copolymer at 60 to 70 ° C. for 7 hours is used. Can be mentioned. It is preferable that the culture broth after the heating and stirring treatment is further cooled to a temperature suitable for the step (a).
＜工程（ｂ）＞
工程（ｂ）は、前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程である。 <Step (b)>
In the step (b), an alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and the pH is adjusted before, at the same time as the adjustment, or after the adjustment. This is a step of adding a surfactant in any of the above.
工程（ｂ）は、前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程である。 <Step (b)>
In the step (b), an alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and the pH is adjusted before, at the same time as the adjustment, or after the adjustment. This is a step of adding a surfactant in any of the above.
工程（ｂ）は、下記の工程（ｂ１）および工程（ｂ２）を含む。
・工程（ｂ１）：前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整する工程
・工程（ｂ２）：界面活性剤を添加する工程
（工程（ｂ１））
工程（ｂ１）は、上述の通り、前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整する工程である。当該工程によれば、菌体由来の不純物（核酸、タンパク質等）を分散および溶解することで、高純度のＰＨＢ共重合体を菌体から分離することができる。 The step (b) includes the following steps (b1) and step (b2).
-Step (b1): A step of adding an alkaline aqueous solution to the culture solution obtained in the above-mentioned step (a) to adjust the pH to 10.0 to 12.0-Step (b2): A step of adding a surfactant. (Step (b1))
As described above, the step (b1) is a step of adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0. According to this step, a high-purity PHB copolymer can be separated from the cells by dispersing and dissolving impurities (nucleic acid, protein, etc.) derived from the cells.
・工程（ｂ１）：前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整する工程
・工程（ｂ２）：界面活性剤を添加する工程
（工程（ｂ１））
工程（ｂ１）は、上述の通り、前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整する工程である。当該工程によれば、菌体由来の不純物（核酸、タンパク質等）を分散および溶解することで、高純度のＰＨＢ共重合体を菌体から分離することができる。 The step (b) includes the following steps (b1) and step (b2).
-Step (b1): A step of adding an alkaline aqueous solution to the culture solution obtained in the above-mentioned step (a) to adjust the pH to 10.0 to 12.0-Step (b2): A step of adding a surfactant. (Step (b1))
As described above, the step (b1) is a step of adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0. According to this step, a high-purity PHB copolymer can be separated from the cells by dispersing and dissolving impurities (nucleic acid, protein, etc.) derived from the cells.
本発明の一実施形態において、アルカリ水溶液は、塩基性化合物を含む水溶液である。アルカリ水溶液に含まれる塩基性化合物としては、特に限定されないが、例えば、水酸化ナトリウム、水酸化カリウム等のアルカリ金属またはアルカリ土類金属の水酸化物；炭酸ナトリウム、炭酸カリウム等の金属炭酸塩；リン酸ナトリウム、リン酸カリウム、リン酸水素ナトリウム、リン酸水素カリウム等の金属リン酸塩または金属リン酸水素塩等が挙げられる。
In one embodiment of the present invention, the alkaline aqueous solution is an aqueous solution containing a basic compound. The basic compound contained in the alkaline aqueous solution is not particularly limited, and is, for example, an alkali metal such as sodium hydroxide or potassium hydroxide or an alkali earth metal hydroxide; a metal carbonate such as sodium carbonate or potassium carbonate; Examples thereof include metal phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate and the like, or metal hydrogen phosphate salts.
本発明の一実施形態において、アルカリ水溶液に含まれる塩基性化合物は、アルカリ金属水酸化物またはアルカリ土類金属水酸化物が好ましく、水酸化ナトリウムがより好ましい。塩基性化合物は、１種を単独で使用してもよく、２種以上を併用してもよい。
In one embodiment of the present invention, the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and more preferably sodium hydroxide. As the basic compound, one kind may be used alone, or two or more kinds may be used in combination.
工程（ｂ１）において、アルカリ水溶液を添加することにより、ｐＨを１０．０～１２．０に調整することが好ましく、ｐＨを１０．２～１１．８に調整することがより好ましく、１０．４～１１．６に調整することがさらに好ましい。ｐＨを１０．０以上に調整することで、菌体成分の分解および溶解ができるという利点を有する。また、ｐＨを１２．０以下に調整することで、意図しない菌体の損傷を防ぐことができる。
In step (b1), the pH is preferably adjusted to 10.0 to 12.0, more preferably 10.2 to 11.8 by adding an alkaline aqueous solution. It is more preferable to adjust to ~ 11.6. Adjusting the pH to 10.0 or higher has the advantage that the bacterial cell components can be decomposed and dissolved. Further, by adjusting the pH to 12.0 or less, unintended damage to the bacterial cells can be prevented.
工程（ｂ１）における温度は、１００℃未満であることが好ましく、８０℃未満であることがより好ましい。下限は特に限定されないが、例えば、４０℃以上であることが好ましい。
The temperature in the step (b1) is preferably less than 100 ° C, more preferably less than 80 ° C. The lower limit is not particularly limited, but is preferably 40 ° C. or higher, for example.
（工程（ｂ２））
工程（ｂ２）は、前記工程（ａ）で得られた培養液に界面活性剤を添加する工程である。当該工程によれば、特に細胞膜を効率的に処理することができ、前記菌体由来の不純物をより多く除去できるため、より高純度のＰＨＢ共重合体を菌体から分離することができる。 (Step (b2))
The step (b2) is a step of adding a surfactant to the culture solution obtained in the step (a). According to this step, the cell membrane can be treated particularly efficiently, and more impurities derived from the cells can be removed, so that a higher purity PHB copolymer can be separated from the cells.
工程（ｂ２）は、前記工程（ａ）で得られた培養液に界面活性剤を添加する工程である。当該工程によれば、特に細胞膜を効率的に処理することができ、前記菌体由来の不純物をより多く除去できるため、より高純度のＰＨＢ共重合体を菌体から分離することができる。 (Step (b2))
The step (b2) is a step of adding a surfactant to the culture solution obtained in the step (a). According to this step, the cell membrane can be treated particularly efficiently, and more impurities derived from the cells can be removed, so that a higher purity PHB copolymer can be separated from the cells.
本発明の一実施形態において、界面活性剤としては、特に限定されないが、例えば、陰イオン界面活性剤、陽イオン界面活性剤、両性界面活性剤、非イオン界面活性剤等が挙げられる。このうち、細胞膜の除去能力が高いとの観点から、陰イオン界面活性が好ましい。これらの１種類を単独で使用してもよく、２種類以上を組み合わせて使用してもよい。
In one embodiment of the present invention, the surfactant is not particularly limited, and examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Of these, anionic surface activity is preferable from the viewpoint of high ability to remove cell membranes. One of these types may be used alone, or two or more types may be used in combination.
陰イオン界面活性剤としては、例えば、アルキル硫酸塩、アルキルベンゼンスルホン酸塩、アルキル硫酸エステル塩、アルケニル硫酸エステル塩、アルキルエーテル硫酸エステル塩、アルケニルエーテル硫酸エステル塩、α－オレフィンスルホン酸塩、α－スルホ脂肪酸塩、α－スルホ脂肪酸塩のエステル、アルキルエーテルカルボン酸塩、アルケニルエーテルカルボン酸塩、アミノ酸型界面活性剤、Ｎ－アシルアミノ酸型界面活性剤等が挙げられる。この中でも、アルキル硫酸エステル塩が好ましく、細胞膜の除去能力が高く、安価であるとの観点から、ドデシル硫酸ナトリウム（ＳＤＳ）が特に好ましい。これらの１種類を単独で使用してもよく、２種類以上を組み合わせて使用してもよい。
Examples of the anionic surfactant include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfates, alkenyl sulfates, alkyl ether sulfates, alkenyl ether sulfates, α-olefin sulfonates, and α-. Examples thereof include a sulfo fatty acid salt, an ester of an α-sulfo fatty acid salt, an alkyl ether carboxylate, an alkenyl ether carboxylate, an amino acid type surfactant, and an N-acyl amino acid type surfactant. Among these, an alkyl sulfate ester salt is preferable, and sodium dodecyl sulfate (SDS) is particularly preferable from the viewpoint of high ability to remove cell membranes and low cost. One of these types may be used alone, or two or more types may be used in combination.
工程（ｂ２）において、添加する界面活性剤の量は特に限定されず、前記培養液に対して、例えば、０．１～５．０重量％であり、０．３～２．５重量％が好ましい。
In the step (b2), the amount of the surfactant to be added is not particularly limited, and is, for example, 0.1 to 5.0% by weight and 0.3 to 2.5% by weight based on the culture solution. preferable.
工程（ｂ２）は、工程（ｂ１）の前に行ってもよいし、同時に行ってもよいし、後に行ってもよい。好ましくは、工程（ｂ２）は、工程（ｂ１）の後に行われる。
The step (b2) may be performed before the step (b1), at the same time, or after the step (b1). Preferably, step (b2) is performed after step (b1).
＜工程（ｂ’）＞
本発明の一実施形態において、本製造方法は、工程（ｂ’）をさらに含み得る。 <Process (b')>
In one embodiment of the invention, the manufacturing method may further comprise step (b').
本発明の一実施形態において、本製造方法は、工程（ｂ’）をさらに含み得る。 <Process (b')>
In one embodiment of the invention, the manufacturing method may further comprise step (b').
工程（ｂ’）は、前記工程（ｂ）において得られた培養液を遠心分離し、上清を除去して、ＰＨＢ共重合体が濃縮されたＰＨＢ共重合体水性懸濁液を得る工程である。すなわち、菌体から分離したＰＨＢ共重合体から不純物を除去し、濃縮および精製する工程である。
The step (b') is a step of centrifuging the culture solution obtained in the step (b) and removing the supernatant to obtain a PHB copolymer aqueous suspension in which the PHB copolymer is concentrated. be. That is, it is a step of removing impurities from the PHB copolymer separated from the bacterial cells, and concentrating and purifying the mixture.
工程（ｂ’）において、前記培養液を遠心分離する方法は特に限定されず、公知の方法を用いることができる。
In the step (b'), the method for centrifuging the culture solution is not particularly limited, and a known method can be used.
工程（ｂ’）において、前記培養液を遠心分離し、上清を除去した後、沈降物に溶液を添加し、再度遠心分離および上清を除去する工程を繰り返し行うことが好ましい。この操作により、より濃縮および精製されたＰＨＢ共重合体水性懸濁液を得ることができる。ここで、上清を除去した後に添加する溶液は、前記培養液と同じｐＨに調整されたアルカリ水溶液であることが好ましい。
In the step (b'), it is preferable to repeat the steps of centrifuging the culture solution, removing the supernatant, adding the solution to the sediment, centrifuging again, and removing the supernatant. By this operation, a more concentrated and purified PHB copolymer aqueous suspension can be obtained. Here, the solution to be added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the culture solution.
工程（ｂ’）により、最終製品に残留する不純物量が概ね決定されるため、これらの不純物は、できる限り低減させた方が好ましい。当然に、用途によっては、最終製品の物性を損なわない限り不純物が混入しても構わないが、医療用用途等、高純度のＰＨＢ共重合体が必要とされる場合は、できる限り不純物を低減させることが好ましい。その際の精製度の指標としては、例えば、ＰＨＢ共重合体水性懸濁液中のタンパク質量が挙げられる。ＰＨＢ共重合体水性懸濁液中のタンパク質量は、後述するＰＨＢ共重合体粉体の残タンパク質量を達成できる量であれば、特に限定されない。当該タンパク質量は、好ましくは、ＰＨＢ共重合体水性懸濁液中のＰＨＢ共重合体重量当たり１００００ｐｐｍ以下、より好ましくは、５０００ｐｐｍ以下、さらに好ましくは、３０００ｐｐｍ以下である。
Since the amount of impurities remaining in the final product is generally determined by the step (b'), it is preferable to reduce these impurities as much as possible. Naturally, depending on the application, impurities may be mixed as long as the physical properties of the final product are not impaired, but if a high-purity PHB copolymer is required for medical applications, etc., the impurities should be reduced as much as possible. It is preferable to let them. As an index of the degree of purification at that time, for example, the amount of protein in the aqueous suspension of the PHB copolymer can be mentioned. The amount of protein in the aqueous suspension of PHB copolymer is not particularly limited as long as the amount of residual protein in the PHB copolymer powder described later can be achieved. The amount of the protein is preferably 10000 ppm or less, more preferably 5000 ppm or less, still more preferably 3000 ppm or less, based on the weight of the PHB copolymer in the aqueous suspension of the PHB copolymer.
工程（ｂ’）において、ＰＨＢ共重合体水性懸濁液を構成する溶媒（「溶媒」は、「水性媒体」とも称する。）は、特に限定されず、水、または水と有機溶媒との混合溶媒であってもよい。また、当該混合溶媒において、有機溶媒の濃度は、使用する有機溶媒の水への溶解度以下であれば特に限定されない。また、有機溶媒は特に限定されないが、例えば、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、ｉｓｏ－ブタノール、ペンタノール、ヘキサノール、ヘプタノール等のアルコール類；アセトン、メチルエチルケトン等のケトン類；テトラヒドロフラン、ジオキサン等のエーテル類；アセトニトリル、プロピオニトリル等のニトリル類；ジメチルホルムアミド、アセトアミド等のアミド類；ジメチルスルホキシド、ピリジン、ピペリジン等が挙げられる。中でも、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、ｉｓｏ－ブタノール、アセトン、メチルエチルケトン、テトラヒドロフラン、ジオキサン、アセトニトリル、プロピオニトリル等が、除去しやすい点から好ましい。また、メタノール、エタノール、１－プロパノール、２－プロパノール、ブタノール、アセトン等が、入手容易であることからより好ましい。さらに、メタノール、エタノール、アセトンが、特に好ましい。
In the step (b'), the solvent constituting the aqueous suspension of the PHB copolymer (the "solvent" is also referred to as "aqueous medium") is not particularly limited, and water or a mixture of water and an organic solvent is not particularly limited. It may be a solvent. Further, in the mixed solvent, the concentration of the organic solvent is not particularly limited as long as it is equal to or less than the solubility of the organic solvent used in water. The organic solvent is not particularly limited, and for example, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol; acetone and methyl ethyl ketone. Ketones such as; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethylsulfoxide, pyridine, piperidine and the like. Of these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile and the like are preferable because they are easy to remove. Further, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone and the like are more preferable because they are easily available. Further, methanol, ethanol and acetone are particularly preferable.
ＰＨＢ共重合体水性懸濁液を構成する水性媒体中の水の含有量は、５重量％以上が好ましく、より好ましくは、１０重量％以上であり、さらに好ましくは、３０重量％以上であり、特に好ましくは、５０重量％以上である。
The content of water in the aqueous medium constituting the aqueous suspension of the PHB copolymer is preferably 5% by weight or more, more preferably 10% by weight or more, still more preferably 30% by weight or more. Particularly preferably, it is 50% by weight or more.
なお、工程（ｂ’）におけるＰＨＢ共重合体水性懸濁液体は、本発明の本質を損なわない限り、他の溶媒、菌体由来の成分、精製時に発生する化合物等を含んでいても構わない。
The PHB copolymer aqueous suspension in step (b') may contain other solvents, bacterial cell-derived components, compounds generated during purification, etc., as long as the essence of the present invention is not impaired. ..
＜工程（ｃ）＞
本製造方法における工程（ｃ）では、工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する。 <Step (c)>
In the step (c) of the present production method, the pH is 7.0 or less, the shear viscosity is 0.005 Pa · s or more, and 0.5 Pa · s from the aqueous suspension obtained in the step (b). Prepare the following aqueous suspension.
本製造方法における工程（ｃ）では、工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する。 <Step (c)>
In the step (c) of the present production method, the pH is 7.0 or less, the shear viscosity is 0.005 Pa · s or more, and 0.5 Pa · s from the aqueous suspension obtained in the step (b). Prepare the following aqueous suspension.
本製造方法の工程（ｃ）に付される前のＰＨＢ共重合体水性懸濁液は、通常、工程（ｂ）を経ることにより、７．０を超えるｐＨを有する。そこで、本製造方法の工程（ｃ）により、上記ＰＨＢ共重合体水性懸濁液のｐＨを７．０以下に調整する。その調整方法は、特に限定されず、例えば、酸を添加する方法等が挙げられる。酸は、特に限定されず、有機酸、無機酸のいずれでもよく、揮発性の有無は問わない。より具体的には、酸としては、例えば、硫酸、塩酸、リン酸、酢酸等が使用できる。
The PHB copolymer aqueous suspension before being subjected to the step (c) of the present production method usually has a pH exceeding 7.0 by going through the step (b). Therefore, the pH of the aqueous suspension of the PHB copolymer is adjusted to 7.0 or less by the step (c) of the present production method. The adjustment method is not particularly limited, and examples thereof include a method of adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid, and may or may not be volatile. More specifically, as the acid, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid and the like can be used.
上記調整工程において調整するＰＨＢ共重合体水性懸濁液のｐＨの上限については、ＰＨＢ共重合体を加熱溶融した時の着色を低減する観点、ならびに、加熱時および／または乾燥時の分子量の安定性を確保する観点から、７．０以下であり、好ましくは、５．０以下であり、より好ましくは、４．０以下である。また、ｐＨの下限については、容器の耐酸性の観点より、好ましくは、１以上であり、より好ましくは、２．０以上であり、さらに好ましくは、３．０以上である。ＰＨＢ共重合体水性懸濁液のｐＨを７．０以下とすることによって、加熱溶融時の着色が低減され、加熱時および／または乾燥時の分子量低下が抑制されたＰＨＢ共重合体が得られる。
Regarding the upper limit of the pH of the aqueous suspension of the PHB copolymer adjusted in the above adjustment step, from the viewpoint of reducing the coloring when the PHB copolymer is heated and melted, and the stability of the molecular weight during heating and / or drying. From the viewpoint of ensuring the property, it is 7.0 or less, preferably 5.0 or less, and more preferably 4.0 or less. The lower limit of pH is preferably 1 or more, more preferably 2.0 or more, still more preferably 3.0 or more, from the viewpoint of acid resistance of the container. By setting the pH of the aqueous suspension of the PHB copolymer to 7.0 or less, it is possible to obtain a PHB copolymer in which coloring during heating and melting is reduced and molecular weight reduction during heating and / or drying is suppressed. ..
本製造方法の工程（ｃ）により調整される水性懸濁液におけるＰＨＢ共重合体の濃度は、３０重量％以上が好ましく、４０重量％以上がより好ましく、５０重量％以上がさらに好ましい。ＰＨＢ共重合体濃度が３０重量％以上であることにより、乾燥ユーティリティーの面から経済的に有利であり、生産性が向上し、次工程（ｄ）で得られる粉体嵩密度が高くなり、その結果、輸送性が向上する。また、ＰＨＢ共重合体の濃度の上限は、最密充填となり、十分な流動性が確保できない可能性があるため、６５重量％以下が好ましく、６０重量％以下がより好ましい。ＰＨＢ共重合体の濃度を調整する方法は、特に限定されず、水性媒体を添加する、および、水性媒体の一部を除去する（例えば、遠心分離した後、上清を取り除く等による）等の方法が挙げられる。ＰＨＢ共重合体の濃度の調整は、工程（ｃ）の段階で実施してもよいし、工程（ｂ）の段階で実施してもよい。
The concentration of the PHB copolymer in the aqueous suspension adjusted by the step (c) of the present production method is preferably 30% by weight or more, more preferably 40% by weight or more, still more preferably 50% by weight or more. When the PHB copolymer concentration is 30% by weight or more, it is economically advantageous in terms of drying utility, productivity is improved, and the powder bulk density obtained in the next step (d) is increased. As a result, transportability is improved. Further, the upper limit of the concentration of the PHB copolymer is preferably close packing, and sufficient fluidity may not be ensured. Therefore, 65% by weight or less is preferable, and 60% by weight or less is more preferable. The method for adjusting the concentration of the PHB copolymer is not particularly limited, and an aqueous medium is added and a part of the aqueous medium is removed (for example, by centrifuging and then removing the supernatant). The method can be mentioned. The adjustment of the concentration of the PHB copolymer may be carried out at the stage of the step (c) or may be carried out at the stage of the step (b).
本発明の一実施形態において、工程（ｃ）により調製される水性懸濁液におけるＰＨＢ共重合体の濃度は、３０～６５重量％が好ましい。
In one embodiment of the present invention, the concentration of the PHB copolymer in the aqueous suspension prepared in step (c) is preferably 30 to 65% by weight.
工程（ｃ）において、ＰＨＢ共重合体水性懸濁液のせん断粘度は、噴霧乾燥機への送液容易性の観点から、０．００５～０．５Ｐａ・ｓであり、０．００７～０．４Ｐａ・ｓであることが好ましく、０．００９～０．３Ｐａ・ｓであることがより好ましい。なお、ＰＨＢ共重合体水性懸濁液のせん断粘度とは、せん断速度１０（１／ｓ）を与えた時の２０℃におけるせん断粘度（Ｐａ・ｓ）を意味する。ＰＨＢ共重合体水性懸濁液のせん断粘度は、実施例に記載の方法により測定される。
In the step (c), the shear viscosity of the aqueous suspension of the PHB copolymer is 0.005 to 0.5 Pa · s and 0.007 to 0. It is preferably 4 Pa · s, and more preferably 0.009 to 0.3 Pa · s. The shear viscosity of the aqueous suspension of PHB copolymer means the shear viscosity (Pa · s) at 20 ° C. when a shear rate of 10 (1 / s) is given. The shear viscosity of the aqueous suspension of PHB copolymer is measured by the method described in Examples.
＜分散剤＞
本発明の一実施形態における前記水性懸濁液は、分散剤を含有していてもよい。すなわち、本発明の一実施形態における前記工程（ｃ）は、ＰＨＢ共重合体に加えて、分散剤をさらに含む水性懸濁液を調製する工程であってもよい。本発明の一実施形態において、前記分散剤を使用する場合は、ｐＨ７．０以下になるように調整する前に、前記分散剤を前記ＰＨＢ共重合体水性懸濁液に添加することが好ましい。本発明の一実施形態における前記ＰＨＢ共重合体水性懸濁液が、分散剤を含有することによって、ＰＨＢ共重合体の生産性および熱安定性を好適に向上させることができる。 <Dispersant>
The aqueous suspension in one embodiment of the present invention may contain a dispersant. That is, the step (c) in one embodiment of the present invention may be a step of preparing an aqueous suspension further containing a dispersant in addition to the PHB copolymer. In one embodiment of the present invention, when the dispersant is used, it is preferable to add the dispersant to the PHB copolymer aqueous suspension before adjusting the pH to 7.0 or less. When the PHB copolymer aqueous suspension in one embodiment of the present invention contains a dispersant, the productivity and thermal stability of the PHB copolymer can be suitably improved.
本発明の一実施形態における前記水性懸濁液は、分散剤を含有していてもよい。すなわち、本発明の一実施形態における前記工程（ｃ）は、ＰＨＢ共重合体に加えて、分散剤をさらに含む水性懸濁液を調製する工程であってもよい。本発明の一実施形態において、前記分散剤を使用する場合は、ｐＨ７．０以下になるように調整する前に、前記分散剤を前記ＰＨＢ共重合体水性懸濁液に添加することが好ましい。本発明の一実施形態における前記ＰＨＢ共重合体水性懸濁液が、分散剤を含有することによって、ＰＨＢ共重合体の生産性および熱安定性を好適に向上させることができる。 <Dispersant>
The aqueous suspension in one embodiment of the present invention may contain a dispersant. That is, the step (c) in one embodiment of the present invention may be a step of preparing an aqueous suspension further containing a dispersant in addition to the PHB copolymer. In one embodiment of the present invention, when the dispersant is used, it is preferable to add the dispersant to the PHB copolymer aqueous suspension before adjusting the pH to 7.0 or less. When the PHB copolymer aqueous suspension in one embodiment of the present invention contains a dispersant, the productivity and thermal stability of the PHB copolymer can be suitably improved.
分散剤としては、特に限定されないが、例えば、アルキレンオキサイド系分散剤、セルロース系分散剤、ポリビニルアルコール（ＰＶＡ）、ソルビタンアルキレート系分散剤等が挙げられる。これらの分散剤を用いることにより、より低い熱風温度で、粒径が大きいＰＨＢ共重合体を得ることができる。分散剤は、１種であってもよく、２種類以上であってもよい。
The dispersant is not particularly limited, and examples thereof include an alkylene oxide-based dispersant, a cellulose-based dispersant, polyvinyl alcohol (PVA), a sorbitan alkylate-based dispersant, and the like. By using these dispersants, a PHB copolymer having a large particle size can be obtained at a lower hot air temperature. The dispersant may be one kind or two or more kinds.
本発明の一実施形態において、分散剤は、アルキレンオキサイド系分散剤、セルロース系分散剤およびポリビニルアルコールからなる群より選択される少なくとも１種である。
In one embodiment of the present invention, the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
本発明の一実施形態において、ＰＨＢ共重合体水性懸濁液のｐＨを７．０以下に調整する際に、ＰＨＢ共重合体の凝集を好適に防ぎ、かつ、粉体加工の際の押出機の軸への付着を好適に抑制する観点より、分散剤は、アルキレンオキサイド系分散剤であることが好ましい。また、アルキレンオキサイド系分散剤にセルロース系分散剤を併用すると、よりＰＨＢ共重合体の凝集を抑制し、さらに乾燥後の粉体が壊れにくくなるバインダーとしても効果が期待できるため、より好ましい。
In one embodiment of the present invention, when the pH of the aqueous suspension of PHB copolymer is adjusted to 7.0 or less, aggregation of the PHB copolymer is suitably prevented, and an extruder for powder processing is used. The dispersant is preferably an alkylene oxide-based dispersant from the viewpoint of preferably suppressing the adhesion of the above to the shaft. Further, it is more preferable to use a cellulose-based dispersant in combination with the alkylene oxide-based dispersant because it can be expected to be effective as a binder that further suppresses aggregation of the PHB copolymer and further prevents the powder after drying from breaking.
本発明の一実施形態において、アルキレンオキサイド系分散剤は、上記の効果を奏する限り特に限定されないが、ポリ（エチレンオキサイド）（ＰＥＯ）のブロックと、ポリ（プロピレンオキサイド）（ＰＰＯ）のブロックとから構成され、ＰＥＯ－ＰＰＯ－ＰＥＯの形態であることが好ましい。
In one embodiment of the present invention, the alkylene oxide-based dispersant is not particularly limited as long as it exhibits the above effects, but is composed of a block of poly (ethylene oxide) (PEO) and a block of poly (propylene oxide) (PPO). It is preferably configured and in the form of PEO-PPO-PEO.
本明細書において、「ポリ（エチレンオキサイド）（ＰＥＯ）のブロック」とは、アルキレンオキサイド系分散剤の構造中、エチレンオキサイド（ＥＯ）が重合して形成された重合体部分を意味する。
As used herein, the term "poly (ethylene oxide) (PEO) block" means a polymer portion formed by polymerizing ethylene oxide (EO) in the structure of an alkylene oxide-based dispersant.
本明細書において、「ポリ（プロピレンオキサイド）（ＰＰＯ）のブロック」とは、アルキレンオキサイド系分散剤の構造中、プロピレンオキサイド（ＰＯ）が重合して形成された重合体部分を意味する。
As used herein, the term "poly (propylene oxide) (PPO) block" means a polymer portion formed by polymerizing propylene oxide (PO) in the structure of an alkylene oxide-based dispersant.
本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量およびＰＥＯ分子量／ＰＰＯ分子量を特定の範囲とすることにより、水性懸濁液の粘度を低く保ち、高い生産性でＰＨＢ共重合体（例えば、ＰＨＢ共重合体粉体）を製造することができる。
In one embodiment of the present invention, by setting the PEO molecular weight and the PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant within a specific range, the viscosity of the aqueous suspension is kept low and the PHB copolymer is highly productive. (For example, PHB copolymer powder) can be produced.
本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量およびＰＥＯ分子量／ＰＰＯ分子量の範囲は、以下の組み合わせであることが好ましい。
In one embodiment of the present invention, the range of PEO molecular weight and PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant is preferably the following combination.
なお、本明細書において、「ＰＥＯ分子量」を「ＥＯ量」と称し、「ＰＰＯ分子量」を「ＰＯ量」と称することもある。
In the present specification, "PEO molecular weight" may be referred to as "EO molecular weight", and "PPO molecular weight" may be referred to as "PO molecular weight".
すなわち、本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量は、１５００以上であればよく、好ましくは、１７５０以上であり、より好ましくは、２０００以上である。また、本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量の上限は、例えば、３００００以下であり、好ましくは、２５０００以下であり、より好ましくは、２００００以下である。
That is, in one embodiment of the present invention, the PEO molecular weight in the alkylene oxide-based dispersant may be 1500 or more, preferably 1750 or more, and more preferably 2000 or more. Further, in one embodiment of the present invention, the upper limit of the PEO molecular weight in the alkylene oxide-based dispersant is, for example, 30,000 or less, preferably 25,000 or less, and more preferably 20,000 or less.
本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量／ＰＰＯ分子量は、０．５以上であることが好ましく、０．６以上であることがより好ましく、０．７以上であることがさらに好ましい。ＰＥＯ分子量／ＰＰＯ分子量の上限は、５．０以下であることが好ましく、４．８以下であることがより好ましく、４．５以下であることがさらに好ましい。
In one embodiment of the present invention, the PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant is preferably 0.5 or more, more preferably 0.6 or more, and 0.7 or more. Is even more preferable. The upper limit of the PEO molecular weight / PPO molecular weight is preferably 5.0 or less, more preferably 4.8 or less, and further preferably 4.5 or less.
本発明の一実施形態において、アルキレンオキサイド系分散剤中のＰＥＯ分子量およびＰＥＯ分子量／ＰＰＯ分子量が上記の範囲内であれば、アルキレンオキサイド系分散剤が親水性を有し、かつ、アルキレンオキサイド系分散剤添加重量に対する分子数が多くなるため、水性懸濁液の分散性を保ちやすい。
In one embodiment of the present invention, if the PEO molecular weight and the PEO molecular weight / PPO molecular weight in the alkylene oxide-based dispersant are within the above ranges, the alkylene oxide-based dispersant has hydrophilicity and the alkylene oxide-based dispersant is dispersed. Since the number of molecules increases with respect to the added weight of the agent, it is easy to maintain the dispersibility of the aqueous suspension.
本発明の一実施形態において、アルキレンオキサイド系分散剤は、ＰＥＯ分子量が１５００以上であり、かつ、ＰＥＯ分子量／ＰＰＯ分子量が０．５～５．０である。
In one embodiment of the present invention, the alkylene oxide-based dispersant has a PEO molecular weight of 1500 or more and a PEO molecular weight / PPO molecular weight of 0.5 to 5.0.
本製造方法の工程（ｃ）において使用されるアルキレンオキサイド系分散剤は、特に限定されず、例えば、市販品を用いることができる。市販品としては、例えば、Ｐｌｕｒｏｎｉｃ（登録商標）１０４００（ＢＡＳＦ社製）、Ｐｌｕｒｏｎｉｃ（登録商標）１０５００（ＢＡＳＦ社製）、Ｇｅｎａｐｏｌ（登録商標）ＰＦ８０（Ｃｌａｒｉａｎｔ社製）、ユニルーブ（登録商標）７０ＤＰ－６００Ｂ（日油社製）、ユニルーブ（登録商標）７０ＤＰ－９５０Ｂ（日油社製）、プロノン（登録商標）２０８（日油社製）、エパン（登録商標）Ｕ１０５（第一工業製薬社製）、エパン（登録商標）Ｕ１０８（第一工業製薬社製）、エパン（登録商標）７５０（第一工業製薬社製）等が挙げられる。
The alkylene oxide-based dispersant used in the step (c) of the present production method is not particularly limited, and for example, a commercially available product can be used. Examples of commercially available products include Pluronic (registered trademark) 10400 (registered trademark) 10400 (registered trademark) 10500 (BASF), Genapol (registered trademark) PF80 (registered trademark), and Unilube (registered trademark) 70DP-. 600B (manufactured by Nichiyu Co., Ltd.), Unilube (registered trademark) 70DP-950B (manufactured by Nichiyu Co., Ltd.), Pronon (registered trademark) 208 (manufactured by Nichiyu Co., Ltd.), Epan (registered trademark) U105 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) , Epan (registered trademark) U108 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Epan (registered trademark) 750 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and the like.
本製造方法の工程（ｃ）における水性懸濁液に対する前記分散剤の添加量は、特に限定されないが、水性懸濁液に含まれる前記ＰＨＢ共重合体の合計１００重量部に対して、０．１～２０重量部が好ましく、０．５～１０重量部がより好ましく、０．７５～５重量部がさらに好ましい。前記分散剤の添加量を上記の範囲とすることにより、ＰＨＢ共重合体水性懸濁液におけるＰＨＢ共重合体の分散安定性がより向上し、噴霧乾燥を効率的に実施でき、その結果、ＰＨＢ共重合体の生産性および熱安定性をより好適に向上させることができる傾向がある。
The amount of the dispersant added to the aqueous suspension in the step (c) of the present production method is not particularly limited, but is 0. 1 to 20 parts by weight is preferable, 0.5 to 10 parts by weight is more preferable, and 0.75 to 5 parts by weight is further preferable. By setting the addition amount of the dispersant within the above range, the dispersion stability of the PHB copolymer in the aqueous suspension of the PHB copolymer is further improved, and spray drying can be efficiently carried out, and as a result, PHB can be carried out. There is a tendency to more preferably improve the productivity and thermal stability of the copolymer.
本発明の一実施形態において、セルロース系分散剤は、本発明の効果を奏するものであれば特に限定されないが、例えば、メチルセルロース（ＭＣ）、エチルセルロース、プロピルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース（ＨＥＣ）、ヒドロキシプロピルセルロース、ヒドロキシエチルメチルセルロース、ヒドロキシプロピルメチルセルロース、ヒドロキシプロピルメチルセルロース（ＨＰＭＣ）、カルボキシメチルセルロース（ＣＭＣ）、カルボキシエチルセルロース、カルボキシプロピルセルロース、カルボキシメチルヒドロキシエチルセルロース、アセチルセルロース、シアノエチルセルロース、セルロース硫酸ナトリウム等が挙げられる。なかでも、メチルセルロース、ヒドロキシプロピルメチルセルロースが、水溶性となる置換度範囲の広さの観点から好ましい。セルロース系分散剤は、１種のみを用いてもよいし、複数を組み合わせて用いてもよい。
In one embodiment of the present invention, the cellulose-based dispersant is not particularly limited as long as it exhibits the effects of the present invention, and for example, methyl cellulose (MC), ethyl cellulose, propyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose (HEC), and the like. Hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC), carboxyethyl cellulose, carboxypropyl cellulose, carboxymethyl hydroxyethyl cellulose, acetyl cellulose, cyanoethyl cellulose, sodium cellulose sulfate and the like can be mentioned. .. Of these, methyl cellulose and hydroxypropyl methyl cellulose are preferable from the viewpoint of a wide range of degree of substitution that makes them water-soluble. Only one type of cellulosic dispersant may be used, or a plurality of cellulosic dispersants may be used in combination.
本製造方法の工程（ｃ）において使用されるセルロース系分散剤は、特に限定されず、例えば、市販品を用いることができる。セルロース系分散剤の市販品としては、例えば、ＭＣＥ－１００（信越化学社製）、ＭＣＥ－４００（信越化学社製）、ＭＣＥ－４０００（信越化学社製）、ＳＦＥ－４００（信越化学社製）、ＳＦＥ－４０００（信越化学社製）、ＳＥ－５０（信越化学社製）、ＮＥ－１００（信越化学社製）等が挙げられる。
The cellulosic dispersant used in the step (c) of this production method is not particularly limited, and for example, a commercially available product can be used. Commercially available products of cellulosic dispersants include, for example, MCE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SFE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.). ), SFE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SE-50 (manufactured by Shin-Etsu Chemical Co., Ltd.), NE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.) and the like.
本製造方法の工程（ｃ）におけるＰＨＢ共重合体水性懸濁液に対するセルロース系分散剤の添加量は、特に限定されないが、水性懸濁液に含まれるＰＨＢ共重合体１００重量部に対して、０．０１～１０重量部が好ましく、０．０５～５重量部がより好ましく、０．０８～３重量部がさらに好ましい。セルロース系分散剤の添加量を上記の範囲とすることにより、本発明の効果を奏することができる。
The amount of the cellulosic dispersant added to the aqueous suspension of the PHB copolymer in the step (c) of the present production method is not particularly limited, but is based on 100 parts by weight of the PHB copolymer contained in the aqueous suspension. 0.01 to 10 parts by weight is preferable, 0.05 to 5 parts by weight is more preferable, and 0.08 to 3 parts by weight is further preferable. The effect of the present invention can be achieved by setting the addition amount of the cellulosic dispersant within the above range.
本発明の一実施形態において、分散剤は、環境問題の観点から、生分解性を有する物質であることが好ましい。
In one embodiment of the present invention, the dispersant is preferably a biodegradable substance from the viewpoint of environmental problems.
本製造方法の工程（ｃ）により得られるＰＨＢ共重合体水性懸濁液におけるＰＨＢ共重合体の体積メジアン径（以下、単に「ＰＨＢ共重合体の体積メジアン径」と称する場合もある。）は、当該ＰＨＢ共重合体の一次粒子の体積メジアン径（以下、「一次粒子径」と称する。）の３０倍以下が好ましく、２０倍以下がより好ましく、１０倍以下がさらに好ましい。ＰＨＢ共重合体の体積メジアン径が一次粒子径の３０倍以下であることにより、ＰＨＢ共重合体水性懸濁液がより優れた流動性を示すため、その後の工程（ｄ）を高効率で実施することができ、ＰＨＢ共重合体の生産性が一層向上する傾向がある。ＰＨＢ共重合体の体積メジアン径は、例えば、ＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定される。
The volume median diameter of the PHB copolymer in the aqueous suspension of the PHB copolymer obtained by the step (c) of the present production method (hereinafter, may be simply referred to as "volume median diameter of the PHB copolymer"). The volume median diameter of the primary particles of the PHB copolymer (hereinafter referred to as "primary particle diameter") is preferably 30 times or less, more preferably 20 times or less, still more preferably 10 times or less. Since the volume median diameter of the PHB copolymer is 30 times or less the primary particle diameter, the PHB copolymer aqueous suspension exhibits better fluidity, and therefore the subsequent step (d) is carried out with high efficiency. This can be done, and the productivity of the PHB copolymer tends to be further improved. The volume median diameter of the PHB copolymer is measured, for example, using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA.
なお、上記のＰＨＢ共重合体の体積メジアン径は、ＰＨＢ共重合体水性懸濁液におけるＰＨＢ共重合体の分散状態の指標とすることができる。上記のＰＨＢ共重合体の体積メジアン径を調整する方法は、特に限定されず、公知の手段（攪拌等）を適用できる。例えば、酸性条件下に曝される等して分散状態が崩れてしまったＰＨＢ共重合体水性懸濁液に対して、当業者が考え得る物理的処理、化学的処理、生物学的処理等を施し、ＰＨＢ共重合体水性懸濁液におけるＰＨＢ共重合体を再度分散状態（例えば、上記のＰＨＢ共重合体の体積メジアン径を有する状態）に復帰させることもできる。
The volume median diameter of the above PHB copolymer can be used as an index of the dispersed state of the PHB copolymer in the aqueous suspension of the PHB copolymer. The method for adjusting the volume median diameter of the PHB copolymer is not particularly limited, and known means (stirring or the like) can be applied. For example, a PHB copolymer aqueous suspension whose dispersed state has collapsed due to exposure to acidic conditions can be subjected to physical treatment, chemical treatment, biological treatment, etc. that can be considered by those skilled in the art. Then, the PHB copolymer in the aqueous suspension of the PHB copolymer can be returned to the dispersed state (for example, the state having the volume median diameter of the above PHB copolymer).
＜工程（ｄ）＞
本製造方法における工程（ｄ）では、工程（ｃ）で調製したＰＨＢ共重合体水性懸濁液を噴霧乾燥する。噴霧乾燥の方法としては、例えば、ＰＨＢ共重合体水性懸濁液を微細な液滴の状態として乾燥機内に供給し、当該乾燥機内で熱風と接触させながら乾燥する方法等が挙げられる。ＰＨＢ共重合体水性懸濁液を微細な液滴の状態で乾燥機内に供給する方法（アトマイザー）は、特に限定されず、回転ディスクを用いる方法、ノズルを用いる方法等の公知の方法が挙げられる。乾燥機内における液滴と熱風の接触方式は、特に限定されず、並流式、向流式、これらを併用する方式等が挙げられる。 <Step (d)>
In the step (d) of the present production method, the PHB copolymer aqueous suspension prepared in the step (c) is spray-dried. Examples of the spray drying method include a method in which a PHB copolymer aqueous suspension is supplied into a dryer in the form of fine droplets and dried while being in contact with hot air in the dryer. The method (atomizer) for supplying the aqueous suspension of the PHB copolymer in the form of fine droplets into the dryer is not particularly limited, and examples thereof include known methods such as a method using a rotating disk and a method using a nozzle. .. The contact method between the droplet and the hot air in the dryer is not particularly limited, and examples thereof include a parallel flow type, a countercurrent type, and a method in which these are used in combination.
本製造方法における工程（ｄ）では、工程（ｃ）で調製したＰＨＢ共重合体水性懸濁液を噴霧乾燥する。噴霧乾燥の方法としては、例えば、ＰＨＢ共重合体水性懸濁液を微細な液滴の状態として乾燥機内に供給し、当該乾燥機内で熱風と接触させながら乾燥する方法等が挙げられる。ＰＨＢ共重合体水性懸濁液を微細な液滴の状態で乾燥機内に供給する方法（アトマイザー）は、特に限定されず、回転ディスクを用いる方法、ノズルを用いる方法等の公知の方法が挙げられる。乾燥機内における液滴と熱風の接触方式は、特に限定されず、並流式、向流式、これらを併用する方式等が挙げられる。 <Step (d)>
In the step (d) of the present production method, the PHB copolymer aqueous suspension prepared in the step (c) is spray-dried. Examples of the spray drying method include a method in which a PHB copolymer aqueous suspension is supplied into a dryer in the form of fine droplets and dried while being in contact with hot air in the dryer. The method (atomizer) for supplying the aqueous suspension of the PHB copolymer in the form of fine droplets into the dryer is not particularly limited, and examples thereof include known methods such as a method using a rotating disk and a method using a nozzle. .. The contact method between the droplet and the hot air in the dryer is not particularly limited, and examples thereof include a parallel flow type, a countercurrent type, and a method in which these are used in combination.
工程（ｄ）における噴霧乾燥の際の乾燥温度は、ＰＨＢ共重合体水性懸濁液の液滴から水性媒体の大半を除去できる温度であればよく、目的とする含水率まで乾燥させることができ、かつ、品質悪化（分子量低下、色調低下等）、溶融等を極力生じさせないような条件で、適宜設定できる。例えば、噴霧乾燥機に吹き込む熱風の温度は、１００～３００℃の範囲で、適宜選択できる。また、乾燥機内の熱風の風量についても、例えば、乾燥機のサイズ等に応じて、適宜設定できる。
The drying temperature at the time of spray drying in the step (d) may be any temperature as long as it can remove most of the aqueous medium from the droplets of the aqueous suspension of the PHB copolymer, and can be dried to the desired moisture content. Moreover, it can be appropriately set under conditions that do not cause deterioration of quality (decrease in molecular weight, decrease in color tone, etc.), melting, etc. as much as possible. For example, the temperature of the hot air blown into the spray dryer can be appropriately selected in the range of 100 to 300 ° C. Further, the amount of hot air in the dryer can be appropriately set according to, for example, the size of the dryer.
本製造方法は、工程（ｄ）の後に、得られたＰＨＢ共重合体（ＰＨＢ共重合体粉体等）をさらに乾燥させる工程（例えば、減圧乾燥に付す工程等）を含んでいてもよい。また、本製造方法は、その他の工程（例えば、ＰＨＢ共重合体水性懸濁液に各種添加物を添加する工程等）を含んでいてもよい。
The present production method may include, after the step (d), a step of further drying the obtained PHB copolymer (PHB copolymer powder or the like) (for example, a step of subjecting to vacuum drying or the like). Further, the present production method may include other steps (for example, a step of adding various additives to the aqueous suspension of PHB copolymer).
本製造方法は、工程（ｄ）の後に、下記の工程（ｅ）および（ｆ）をさらに含んでもよい。これらの工程によれば、ＰＨＢ共重合体に含まれる菌体残渣分に由来する窒素含有量を低くすることができ、純度の高いＰＨＢ共重合体を得ることができる。
The present manufacturing method may further include the following steps (e) and (f) after the step (d). According to these steps, the nitrogen content derived from the bacterial cell residue contained in the PHB copolymer can be reduced, and a high-purity PHB copolymer can be obtained.
＜工程（ｅ）＞
工程（ｅ）は、前記工程（ｄ）で得られたＰＨＢ共重合体粉体を洗浄する工程である。工程（ｅ）は、下記の工程（ｅ１）および工程（ｅ２）を含むことが好ましい。
・工程（ｅ１）：前記工程（ｅ）で得られたＰＨＢ共重合体粉体を所定の濃度になるように純水に分散させて分散スラリーを調製した後、アルカリ水溶液を加えて、該分散スラリーｐＨを調整し、洗浄液と共に撹拌する工程
・工程（ｅ２）：前記工程（ｅ１）で得られた分散スラリーにアルカリ水溶液を加え、遠心分離し、上澄を除去する工程。 <Step (e)>
The step (e) is a step of washing the PHB copolymer powder obtained in the step (d). The step (e) preferably includes the following steps (e1) and step (e2).
Step (e1): The PHB copolymer powder obtained in the step (e) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added to disperse the dispersion. Step / Step (e2) of adjusting the slurry pH and stirring with the cleaning liquid: A step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), centrifuging and removing the supernatant.
工程（ｅ）は、前記工程（ｄ）で得られたＰＨＢ共重合体粉体を洗浄する工程である。工程（ｅ）は、下記の工程（ｅ１）および工程（ｅ２）を含むことが好ましい。
・工程（ｅ１）：前記工程（ｅ）で得られたＰＨＢ共重合体粉体を所定の濃度になるように純水に分散させて分散スラリーを調製した後、アルカリ水溶液を加えて、該分散スラリーｐＨを調整し、洗浄液と共に撹拌する工程
・工程（ｅ２）：前記工程（ｅ１）で得られた分散スラリーにアルカリ水溶液を加え、遠心分離し、上澄を除去する工程。 <Step (e)>
The step (e) is a step of washing the PHB copolymer powder obtained in the step (d). The step (e) preferably includes the following steps (e1) and step (e2).
Step (e1): The PHB copolymer powder obtained in the step (e) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added to disperse the dispersion. Step / Step (e2) of adjusting the slurry pH and stirring with the cleaning liquid: A step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), centrifuging and removing the supernatant.
（工程（ｅ１））
工程（ｅ１）は、上述の通り、前記工程（ｄ）で得られたＰＨＢ共重合体粉体を所定の濃度になるように純水に分散させて分散スラリーを調製した後、アルカリ水溶液を加えて、該分散スラリーｐＨを調整し、洗浄液と共に撹拌する工程である。撹拌する際には、液を昇温してもよい。 (Step (e1))
In the step (e1), as described above, the PHB copolymer powder obtained in the step (d) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added. This is a step of adjusting the pH of the dispersed slurry and stirring it together with the washing liquid. When stirring, the temperature of the liquid may be raised.
工程（ｅ１）は、上述の通り、前記工程（ｄ）で得られたＰＨＢ共重合体粉体を所定の濃度になるように純水に分散させて分散スラリーを調製した後、アルカリ水溶液を加えて、該分散スラリーｐＨを調整し、洗浄液と共に撹拌する工程である。撹拌する際には、液を昇温してもよい。 (Step (e1))
In the step (e1), as described above, the PHB copolymer powder obtained in the step (d) is dispersed in pure water so as to have a predetermined concentration to prepare a dispersed slurry, and then an alkaline aqueous solution is added. This is a step of adjusting the pH of the dispersed slurry and stirring it together with the washing liquid. When stirring, the temperature of the liquid may be raised.
工程（ｅ１）における分散スラリーの所定の濃度は、特に限定されないが、分散するタンクのサイズをできるだけ小さくし、かつ、スラリーの流動性がある範囲の観点から、２０～４０％であることが好ましく、３０～４０％であることが好ましい。
The predetermined concentration of the dispersed slurry in the step (e1) is not particularly limited, but is preferably 20 to 40% from the viewpoint of minimizing the size of the tank to be dispersed and the fluidity of the slurry. , 30-40% is preferable.
工程（ｅ１）において、アルカリ水溶液は、特に限定されないが、（工程（ｂ１））にて説明したアルカリ水溶液が用いられ得る。分散スラリーのｐＨは、例えば、５．０～１４．０に調整されることが好ましい。
In the step (e1), the alkaline aqueous solution is not particularly limited, but the alkaline aqueous solution described in (step (b1)) can be used. The pH of the dispersed slurry is preferably adjusted to, for example, 5.0 to 14.0.
工程（ｅ１）の洗浄における撹拌時間は特に限定されないが、数時間であってもよい。ＰＨＢ共重合体を十分に洗浄する観点より、撹拌時間は１～１２時間であってよく、２～１０時間であってもよい。
The stirring time in the washing of the step (e1) is not particularly limited, but may be several hours. From the viewpoint of sufficiently washing the PHB copolymer, the stirring time may be 1 to 12 hours or 2 to 10 hours.
工程（ｅ１）における分散スラリーは、硫酸ナトリウムをさらに含んでもよい。
The dispersed slurry in the step (e1) may further contain sodium sulfate.
工程（ｅ１）における洗浄液は、アルカリ性タンパク質分解酵素および／または溶菌酵素を含んでもよい。アルカリ性タンパク質分解酵素の種類は特に限定されないが、本明細書の（アルカリ性タンパク質分解酵素）で記載したものを用いてもよい。また、本工程で用いられるアルカリ性タンパク質分解酵素は、工程（ａ）で用いたアルカリ性タンパク質分解酵素と同じであってもよく、異なっていてもよい。また、溶菌酵素の種類は特に限定されないが、工程（ａ）についての説明にて記載した溶菌酵素であってもよい。
The cleaning solution in the step (e1) may contain an alkaline proteolytic enzyme and / or a lytic enzyme. The type of the alkaline proteolytic enzyme is not particularly limited, but the one described in (Alkaline Proteolytic Enzyme) of the present specification may be used. Further, the alkaline proteolytic enzyme used in this step may be the same as or different from the alkaline proteolytic enzyme used in step (a). The type of lytic enzyme is not particularly limited, but may be the lytic enzyme described in the description of step (a).
（工程（ｅ２））
工程（ｅ２）は、上述の通り、前記工程（ｅ１）で得られた分散スラリーにアルカリ水溶液を加え、ＰＨＢ共重合体粉体を分離し、上清を除去する工程である。 (Step (e2))
As described above, the step (e2) is a step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), separating the PHB copolymer powder, and removing the supernatant.
工程（ｅ２）は、上述の通り、前記工程（ｅ１）で得られた分散スラリーにアルカリ水溶液を加え、ＰＨＢ共重合体粉体を分離し、上清を除去する工程である。 (Step (e2))
As described above, the step (e2) is a step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), separating the PHB copolymer powder, and removing the supernatant.
工程（ｅ２）において、アルカリ水溶液は、特に限定されないが、工程（ｅ１）で用いたアルカリ水溶液と同じであってもよく、異なるものであってもよい。
In the step (e2), the alkaline aqueous solution is not particularly limited, but may be the same as or different from the alkaline aqueous solution used in the step (e1).
工程（ｅ２）において、ＰＨＢ共重合体粉体を分離する方法は特に限定されず、公知の方法を用いることができる。例えば、遠心分離、フィルター脱水、液体サイクロンによる分離を用いることができる。
In the step (e2), the method for separating the PHB copolymer powder is not particularly limited, and a known method can be used. For example, centrifugation, filter dehydration, and liquid cyclone separation can be used.
工程（ｅ２）において、前記分散スラリーにアルカリ水溶液を添加し、遠心分離し、上清を除去する工程を繰り返し行うことが好ましい。この操作により、より濃縮および精製されたＰＨＢ共重合体を得ることができる。
In the step (e2), it is preferable to repeat the steps of adding an alkaline aqueous solution to the dispersed slurry, centrifuging the dispersion, and removing the supernatant. By this operation, a more concentrated and purified PHB copolymer can be obtained.
＜工程（ｆ）＞
工程（ｆ）は、前記工程（ｅ）で得られたポリヒドロキシアルカン酸粉体を脱水および／または乾燥する工程である。脱水および／または乾燥には、例えば、噴霧乾燥機、流動層乾燥機、ドラムドライヤー等が用いられるが、操作が簡便であることから、好ましくは、噴霧乾燥機が用いられる。噴霧乾燥の方法、乾燥温度としては、例えば、工程（ｄ）にて説明した方法、温度を用いてもよい。 <Step (f)>
The step (f) is a step of dehydrating and / or drying the polyhydroxyalkanoic acid powder obtained in the step (e). For dehydration and / or drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer, or the like is used, but a spray dryer is preferably used because of its simple operation. As the spray drying method and drying temperature, for example, the method and temperature described in step (d) may be used.
工程（ｆ）は、前記工程（ｅ）で得られたポリヒドロキシアルカン酸粉体を脱水および／または乾燥する工程である。脱水および／または乾燥には、例えば、噴霧乾燥機、流動層乾燥機、ドラムドライヤー等が用いられるが、操作が簡便であることから、好ましくは、噴霧乾燥機が用いられる。噴霧乾燥の方法、乾燥温度としては、例えば、工程（ｄ）にて説明した方法、温度を用いてもよい。 <Step (f)>
The step (f) is a step of dehydrating and / or drying the polyhydroxyalkanoic acid powder obtained in the step (e). For dehydration and / or drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer, or the like is used, but a spray dryer is preferably used because of its simple operation. As the spray drying method and drying temperature, for example, the method and temperature described in step (d) may be used.
〔３．ＰＨＢ共重合体粉体〕
（中間体）
本発明の一実施形態に係るＰＨＢ共重合体粉体（以下、「本第１のＰＨＢ共重合体粉体」と称する。）は、ＰＨＢ共重合体と、ペプチドグリカンと、分散剤と、を含み、前記ＰＨＢ共重合体が、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。本第１のＰＨＢ共重合体粉体は、３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が高いＰＨＢ共重合体であるとともに、嵩密度が高いため、種々の分野において極めて有用である。 [3. PHB copolymer powder]
(Intermediate)
The PHB copolymer powder according to one embodiment of the present invention (hereinafter, referred to as “the first PHB copolymer powder”) contains a PHB copolymer, a peptide glycan, and a dispersant. , The PHB copolymer has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol), and a bulk density of 0.45 g / mL or more. A PHB copolymer powder having a median particle size of 80 to 200 μm. The first PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units, and has a high bulk density, so that it is extremely useful in various fields.
（中間体）
本発明の一実施形態に係るＰＨＢ共重合体粉体（以下、「本第１のＰＨＢ共重合体粉体」と称する。）は、ＰＨＢ共重合体と、ペプチドグリカンと、分散剤と、を含み、前記ＰＨＢ共重合体が、３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。本第１のＰＨＢ共重合体粉体は、３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が高いＰＨＢ共重合体であるとともに、嵩密度が高いため、種々の分野において極めて有用である。 [3. PHB copolymer powder]
(Intermediate)
The PHB copolymer powder according to one embodiment of the present invention (hereinafter, referred to as “the first PHB copolymer powder”) contains a PHB copolymer, a peptide glycan, and a dispersant. , The PHB copolymer has a composition ratio of hydroxyalkanoate units other than 3HB units / 3HB units of 80/20 to 88/12 (mol / mol), and a bulk density of 0.45 g / mL or more. A PHB copolymer powder having a median particle size of 80 to 200 μm. The first PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units, and has a high bulk density, so that it is extremely useful in various fields.
本実施形態において、「ＰＨＢ共重合体」、「ペプチドグリカン」、および「分散剤」については、上記したものが援用される。
In the present embodiment, the above-mentioned substances are used as the "PHB copolymer", "peptidoglycan", and "dispersant".
本第１のＰＨＢ共重合体粉体は、ＰＨＢ共重合体を含む。本第１のＰＨＢ共重合体粉体中のＰＨＢ共重合体の含有量は、特に限定されないが、例えば、９０～９９重量％であり、９３～９８重量％が好ましく、９５～９７重量％がより好ましい。ＰＨＢ共重合体の含有量を上記範囲とすることにより、流動性などのＰＨＢＨの物理的な特性を失わないとの利点を有する。
The first PHB copolymer powder contains a PHB copolymer. The content of the PHB copolymer in the first PHB copolymer powder is not particularly limited, but is, for example, 90 to 99% by weight, preferably 93 to 98% by weight, and 95 to 97% by weight. More preferred. By setting the content of the PHB copolymer in the above range, there is an advantage that the physical properties of PHBH such as fluidity are not lost.
本第１のＰＨＢ共重合体粉体は、ペプチドグリカンを含む。本第１のＰＨＢ共重合体粉体中のペプチドグリカンの含有量は、特に限定されないが、ＰＰＨＢ共重合体粉体を構成するＰＨＢ共重合体１００重量部に対して、０．１～１．５重量部（ｐｈｒ）が好ましく、０．３～１．３重量部（ｐｈｒ）がより好ましい。ペプチドグリカンの含有量を上記範囲とすることにより、ＰＨＢ共重合体粉体の嵩密度が向上する傾向がある。本第１のＰＨＢ共重合体粉体中のペプチドグリカンの含有量は、実施例に記載の方法により測定される。
The first PHB copolymer powder contains peptidoglycan. The content of peptidoglycan in the first PHB copolymer powder is not particularly limited, but is 0.1 to 1.5 with respect to 100 parts by weight of the PHB copolymer constituting the PPHB copolymer powder. The part by weight (phr) is preferable, and 0.3 to 1.3 parts by weight (phr) is more preferable. By setting the content of peptidoglycan in the above range, the bulk density of the PHB copolymer powder tends to be improved. The content of peptidoglycan in the first PHB copolymer powder is measured by the method described in Examples.
本第１のＰＨＢ共重合体粉体は、分散剤を含む。分散剤は、好ましくは、アルキレンオキサイド系分散剤である。ＰＨＢ共重合体粉体中の分散剤の含有量は、特に限定されないが、ＰＨＢ共重合体粉体を構成するＰＨＢ共重合体１００重量部に対して、０．１～２０重量部が好ましく、０．５～１０重量部がより好ましく、０．７５～５重量部がさらに好ましい。分散剤の添加量を上記範囲とすることにより、ＰＨＢ共重合体粉体の生産性が向上する傾向がある。
The first PHB copolymer powder contains a dispersant. The dispersant is preferably an alkylene oxide-based dispersant. The content of the dispersant in the PHB copolymer powder is not particularly limited, but is preferably 0.1 to 20 parts by weight with respect to 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. 0.5 to 10 parts by weight is more preferable, and 0.75 to 5 parts by weight is further preferable. By setting the addition amount of the dispersant within the above range, the productivity of the PHB copolymer powder tends to be improved.
本第１のＰＨＢ共重合体粉体の嵩密度は、粉体輸送性が高くなるという観点から、０．４５ｇ／ｍＬ以上であり、０．４８ｇ／ｍＬ以上であることが好ましく、０．５０ｇ／ｍＬ以上であることがより好ましい。なお、上限は特に限定されないが、粉体輸送性の観点から、例えば、０．５５ｇ／ｍＬを挙げることができる。本第１のＰＨＢ共重合体粉体の嵩密度は、実施例に記載の方法により測定される。
The bulk density of the first PHB copolymer powder is 0.45 g / mL or more, preferably 0.48 g / mL or more, preferably 0.50 g, from the viewpoint of increasing powder transportability. More preferably, it is / mL or more. The upper limit is not particularly limited, but from the viewpoint of powder transportability, for example, 0.55 g / mL can be mentioned. The bulk density of the first PHB copolymer powder is measured by the method described in Examples.
本第１のＰＨＢ共重合体粉体のメジアン粒子径は、優れた流動性が達成されるという観点から、８０～２００μｍであり、１００～１８０μｍが好ましく、１０５～１６０μｍがより好ましい。本第１のＰＨＢ共重合体粉体のメジアン粒子径は、実施例に記載の方法により測定される。
The median particle size of the first PHB copolymer powder is 80 to 200 μm, preferably 100 to 180 μm, and more preferably 105 to 160 μm from the viewpoint of achieving excellent fluidity. The median particle size of the first PHB copolymer powder is measured by the method described in Examples.
本第１のＰＨＢ共重合体粉体の純度は、上限値に特に規定はないが、９８％未満である。下限は、ＰＨＢ共重合体の物理的性質を損なわない観点から、８５％以上であり、９０％以上が好ましく、９２％以上がより好ましい。なお、ＰＨＢ共重合体粉体の純度とは、ＰＨＢ共重合体粉体中のＰＨＢ共重合体の含有量（重量％）を意味する。本第１のＰＨＢ共重合体粉体の純度は、実施例に記載の方法により測定される。
The purity of the first PHB copolymer powder is less than 98%, although the upper limit is not particularly specified. The lower limit is 85% or more, preferably 90% or more, and more preferably 92% or more, from the viewpoint of not impairing the physical properties of the PHB copolymer. The purity of the PHB copolymer powder means the content (% by weight) of the PHB copolymer in the PHB copolymer powder. The purity of the first PHB copolymer powder is measured by the method described in Examples.
本発明の一実施形態において、本第１のＰＨＢ共重合体粉体の残タンパク質量は、着色の観点から、０．１～１．０ｐｈｒであることが好ましく、０．１２～０．５ｐｈｒであることがより好ましく、０．１５～０．３ｐｈｒであることがさらに好ましい。なお、残タンパク質量とは、ＰＨＢ共重合体粉体に残存するタンパク質の量を意味し、ＰＨＢ共重合体粉体中のＰＨＢ共重合体１００重量部に対する量として表される。本第１のＰＨＢ共重合体粉体の残タンパク質量は、実施例に記載の方法により測定される。
In one embodiment of the present invention, the amount of residual protein in the first PHB copolymer powder is preferably 0.1 to 1.0 phr, preferably 0.12 to 0.5 phr, from the viewpoint of coloring. It is more preferably 0.15 to 0.3 phr, and even more preferably 0.15 to 0.3 phr. The amount of residual protein means the amount of protein remaining in the PHB copolymer powder, and is expressed as an amount with respect to 100 parts by weight of the PHB copolymer in the PHB copolymer powder. The amount of residual protein in the first PHB copolymer powder is measured by the method described in Examples.
また、本第１のＰＨＢ共重合体粉体は、本発明の効果を奏する限り、製造の過程で生じた、または除去されなかった種々の成分を含んでいてもよい。
Further, the first PHB copolymer powder may contain various components generated or not removed in the manufacturing process as long as the effect of the present invention is exhibited.
本発明の一実施形態において、本第１のＰＨＢ共重合体粉体は、後述する最終生成物に対する中間体であり、上記した本製造方法（例えば、工程（ａ）～（ｄ））により製造される。
In one embodiment of the present invention, the first PHB copolymer powder is an intermediate to the final product described later, and is produced by the above-mentioned production method (for example, steps (a) to (d)). Will be done.
（最終生成物）
本発明の一実施形態に係るＰＨＢ共重合体粉体（以下、「本第２のＰＨＢ共重合体粉体」と称する。）は、ＰＨＢ共重合体と、窒素化合物と、を含み、前記ＰＨＢ共重合体が、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。本第２のＰＨＢ共重合体粉体は、３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が高いＰＨＢ共重合体であるとともに、嵩密度が高く、かつ、純度が高いため、種々の分野において極めて有用である。 (Final product)
The PHB copolymer powder according to one embodiment of the present invention (hereinafter, referred to as “the second PHB copolymer powder”) contains a PHB copolymer and a nitrogen compound, and the PHB. The copolymer has a composition ratio of 80/20 to 88/12 (mol / mol) of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit, and a bulk density of 0.45 g /. A PHB copolymer powder having a size of mL or more and a median particle size of 80 to 200 μm. The second PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate units, and has a high bulk density and high purity. It is extremely useful in the field of.
本発明の一実施形態に係るＰＨＢ共重合体粉体（以下、「本第２のＰＨＢ共重合体粉体」と称する。）は、ＰＨＢ共重合体と、窒素化合物と、を含み、前記ＰＨＢ共重合体が、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ＰＨＢ共重合体粉体である。本第２のＰＨＢ共重合体粉体は、３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が高いＰＨＢ共重合体であるとともに、嵩密度が高く、かつ、純度が高いため、種々の分野において極めて有用である。 (Final product)
The PHB copolymer powder according to one embodiment of the present invention (hereinafter, referred to as “the second PHB copolymer powder”) contains a PHB copolymer and a nitrogen compound, and the PHB. The copolymer has a composition ratio of 80/20 to 88/12 (mol / mol) of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit, and a bulk density of 0.45 g /. A PHB copolymer powder having a size of mL or more and a median particle size of 80 to 200 μm. The second PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate units, and has a high bulk density and high purity. It is extremely useful in the field of.
本明細書において、「窒素化合物」とは、少なくとも窒素を含有する化合物を意味する。窒素化合物は、ＰＨＢ共重合体粉体に残存するタンパク質や細胞壁の成分であるペプチドグリカンの分解物である。
As used herein, the term "nitrogen compound" means a compound containing at least nitrogen. The nitrogen compound is a decomposition product of the protein remaining in the PHB copolymer powder and peptidoglycan which is a component of the cell wall.
本実施形態において、「ＰＨＢ共重合体」、および「窒素化合物」については、上記したものが援用される。また、「嵩密度」、および「メジアン粒子径」については、上記（中間体）の項で記載したものが援用される。
In the present embodiment, the above-mentioned ones are used as the "PHB copolymer" and the "nitrogen compound". As for "bulk density" and "median particle size", those described in the above (intermediate) section are used.
本第２のＰＨＢ共重合体粉体は、窒素化合物を含む。本第２のＰＨＢ共重合体粉体中の全窒素量は、特に限定されないが、ＰＨＢ共重合体粉体を構成するＰＨＢ共重合体１００重量部に対して、０．０１０～０．０７５重量部（ｐｈｒ）が好ましく、０．０１～０．０６重量部（ｐｈｒ）がより好ましい。全窒素量を上記範囲とすることにより、ＰＨＢ共重合体粉体の純度が向上する傾向がある。本第２のＰＨＢ共重合体粉体中の全窒素量は、実施例に記載の方法により測定される。
The second PHB copolymer powder contains a nitrogen compound. The total amount of nitrogen in the second PHB copolymer powder is not particularly limited, but is 0.010 to 0.075 weight with respect to 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. The part (phr) is preferable, and 0.01 to 0.06 parts by weight (phr) is more preferable. By setting the total amount of nitrogen in the above range, the purity of the PHB copolymer powder tends to be improved. The total amount of nitrogen in the second PHB copolymer powder is measured by the method described in Examples.
また、本第２のＰＨＢ共重合体粉体は、本発明の効果を奏する限り、製造の過程で生じた、または除去されなかった種々の成分を含んでいてもよい。
Further, the second PHB copolymer powder may contain various components generated or not removed in the manufacturing process as long as the effect of the present invention is exhibited.
本発明の一実施形態において、本第２のＰＨＢ共重合体粉体は、最終生成物であり、上記した本製造方法（例えば、工程（ａ）～（ｆ））により製造される。
In one embodiment of the present invention, the second PHB copolymer powder is a final product and is produced by the above-mentioned production method (for example, steps (a) to (f)).
本ＰＨＢ共重合体粉体は、紙、フィルム、シート、チューブ、板、棒、容器（例えば、ボトル容器等）、袋、部品等、種々の用途に利用できる。
This PHB copolymer powder can be used for various purposes such as paper, film, sheet, tube, plate, rod, container (for example, bottle container, etc.), bag, parts, and the like.
本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。
The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the embodiment obtained by appropriately combining the technical means disclosed in the different embodiments. Is also included in the technical scope of the present invention.
すなわち、本発明の一実施形態は、以下である。
＜１＞ポリヒドロキシ酪酸共重合体を製造する方法であって、
前記ポポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
（ａ）前記ポリヒドロキシ酪酸共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、
（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、
（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および
（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、
を含む、ポリヒドロキシ酪酸共重合体の製造方法。
＜２＞前記工程（ｄ）の後に、
（ｅ）前記工程（ｄ）で得られたポリヒドロキシ酪酸共重合体粉体を洗浄する工程、および
（ｆ）前記工程（ｅ）で得られたポリヒドロキシ酪酸共重合体粉体を脱水および／または乾燥する工程
をさらに含む、＜１＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜３＞前記３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位が、３－ヒドロキシヘキサノエート単位である、＜１＞または＜２＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜４＞前記工程（ｂ）の界面活性剤がドデシル硫酸ナトリウムである、＜１＞～＜３＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜５＞前記水性懸濁液が、分散剤をさらに含む、＜１＞～＜４＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜６＞前記分散剤が、アルキレンオキサイド系分散剤、セルロース系分散剤およびポリビニルアルコールからなる群より選択される少なくとも１種である、＜５＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜７＞前記工程（ｃ）で調製する水性懸濁液におけるポリヒドロキシ酪酸共重合体の濃度が、３０～６５重量％である、＜１＞～＜６＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜８＞前記工程（ｅ）の洗浄液がアルカリ性タンパク質分解酵素および／または溶菌酵素を含む、＜２＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜９＞ポリヒドロキシ酪酸共重合体と、ペプチドグリカンと、分散剤と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。
＜１０＞前記ペプチドグリカン含有量が０．１～１．５ｐｈｒである、＜９＞に記載のポリヒドロキシ酪酸共重合体粉体。
＜１１＞残タンパク質量が０．１～１．０ｐｈｒである、＜８＞または＜９＞に記載のポリヒドロキシ酪酸共重合体粉体。
＜１２＞ポリヒドロキシ酪酸共重合体と、窒素化合物と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。
＜１３＞前記ポリヒドロキシ酪酸共重合体粉体中の全窒素量が０．０１０～０．０７５ｐｈｒである、＜１２＞に記載のポリヒドロキシ酪酸共重合体粉体。 That is, one embodiment of the present invention is as follows.
<1> A method for producing a polyhydroxybutyrate copolymer.
The popopolyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
(A) A step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the cells.
(B) An alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and either before the adjustment, at the same time as the adjustment, or after the adjustment. In the process of adding a surfactant,
(C) From the aqueous suspension obtained in the step (b), an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less. And (d) the step of spray-drying the aqueous suspension prepared in the above step (c),
A method for producing a polyhydroxybutyric acid copolymer.
<2> After the step (d),
(E) The step of washing the polyhydroxybutyrate copolymer powder obtained in the step (d), and (f) the polyhydroxybutyrate copolymer powder obtained in the step (e) of dehydration and / The method for producing a polyhydroxybutyrate copolymer according to <1>, further comprising a step of drying.
<3> The method for producing a polyhydroxybutyric acid copolymer according to <1> or <2>, wherein the hydroxyalkanoate unit other than the 3-hydroxybutyrate unit is a 3-hydroxyhexanoate unit.
<4> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <3>, wherein the surfactant in the step (b) is sodium dodecyl sulfate.
<5> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <4>, wherein the aqueous suspension further contains a dispersant.
<6> The method for producing a polyhydroxybutyric acid copolymer according to <5>, wherein the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
<7> The polyhydroxybutyric acid according to any one of <1> to <6>, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight. Method for producing a copolymer.
<8> The method for producing a polyhydroxybutyric acid copolymer according to <2>, wherein the cleaning solution of the step (e) contains an alkaline proteolytic enzyme and / or a lytic enzyme.
<9> Containing a polyhydroxybutyrate copolymer, peptidoglycan, and a dispersant,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm.
<10> The polyhydroxybutyrate copolymer powder according to <9>, wherein the peptidoglycan content is 0.1 to 1.5 phr.
<11> The polyhydroxybutyrate copolymer powder according to <8> or <9>, wherein the amount of residual protein is 0.1 to 1.0 phr.
<12> Containing a polyhydroxybutyrate copolymer and a nitrogen compound,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm.
<13> The polyhydroxybutyrate copolymer powder according to <12>, wherein the total amount of nitrogen in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr.
＜１＞ポリヒドロキシ酪酸共重合体を製造する方法であって、
前記ポポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
（ａ）前記ポリヒドロキシ酪酸共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、
（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、
（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および
（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、
を含む、ポリヒドロキシ酪酸共重合体の製造方法。
＜２＞前記工程（ｄ）の後に、
（ｅ）前記工程（ｄ）で得られたポリヒドロキシ酪酸共重合体粉体を洗浄する工程、および
（ｆ）前記工程（ｅ）で得られたポリヒドロキシ酪酸共重合体粉体を脱水および／または乾燥する工程
をさらに含む、＜１＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜３＞前記３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位が、３－ヒドロキシヘキサノエート単位である、＜１＞または＜２＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜４＞前記工程（ｂ）の界面活性剤がドデシル硫酸ナトリウムである、＜１＞～＜３＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜５＞前記水性懸濁液が、分散剤をさらに含む、＜１＞～＜４＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜６＞前記分散剤が、アルキレンオキサイド系分散剤、セルロース系分散剤およびポリビニルアルコールからなる群より選択される少なくとも１種である、＜５＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜７＞前記工程（ｃ）で調製する水性懸濁液におけるポリヒドロキシ酪酸共重合体の濃度が、３０～６５重量％である、＜１＞～＜６＞のいずれかに記載のポリヒドロキシ酪酸共重合体の製造方法。
＜８＞前記工程（ｅ）の洗浄液がアルカリ性タンパク質分解酵素および／または溶菌酵素を含む、＜２＞に記載のポリヒドロキシ酪酸共重合体の製造方法。
＜９＞ポリヒドロキシ酪酸共重合体と、ペプチドグリカンと、分散剤と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。
＜１０＞前記ペプチドグリカン含有量が０．１～１．５ｐｈｒである、＜９＞に記載のポリヒドロキシ酪酸共重合体粉体。
＜１１＞残タンパク質量が０．１～１．０ｐｈｒである、＜８＞または＜９＞に記載のポリヒドロキシ酪酸共重合体粉体。
＜１２＞ポリヒドロキシ酪酸共重合体と、窒素化合物と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。
＜１３＞前記ポリヒドロキシ酪酸共重合体粉体中の全窒素量が０．０１０～０．０７５ｐｈｒである、＜１２＞に記載のポリヒドロキシ酪酸共重合体粉体。 That is, one embodiment of the present invention is as follows.
<1> A method for producing a polyhydroxybutyrate copolymer.
The popopolyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
(A) A step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the cells.
(B) An alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and either before the adjustment, at the same time as the adjustment, or after the adjustment. In the process of adding a surfactant,
(C) From the aqueous suspension obtained in the step (b), an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less. And (d) the step of spray-drying the aqueous suspension prepared in the above step (c),
A method for producing a polyhydroxybutyric acid copolymer.
<2> After the step (d),
(E) The step of washing the polyhydroxybutyrate copolymer powder obtained in the step (d), and (f) the polyhydroxybutyrate copolymer powder obtained in the step (e) of dehydration and / The method for producing a polyhydroxybutyrate copolymer according to <1>, further comprising a step of drying.
<3> The method for producing a polyhydroxybutyric acid copolymer according to <1> or <2>, wherein the hydroxyalkanoate unit other than the 3-hydroxybutyrate unit is a 3-hydroxyhexanoate unit.
<4> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <3>, wherein the surfactant in the step (b) is sodium dodecyl sulfate.
<5> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <4>, wherein the aqueous suspension further contains a dispersant.
<6> The method for producing a polyhydroxybutyric acid copolymer according to <5>, wherein the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
<7> The polyhydroxybutyric acid according to any one of <1> to <6>, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight. Method for producing a copolymer.
<8> The method for producing a polyhydroxybutyric acid copolymer according to <2>, wherein the cleaning solution of the step (e) contains an alkaline proteolytic enzyme and / or a lytic enzyme.
<9> Containing a polyhydroxybutyrate copolymer, peptidoglycan, and a dispersant,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm.
<10> The polyhydroxybutyrate copolymer powder according to <9>, wherein the peptidoglycan content is 0.1 to 1.5 phr.
<11> The polyhydroxybutyrate copolymer powder according to <8> or <9>, wherein the amount of residual protein is 0.1 to 1.0 phr.
<12> Containing a polyhydroxybutyrate copolymer and a nitrogen compound,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm.
<13> The polyhydroxybutyrate copolymer powder according to <12>, wherein the total amount of nitrogen in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr.
本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。
The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. Is also included in the technical scope of the present invention.
以下、本発明を実施例に基づいてより詳細に説明するが、本発明はこれら実施例に限定されるものではない。なお、実施例において、「ＰＨＢ共重合体」としては「ＰＨＢＨ」を用いており、「ＰＨＢ共重合体」を「ＰＨＢＨ」と読み替えることができる。
Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples. In the examples, "PHBH" is used as the "PHB copolymer", and the "PHB copolymer" can be read as "PHBH".
〔測定および評価方法〕
実施例および比較例における測定および評価を、以下の方法で行った。 [Measurement and evaluation method]
Measurements and evaluations in Examples and Comparative Examples were performed by the following methods.
実施例および比較例における測定および評価を、以下の方法で行った。 [Measurement and evaluation method]
Measurements and evaluations in Examples and Comparative Examples were performed by the following methods.
（体積メジアン径）
ＰＨＢ共重合体水性懸濁液中の体積メジアン径は、ＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定した。 (Volume median diameter)
The volume median diameter in the aqueous suspension of PHB copolymer was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 manufactured by HORIBA.
ＰＨＢ共重合体水性懸濁液中の体積メジアン径は、ＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定した。 (Volume median diameter)
The volume median diameter in the aqueous suspension of PHB copolymer was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 manufactured by HORIBA.
（ＰＨＢ共重合体水性懸濁液のせん断粘度）
ＰＨＢ共重合体水性懸濁液のせん断粘度は、以下の方法により測定した。具体的には、Ａｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて、同軸２重円筒にてせん断粘度を測定した。ＰＨＢ共重合体水性懸濁液を２０ｍＬ円筒に投入し、液温度が１５℃になるまで、せん断速度１０（１／ｓ）の条件化で冷却した。その後、２０℃まで液温度を昇温し、目的の液温度に到達後、トルクの時間変化が１％未満になった際の粘度を測定した。 (Shear viscosity of aqueous suspension of PHB copolymer)
The shear viscosity of the aqueous suspension of PHB copolymer was measured by the following method. Specifically, the shear viscosity was measured with a coaxial double cylinder using MCR302 manufactured by Antonio Par. The aqueous suspension of the PHB copolymer was placed in a 20 mL cylinder and cooled under the condition of a shear rate of 10 (1 / s) until the liquid temperature reached 15 ° C. Then, the liquid temperature was raised to 20 ° C., and after reaching the target liquid temperature, the viscosity when the time change of the torque became less than 1% was measured.
ＰＨＢ共重合体水性懸濁液のせん断粘度は、以下の方法により測定した。具体的には、Ａｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて、同軸２重円筒にてせん断粘度を測定した。ＰＨＢ共重合体水性懸濁液を２０ｍＬ円筒に投入し、液温度が１５℃になるまで、せん断速度１０（１／ｓ）の条件化で冷却した。その後、２０℃まで液温度を昇温し、目的の液温度に到達後、トルクの時間変化が１％未満になった際の粘度を測定した。 (Shear viscosity of aqueous suspension of PHB copolymer)
The shear viscosity of the aqueous suspension of PHB copolymer was measured by the following method. Specifically, the shear viscosity was measured with a coaxial double cylinder using MCR302 manufactured by Antonio Par. The aqueous suspension of the PHB copolymer was placed in a 20 mL cylinder and cooled under the condition of a shear rate of 10 (1 / s) until the liquid temperature reached 15 ° C. Then, the liquid temperature was raised to 20 ° C., and after reaching the target liquid temperature, the viscosity when the time change of the torque became less than 1% was measured.
（ペプチドグリカン量）
ＰＨＢ共重合体粉体中に残存するペプチドグリカン量は、以下の方法により測定した。具体的には、まず、ペプチドグリカン標準品５０μＬをマイクロプレートに入れた。次いで、ＳＬＰ－ＨＳシングル試薬セット（Ｗａｋｏ ＬＡＬシステム）の試薬５０μＬを上記マイクロプレートに入れた。６５０ｎｍの波長で１５秒毎に吸光度を測定し、測定開始から３時間後の吸光度を最終吸光度として、吸光度が最終吸光度の０．４倍になる時間を測定した。同様に、ペプチドグリカン標準品を１０００～１００００倍に蒸留水で希釈して、上記と同様の操作を行い、検量線を作成した。その後、工程（ｃ）で調製したＰＨＢ共重合体水性懸濁液を１０００～１００００倍に蒸留水で希釈し、希釈した液のうち５０μＬをマイクロプレートに入れた。次いで、ＳＬＰ－ＨＳシングル試薬セット（Ｗａｋｏ ＬＡＬシステム）の試薬５０μＬを上記マイクロプレートに入れた。６５０ｎｍの波長で１５秒毎に吸光度を測定し、測定開始から３時間後の吸光度を最終吸光度として、吸光度が最終吸光度の０．４倍になる時間からペプチドグリカン量を算出した。 (Amount of peptidoglycan)
The amount of peptidoglycan remaining in the PHB copolymer powder was measured by the following method. Specifically, first, 50 μL of peptidoglycan standard product was placed in a microplate. Then, 50 μL of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the time at which the absorbance became 0.4 times the final absorbance was measured, with the absorbance 3 hours after the start of the measurement as the final absorbance. Similarly, the standard peptidoglycan was diluted 1000 to 10000 times with distilled water, and the same operation as above was carried out to prepare a calibration curve. Then, the aqueous suspension of PHB copolymer prepared in the step (c) was diluted 1000 to 10000 times with distilled water, and 50 μL of the diluted solution was placed in a microplate. Then, 50 μL of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the amount of peptide glycan was calculated from the time when the absorbance became 0.4 times the final absorbance, with the absorbance 3 hours after the start of measurement as the final absorbance.
ＰＨＢ共重合体粉体中に残存するペプチドグリカン量は、以下の方法により測定した。具体的には、まず、ペプチドグリカン標準品５０μＬをマイクロプレートに入れた。次いで、ＳＬＰ－ＨＳシングル試薬セット（Ｗａｋｏ ＬＡＬシステム）の試薬５０μＬを上記マイクロプレートに入れた。６５０ｎｍの波長で１５秒毎に吸光度を測定し、測定開始から３時間後の吸光度を最終吸光度として、吸光度が最終吸光度の０．４倍になる時間を測定した。同様に、ペプチドグリカン標準品を１０００～１００００倍に蒸留水で希釈して、上記と同様の操作を行い、検量線を作成した。その後、工程（ｃ）で調製したＰＨＢ共重合体水性懸濁液を１０００～１００００倍に蒸留水で希釈し、希釈した液のうち５０μＬをマイクロプレートに入れた。次いで、ＳＬＰ－ＨＳシングル試薬セット（Ｗａｋｏ ＬＡＬシステム）の試薬５０μＬを上記マイクロプレートに入れた。６５０ｎｍの波長で１５秒毎に吸光度を測定し、測定開始から３時間後の吸光度を最終吸光度として、吸光度が最終吸光度の０．４倍になる時間からペプチドグリカン量を算出した。 (Amount of peptidoglycan)
The amount of peptidoglycan remaining in the PHB copolymer powder was measured by the following method. Specifically, first, 50 μL of peptidoglycan standard product was placed in a microplate. Then, 50 μL of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the time at which the absorbance became 0.4 times the final absorbance was measured, with the absorbance 3 hours after the start of the measurement as the final absorbance. Similarly, the standard peptidoglycan was diluted 1000 to 10000 times with distilled water, and the same operation as above was carried out to prepare a calibration curve. Then, the aqueous suspension of PHB copolymer prepared in the step (c) was diluted 1000 to 10000 times with distilled water, and 50 μL of the diluted solution was placed in a microplate. Then, 50 μL of the reagent of the SLP-HS single reagent set (Wako LAL system) was placed in the above microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the amount of peptide glycan was calculated from the time when the absorbance became 0.4 times the final absorbance, with the absorbance 3 hours after the start of measurement as the final absorbance.
（残タンパク質量）
ＰＨＢ共重合体粉体の残タンパク質量は、ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて測定した。具体的には、１０ｍｇのＰＨＢＨ粉体を１４ｍＬのファルコンチューブに投入し、上記試薬２ｍＬを添加し、６０℃で３０分間振とうした。３０分後に冷却し、５６２ｎｍの波長の吸光度を測定した。 (Amount of remaining protein)
The amount of residual protein in the PHB copolymer powder was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was put into a 14 mL falcon tube, 2 mL of the above reagent was added, and the mixture was shaken at 60 ° C. for 30 minutes. After 30 minutes, it was cooled and the absorbance at a wavelength of 562 nm was measured.
ＰＨＢ共重合体粉体の残タンパク質量は、ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて測定した。具体的には、１０ｍｇのＰＨＢＨ粉体を１４ｍＬのファルコンチューブに投入し、上記試薬２ｍＬを添加し、６０℃で３０分間振とうした。３０分後に冷却し、５６２ｎｍの波長の吸光度を測定した。 (Amount of remaining protein)
The amount of residual protein in the PHB copolymer powder was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was put into a 14 mL falcon tube, 2 mL of the above reagent was added, and the mixture was shaken at 60 ° C. for 30 minutes. After 30 minutes, it was cooled and the absorbance at a wavelength of 562 nm was measured.
（嵩密度）
ＰＨＢ共重合体粉体の嵩密度は、JIS K 7365 : 1999に基づき、嵩比重測定器（蔵持科学器械製）を用いて測定した。 (The bulk density)
The bulk density of the PHB copolymer powder was measured using a bulk specific gravity measuring instrument (manufactured by Kuramochi Kagaku Kikai) based on JIS K 7365: 1999.
ＰＨＢ共重合体粉体の嵩密度は、JIS K 7365 : 1999に基づき、嵩比重測定器（蔵持科学器械製）を用いて測定した。 (The bulk density)
The bulk density of the PHB copolymer powder was measured using a bulk specific gravity measuring instrument (manufactured by Kuramochi Kagaku Kikai) based on JIS K 7365: 1999.
（メジアン粒子径）
ＰＨＢ共重合体粉体のメジアン粒子径は、以下の方法により測定した。具体的には、メジアン粒子径は、レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０（ＨＯＲＩＢＡ社）を用いて測定した。イオン交換水２０ｍＬに、界面活性剤としてドデシル硫酸ナトリウム０．０５ｇを加えて、界面活性剤水溶液を得た。次いで、上記界面活性剤水溶液に、測定対象のＰＨＢ共重合体粉体０．２ｇを加えて、上記ＰＨＢ共重合体粉体を上記界面活性剤水溶液中に分散させ、測定用の分散液を得た。調製した分散液を、上記レーザ回折／散乱式粒子径分布測定装置に導入し、測定を行った。 (Median particle size)
The median particle size of the PHB copolymer powder was measured by the following method. Specifically, the median particle size was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 (HORIBA). To 20 mL of ion-exchanged water, 0.05 g of sodium dodecyl sulfate was added as a surfactant to obtain an aqueous surfactant solution. Next, 0.2 g of the PHB copolymer powder to be measured was added to the aqueous surfactant solution, and the PHB copolymer powder was dispersed in the aqueous surfactant solution to obtain a dispersion for measurement. rice field. The prepared dispersion was introduced into the laser diffraction / scattering type particle size distribution measuring device and measured.
ＰＨＢ共重合体粉体のメジアン粒子径は、以下の方法により測定した。具体的には、メジアン粒子径は、レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０（ＨＯＲＩＢＡ社）を用いて測定した。イオン交換水２０ｍＬに、界面活性剤としてドデシル硫酸ナトリウム０．０５ｇを加えて、界面活性剤水溶液を得た。次いで、上記界面活性剤水溶液に、測定対象のＰＨＢ共重合体粉体０．２ｇを加えて、上記ＰＨＢ共重合体粉体を上記界面活性剤水溶液中に分散させ、測定用の分散液を得た。調製した分散液を、上記レーザ回折／散乱式粒子径分布測定装置に導入し、測定を行った。 (Median particle size)
The median particle size of the PHB copolymer powder was measured by the following method. Specifically, the median particle size was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 (HORIBA). To 20 mL of ion-exchanged water, 0.05 g of sodium dodecyl sulfate was added as a surfactant to obtain an aqueous surfactant solution. Next, 0.2 g of the PHB copolymer powder to be measured was added to the aqueous surfactant solution, and the PHB copolymer powder was dispersed in the aqueous surfactant solution to obtain a dispersion for measurement. rice field. The prepared dispersion was introduced into the laser diffraction / scattering type particle size distribution measuring device and measured.
（純度）
ＰＨＢ共重合体粉体の純度は、以下の方法により測定した。具体的には、ＴＧ－ＤＴＡ（２０００ＳＥ NIETZSCHE社製）にて、ＰＨＢＨ乾燥粉体を約１０ｍｇ（重量をＷとする。）を１０℃／ｍｉｎで５０℃から５００℃まで窒素雰囲気下で昇温し、温度とサンプル重量のグラフを作成した。２８０－２８５℃の重量減少の傾きをもつ１次直線と４００－４５０℃の重量減少の傾きをもつ１次直線との交点Ａ、２８０－２８５℃の重量減少の傾きをもつ１次直線と１５０－２００℃の重量減少の傾きをもつ１次直線との交点Ｂの重量をグラフから算出した。交点Ａと交点Ｂの重量差がＰＨＢＨ量としてＷ‘を算出し、Ｗ’／ＷをＰＨＢＨの純度とした。 (purity)
The purity of the PHB copolymer powder was measured by the following method. Specifically, in TG-DTA (manufactured by 2000SE NIETZSCHE), about 10 mg of PHBH dry powder (weight is W) is heated at 10 ° C / min from 50 ° C to 500 ° C in a nitrogen atmosphere. Then, a graph of temperature and sample weight was created. Intersection A of a linear line with a weight loss slope of 280-285 ° C. and a linear line with a weight loss slope of 400-450 ° C. A, a linear line with a weight loss slope of 280-285 ° C. and 150 The weight of the intersection B with the linear straight line having a slope of weight reduction of −200 ° C. was calculated from the graph. W'was calculated as the amount of PHBH by the weight difference between the intersection A and the intersection B, and W'/ W was defined as the purity of PHBH.
ＰＨＢ共重合体粉体の純度は、以下の方法により測定した。具体的には、ＴＧ－ＤＴＡ（２０００ＳＥ NIETZSCHE社製）にて、ＰＨＢＨ乾燥粉体を約１０ｍｇ（重量をＷとする。）を１０℃／ｍｉｎで５０℃から５００℃まで窒素雰囲気下で昇温し、温度とサンプル重量のグラフを作成した。２８０－２８５℃の重量減少の傾きをもつ１次直線と４００－４５０℃の重量減少の傾きをもつ１次直線との交点Ａ、２８０－２８５℃の重量減少の傾きをもつ１次直線と１５０－２００℃の重量減少の傾きをもつ１次直線との交点Ｂの重量をグラフから算出した。交点Ａと交点Ｂの重量差がＰＨＢＨ量としてＷ‘を算出し、Ｗ’／ＷをＰＨＢＨの純度とした。 (purity)
The purity of the PHB copolymer powder was measured by the following method. Specifically, in TG-DTA (manufactured by 2000SE NIETZSCHE), about 10 mg of PHBH dry powder (weight is W) is heated at 10 ° C / min from 50 ° C to 500 ° C in a nitrogen atmosphere. Then, a graph of temperature and sample weight was created. Intersection A of a linear line with a weight loss slope of 280-285 ° C. and a linear line with a weight loss slope of 400-450 ° C. A, a linear line with a weight loss slope of 280-285 ° C. and 150 The weight of the intersection B with the linear straight line having a slope of weight reduction of −200 ° C. was calculated from the graph. W'was calculated as the amount of PHBH by the weight difference between the intersection A and the intersection B, and W'/ W was defined as the purity of PHBH.
（全窒素量測定）
ＰＨＢ共重合体紛体の全窒素量は、微量全窒素分析装置ＴＮ－２１００Ｈ（日東精工アナリテック社）を用いて測定した。 (Measurement of total nitrogen content)
The total nitrogen content of the PHB copolymer powder was measured using a trace total nitrogen analyzer TN-2100H (Nittoseiko Analytech Co., Ltd.).
ＰＨＢ共重合体紛体の全窒素量は、微量全窒素分析装置ＴＮ－２１００Ｈ（日東精工アナリテック社）を用いて測定した。 (Measurement of total nitrogen content)
The total nitrogen content of the PHB copolymer powder was measured using a trace total nitrogen analyzer TN-2100H (Nittoseiko Analytech Co., Ltd.).
〔実施例１〕
（菌体培養液の調製）
国際公開第ＷＯ２０１９／１４２７１７号に記載のラルストニア・ユートロファを、同文献の段落〔００４１〕～〔００４８〕に記載の方法で培養し、ＰＨＢ共重合体を含有する菌体を含む菌体培養液を得た。なお、ラルストニア・ユートロファは、現在では、カプリアビダス・ネカトールに分類されている。ＰＨＢ共重合体の繰り返し単位の組成比（３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比）は８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であった。 [Example 1]
(Preparation of cell culture solution)
Ralstonia eutropha described in International Publication No. WO2019 / 142717 is cultured by the method described in paragraphs [0041] to [0048] of the same document, and a cell culture solution containing cells containing a PHB copolymer is prepared. Obtained. Ralstonia eutropha is now classified as Cupriavidus necator. The composition ratio of the repeating unit of the PHB copolymer (the composition ratio of the hydroxy alkanoate unit other than 3HB unit / 3HB unit) was 80/20 to 88/12 (mol / mol).
（菌体培養液の調製）
国際公開第ＷＯ２０１９／１４２７１７号に記載のラルストニア・ユートロファを、同文献の段落〔００４１〕～〔００４８〕に記載の方法で培養し、ＰＨＢ共重合体を含有する菌体を含む菌体培養液を得た。なお、ラルストニア・ユートロファは、現在では、カプリアビダス・ネカトールに分類されている。ＰＨＢ共重合体の繰り返し単位の組成比（３ＨＢ単位／３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比）は８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であった。 [Example 1]
(Preparation of cell culture solution)
Ralstonia eutropha described in International Publication No. WO2019 / 142717 is cultured by the method described in paragraphs [0041] to [0048] of the same document, and a cell culture solution containing cells containing a PHB copolymer is prepared. Obtained. Ralstonia eutropha is now classified as Cupriavidus necator. The composition ratio of the repeating unit of the PHB copolymer (the composition ratio of the hydroxy alkanoate unit other than 3HB unit / 3HB unit) was 80/20 to 88/12 (mol / mol).
（不活化）
上記で得られた菌体培養液を内温６０～７０℃で７時間加熱および攪拌処理し、滅菌処理を行い、その後５５℃まで冷却することで、不活化培養液を得た。 (Inactivated)
The cell culture broth obtained above was heated and stirred at an internal temperature of 60 to 70 ° C. for 7 hours, sterilized, and then cooled to 55 ° C. to obtain an inactivated culture broth.
上記で得られた菌体培養液を内温６０～７０℃で７時間加熱および攪拌処理し、滅菌処理を行い、その後５５℃まで冷却することで、不活化培養液を得た。 (Inactivated)
The cell culture broth obtained above was heated and stirred at an internal temperature of 60 to 70 ° C. for 7 hours, sterilized, and then cooled to 55 ° C. to obtain an inactivated culture broth.
（酵素処理）
上記不活化培養液に対して３０％水酸化ナトリウムを用いてｐＨを８．５±０．２に調整した。その後、タンパク質分解酵素であるアルカラーゼ（Ｎｏｖｏｚｙｍｅｓ社製）を０．２ｐｈｒ添加し、５０℃で３０％水酸化ナトリウムにてｐＨ８．５にコントロールしながら、２時間以上ホールドした。 (Enzyme treatment)
The pH of the inactivated culture solution was adjusted to 8.5 ± 0.2 with 30% sodium hydroxide. Then, 0.2 phr of alcalase (manufactured by Novozymes), which is a proteolytic enzyme, was added, and the mixture was held for 2 hours or more while controlling the pH to 8.5 with 30% sodium hydroxide at 50 ° C.
上記不活化培養液に対して３０％水酸化ナトリウムを用いてｐＨを８．５±０．２に調整した。その後、タンパク質分解酵素であるアルカラーゼ（Ｎｏｖｏｚｙｍｅｓ社製）を０．２ｐｈｒ添加し、５０℃で３０％水酸化ナトリウムにてｐＨ８．５にコントロールしながら、２時間以上ホールドした。 (Enzyme treatment)
The pH of the inactivated culture solution was adjusted to 8.5 ± 0.2 with 30% sodium hydroxide. Then, 0.2 phr of alcalase (manufactured by Novozymes), which is a proteolytic enzyme, was added, and the mixture was held for 2 hours or more while controlling the pH to 8.5 with 30% sodium hydroxide at 50 ° C.
（溶菌、濃縮）
上記で得られた酵素処理液に対してアルカリ水溶液を添加し、ｐＨを１１．５以上に調整し、４５℃以上で４時間以上保持した。その後、０．６～１．０ｗｔ％のドデシル硫酸ナトリウム（花王製）を添加し、かつ、ｐＨが１２前後になるように３０％水酸化ナトリウムを添加するとともに、アルカリ水溶液（ｐＨ１０～１１．５）で２倍に希釈した。これを遠心分離した後、上清を除去して２倍濃縮した。この濃縮したＰＨＢ共重合体の水性懸濁液に、除去した上清と同量の水酸化ナトリウム水溶液（ｐＨ１１）を添加して遠心分離し、上清を除去した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を７回繰り返した。このＰＨＢ共重合体水性懸濁液を４倍濃縮して、ＰＨＢ共重合体濃度が５２重量％以上になるように調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、１．９μｍであった。 (Lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above, the pH was adjusted to 11.5 or higher, and the pH was maintained at 45 ° C. or higher for 4 hours or longer. Then, 0.6 to 1.0 wt% sodium dodecyl sulfate (manufactured by Kao) is added, and 30% sodium hydroxide is added so that the pH becomes around 12, and an alkaline aqueous solution (pH 10 to 11.5) is added. ) Was diluted 2-fold. After centrifuging this, the supernatant was removed and concentrated twice. To the aqueous suspension of the concentrated PHB copolymer, the same amount of sodium hydroxide aqueous solution (pH 11) as the removed supernatant was added and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated 7 times. The PHB copolymer aqueous suspension was concentrated 4-fold to adjust the PHB copolymer concentration to 52% by weight or more. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 μm.
上記で得られた酵素処理液に対してアルカリ水溶液を添加し、ｐＨを１１．５以上に調整し、４５℃以上で４時間以上保持した。その後、０．６～１．０ｗｔ％のドデシル硫酸ナトリウム（花王製）を添加し、かつ、ｐＨが１２前後になるように３０％水酸化ナトリウムを添加するとともに、アルカリ水溶液（ｐＨ１０～１１．５）で２倍に希釈した。これを遠心分離した後、上清を除去して２倍濃縮した。この濃縮したＰＨＢ共重合体の水性懸濁液に、除去した上清と同量の水酸化ナトリウム水溶液（ｐＨ１１）を添加して遠心分離し、上清を除去した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を７回繰り返した。このＰＨＢ共重合体水性懸濁液を４倍濃縮して、ＰＨＢ共重合体濃度が５２重量％以上になるように調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、１．９μｍであった。 (Lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above, the pH was adjusted to 11.5 or higher, and the pH was maintained at 45 ° C. or higher for 4 hours or longer. Then, 0.6 to 1.0 wt% sodium dodecyl sulfate (manufactured by Kao) is added, and 30% sodium hydroxide is added so that the pH becomes around 12, and an alkaline aqueous solution (pH 10 to 11.5) is added. ) Was diluted 2-fold. After centrifuging this, the supernatant was removed and concentrated twice. To the aqueous suspension of the concentrated PHB copolymer, the same amount of sodium hydroxide aqueous solution (pH 11) as the removed supernatant was added and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated 7 times. The PHB copolymer aqueous suspension was concentrated 4-fold to adjust the PHB copolymer concentration to 52% by weight or more. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 μm.
（造粒）
上記で得られたＰＨＢ共重合体水性懸濁液にエチレンオキサイド／プロピレンオキサイド共重合体非イオン性分散剤（ポリエチレンオキサイド分子量８０００、ポリプロピレンオキサイド分子量２０００、商品名プロノン２０８）を０．９５ｐｈｒ（水性懸濁液中に存在するＰＨＢ共重合体１００重量部に対して０．９５重量部）添加した。次いで、メチルセルロース（商品名ＳＭ-１５）を０．３ｐｈｒ添加した。この液を撹拌し、液温度を６０℃まで上昇させ、ｐＨが４．０になるように１０％硫酸を添加し、１２０分間撹拌を継続して、ＰＨＢ共重合体水性懸濁液を得た。その後、前記混合物の固形分濃度を、水を加えることにより５０質量％に調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、１．９μｍであった。さらに、前記ＰＨＢ共重合体水性懸濁液のせん断粘度をＡｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて測定したところ、０．１Ｐａ・ｓであった。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１１５℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、洗浄前ＰＨＢ共重合体粉体を得た。得られた洗浄前ＰＨＢ共重合体粉体に含まれるペプチドグリカン量は、１．０４ｐｈｒ、残タンパク質量は０．１８ｐｈｒ、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５１ｇ/ｍＬ、メジアン粒子径は１１０μｍ、純度は９７％、全窒素量は０．０９９ｐｈｒであった。 (Granulation)
An ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added to the PHB copolymer aqueous suspension obtained above at 0.95 phr (aqueous suspension). 0.95 parts by weight) was added to 100 parts by weight of the PHB copolymer present in the turbid liquid. Then, 0.3 phr of methyl cellulose (trade name SM-15) was added. This liquid was stirred, the liquid temperature was raised to 60 ° C., 10% sulfuric acid was added so that the pH became 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. .. Then, the solid content concentration of the mixture was adjusted to 50% by mass by adding water. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 μm. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa · s. The obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder before washing was obtained. The amount of peptidoglycan contained in the obtained pre-washed PHB copolymer powder was 1.04 phr, the amount of residual protein was 0.18 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g / mL, and the median particles. The diameter was 110 μm, the purity was 97%, and the total amount of nitrogen was 0.099 phr.
上記で得られたＰＨＢ共重合体水性懸濁液にエチレンオキサイド／プロピレンオキサイド共重合体非イオン性分散剤（ポリエチレンオキサイド分子量８０００、ポリプロピレンオキサイド分子量２０００、商品名プロノン２０８）を０．９５ｐｈｒ（水性懸濁液中に存在するＰＨＢ共重合体１００重量部に対して０．９５重量部）添加した。次いで、メチルセルロース（商品名ＳＭ-１５）を０．３ｐｈｒ添加した。この液を撹拌し、液温度を６０℃まで上昇させ、ｐＨが４．０になるように１０％硫酸を添加し、１２０分間撹拌を継続して、ＰＨＢ共重合体水性懸濁液を得た。その後、前記混合物の固形分濃度を、水を加えることにより５０質量％に調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、１．９μｍであった。さらに、前記ＰＨＢ共重合体水性懸濁液のせん断粘度をＡｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて測定したところ、０．１Ｐａ・ｓであった。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１１５℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、洗浄前ＰＨＢ共重合体粉体を得た。得られた洗浄前ＰＨＢ共重合体粉体に含まれるペプチドグリカン量は、１．０４ｐｈｒ、残タンパク質量は０．１８ｐｈｒ、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５１ｇ/ｍＬ、メジアン粒子径は１１０μｍ、純度は９７％、全窒素量は０．０９９ｐｈｒであった。 (Granulation)
An ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added to the PHB copolymer aqueous suspension obtained above at 0.95 phr (aqueous suspension). 0.95 parts by weight) was added to 100 parts by weight of the PHB copolymer present in the turbid liquid. Then, 0.3 phr of methyl cellulose (trade name SM-15) was added. This liquid was stirred, the liquid temperature was raised to 60 ° C., 10% sulfuric acid was added so that the pH became 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. .. Then, the solid content concentration of the mixture was adjusted to 50% by mass by adding water. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 1.9 μm. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa · s. The obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder before washing was obtained. The amount of peptidoglycan contained in the obtained pre-washed PHB copolymer powder was 1.04 phr, the amount of residual protein was 0.18 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g / mL, and the median particles. The diameter was 110 μm, the purity was 97%, and the total amount of nitrogen was 0.099 phr.
（洗浄１）
上記で得られた洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、その分散スラリーに硫酸ナトリウムを１ｐｈｒ添加した。その後、３０％水酸化ナトリウムで分散スラリーのｐＨを６になるように調整した。分散スラリーを５０℃まで昇温し、卵白リゾチーム（卵白リゾチームＦＧ、長瀬産業製）を０．００５０ｐｈｒ添加し２時間撹拌した。 (Washing 1)
The pre-washed PHB copolymer powder obtained above was dispersed in pure water so that the PHB copolymer concentration was 37.5%, and 1 phr of sodium sulfate was added to the dispersed slurry. Then, the pH of the dispersed slurry was adjusted to 6 with 30% sodium hydroxide. The temperature of the dispersed slurry was raised to 50 ° C., 0.0050 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.) was added, and the mixture was stirred for 2 hours.
上記で得られた洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、その分散スラリーに硫酸ナトリウムを１ｐｈｒ添加した。その後、３０％水酸化ナトリウムで分散スラリーのｐＨを６になるように調整した。分散スラリーを５０℃まで昇温し、卵白リゾチーム（卵白リゾチームＦＧ、長瀬産業製）を０．００５０ｐｈｒ添加し２時間撹拌した。 (Washing 1)
The pre-washed PHB copolymer powder obtained above was dispersed in pure water so that the PHB copolymer concentration was 37.5%, and 1 phr of sodium sulfate was added to the dispersed slurry. Then, the pH of the dispersed slurry was adjusted to 6 with 30% sodium hydroxide. The temperature of the dispersed slurry was raised to 50 ° C., 0.0050 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.) was added, and the mixture was stirred for 2 hours.
（洗浄２）
上記で得られた分散スラリーに３０％水酸化ナトリウムを加え、分散スラリーのｐＨが１１になるように調整した。その後、分散スラリーと同様の重量のｐＨ１１の水酸化ナトリウム水溶液を分散スラリーに添加し、遠心分離し、上澄みを排除した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を３回繰り返した。 (Washing 2)
30% sodium hydroxide was added to the dispersed slurry obtained above to adjust the pH of the dispersed slurry to 11. Then, a sodium hydroxide aqueous solution having the same weight as that of the dispersed slurry was added to the dispersed slurry and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated three times.
上記で得られた分散スラリーに３０％水酸化ナトリウムを加え、分散スラリーのｐＨが１１になるように調整した。その後、分散スラリーと同様の重量のｐＨ１１の水酸化ナトリウム水溶液を分散スラリーに添加し、遠心分離し、上澄みを排除した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を３回繰り返した。 (Washing 2)
30% sodium hydroxide was added to the dispersed slurry obtained above to adjust the pH of the dispersed slurry to 11. Then, a sodium hydroxide aqueous solution having the same weight as that of the dispersed slurry was added to the dispersed slurry and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated three times.
（乾燥）
上記で得られた分散スラリーに１０％硫酸を加え、分散スラリーのｐＨが４になるように調整した。その後、分散スラリー中のＰＨＢ共重合体量濃度が３５％になるように加水した。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１６０℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、ＰＨＢ共重合体粉体を得た。得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０５９ｐｈｒであり、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ／ｍＬ、メジアン粒子径は１０２μｍであった。 (Dry)
10% sulfuric acid was added to the dispersed slurry obtained above to adjust the pH of the dispersed slurry to 4. Then, water was added so that the concentration of the PHB copolymer in the dispersed slurry was 35%. The obtained aqueous suspension of PHB copolymer was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 160 ° C, exhaust air temperature: 75 ° C, rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder was obtained. The total amount of nitrogen contained in the obtained PHB copolymer powder was 0.059 phr, the bulk density of the PHB copolymer powder before washing was 0.50 g / mL, and the median particle size was 102 μm.
上記で得られた分散スラリーに１０％硫酸を加え、分散スラリーのｐＨが４になるように調整した。その後、分散スラリー中のＰＨＢ共重合体量濃度が３５％になるように加水した。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１６０℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、ＰＨＢ共重合体粉体を得た。得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０５９ｐｈｒであり、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ／ｍＬ、メジアン粒子径は１０２μｍであった。 (Dry)
10% sulfuric acid was added to the dispersed slurry obtained above to adjust the pH of the dispersed slurry to 4. Then, water was added so that the concentration of the PHB copolymer in the dispersed slurry was 35%. The obtained aqueous suspension of PHB copolymer was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 160 ° C, exhaust air temperature: 75 ° C, rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder was obtained. The total amount of nitrogen contained in the obtained PHB copolymer powder was 0.059 phr, the bulk density of the PHB copolymer powder before washing was 0.50 g / mL, and the median particle size was 102 μm.
〔実施例２〕
（造粒）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体を得た。洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、その分散スラリーに硫酸ナトリウムを１ｐｈｒ添加した。その後、３０％水酸化ナトリウムで分散スラリーのｐＨを１０．５になるように調整した。エスペラーゼ（Ｎｏｖｏｚｙｍｅ社）を０．０５ｐｈｒ添加し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０７０ｐｈｒであり、ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ/ｍＬ、メジアン粒子径は１０８μｍであった。 [Example 2]
Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation). The PHB copolymer powder before washing was dispersed in pure water so that the PHB copolymer concentration was 37.5%, and 1 phr of sodium sulfate was added to the dispersed slurry. Then, the pH of the dispersed slurry was adjusted to 10.5 with 30% sodium hydroxide. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.070 phr, and the bulk of the PHB copolymer powder was obtained. The density was 0.50 g / mL and the polymer particle size was 108 μm.
（造粒）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体を得た。洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、その分散スラリーに硫酸ナトリウムを１ｐｈｒ添加した。その後、３０％水酸化ナトリウムで分散スラリーのｐＨを１０．５になるように調整した。エスペラーゼ（Ｎｏｖｏｚｙｍｅ社）を０．０５ｐｈｒ添加し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０７０ｐｈｒであり、ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ/ｍＬ、メジアン粒子径は１０８μｍであった。 [Example 2]
Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation). The PHB copolymer powder before washing was dispersed in pure water so that the PHB copolymer concentration was 37.5%, and 1 phr of sodium sulfate was added to the dispersed slurry. Then, the pH of the dispersed slurry was adjusted to 10.5 with 30% sodium hydroxide. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.070 phr, and the bulk of the PHB copolymer powder was obtained. The density was 0.50 g / mL and the polymer particle size was 108 μm.
〔実施例３〕
（洗浄１）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体の分散スラリーを得た。得られた分散スラリーに３０％水酸化ナトリウムを加え、分散スラリーのｐＨを１０．５になるように調整した。エスペラーゼ（Ｎｏｖｏｚｙｍｅ社）を０．０５ｐｈｒ添加し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０３３ｐｈｒであり、ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ／ｍＬ、メジアン粒子径は１１５μｍであった。 [Example 3]
Up to (washing 1), a dispersed slurry of PHB copolymer powder before washing was obtained by the same method as in Example 1. 30% sodium hydroxide was added to the obtained dispersed slurry to adjust the pH of the dispersed slurry to 10.5. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.033 phr, and the bulk of the PHB copolymer powder was obtained. The density was 0.50 g / mL and the polymer particle size was 115 μm.
（洗浄１）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体の分散スラリーを得た。得られた分散スラリーに３０％水酸化ナトリウムを加え、分散スラリーのｐＨを１０．５になるように調整した。エスペラーゼ（Ｎｏｖｏｚｙｍｅ社）を０．０５ｐｈｒ添加し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０３３ｐｈｒであり、ＰＨＢ共重合体粉体の嵩密度は０．５０ｇ／ｍＬ、メジアン粒子径は１１５μｍであった。 [Example 3]
Up to (washing 1), a dispersed slurry of PHB copolymer powder before washing was obtained by the same method as in Example 1. 30% sodium hydroxide was added to the obtained dispersed slurry to adjust the pH of the dispersed slurry to 10.5. Esperase (Novozymes) was added at 0.05 phr and stirred for 2 hours. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.033 phr, and the bulk of the PHB copolymer powder was obtained. The density was 0.50 g / mL and the polymer particle size was 115 μm.
〔実施例４〕
（造粒）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体を得た。洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、３０％水酸化ナトリウムを加え、ｐＨが１１になるように調整し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０９８ｐｈｒであり、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５１ｇ／ｍＬ、メジアン粒子径は１１０μｍであった。 [Example 4]
Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation). Before washing PHB copolymer powder was dispersed in pure water so that the PHB copolymer concentration was 37.5%, 30% sodium hydroxide was added, and the pH was adjusted to 11 for 2 hours. Stirred. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.098 phr, and the PHB copolymer powder before washing was obtained. The bulk density was 0.51 g / mL, and the polymer particle size was 110 μm.
（造粒）までは実施例１と同じ方法で洗浄前ＰＨＢ共重合体粉体を得た。洗浄前ＰＨＢ共重合体粉体をＰＨＢ共重合体濃度が３７．５％になるように、純水に分散させ、３０％水酸化ナトリウムを加え、ｐＨが１１になるように調整し、２時間攪拌した。その後、実施例１に記載の（洗浄２）以降の操作を実施したところ、得られたＰＨＢ共重合体粉体に含まれる全窒素量は０．０９８ｐｈｒであり、洗浄前ＰＨＢ共重合体粉体の嵩密度は０．５１ｇ／ｍＬ、メジアン粒子径は１１０μｍであった。 [Example 4]
Pre-cleaning PHB copolymer powder was obtained by the same method as in Example 1 until (granulation). Before washing PHB copolymer powder was dispersed in pure water so that the PHB copolymer concentration was 37.5%, 30% sodium hydroxide was added, and the pH was adjusted to 11 for 2 hours. Stirred. After that, when the operations after (washing 2) described in Example 1 were carried out, the total amount of nitrogen contained in the obtained PHB copolymer powder was 0.098 phr, and the PHB copolymer powder before washing was obtained. The bulk density was 0.51 g / mL, and the polymer particle size was 110 μm.
〔比較例１〕
（不活化）までは、実施例１と同様の方法で不活化培養液を得た。 [Comparative Example 1]
Until (inactivation), an inactivated culture solution was obtained in the same manner as in Example 1.
（不活化）までは、実施例１と同様の方法で不活化培養液を得た。 [Comparative Example 1]
Until (inactivation), an inactivated culture solution was obtained in the same manner as in Example 1.
（酵素処理）
上記で得られた不活化培養液に対してペプチドグリカンの分解酵素である卵白リゾチーム（卵白リゾチームＦＧ、長瀬産業製）を０．０１ｐｈｒ添加し、５０℃で２時間ホールドした。次いで、３０％水酸化ナトリウムを用いて、ｐＨを８．５±０．２に調整した。その後、タンパク質分解酵素であるアルカラーゼ（Ｎｏｖｏｚｙｍｅｓ社製）を添加し、５０℃で３０％水酸化ナトリウムにてｐＨ８．５にコントロールしながら、２時間以上ホールドした。 (Enzyme treatment)
To the inactivated culture solution obtained above, 0.01 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.), which is a degrading enzyme of peptidoglycan, was added and held at 50 ° C. for 2 hours. The pH was then adjusted to 8.5 ± 0.2 with 30% sodium hydroxide. Then, alcalase (manufactured by Novozymes), which is a proteolytic enzyme, was added, and the mixture was held for 2 hours or more while controlling the pH to 8.5 with 30% sodium hydroxide at 50 ° C.
上記で得られた不活化培養液に対してペプチドグリカンの分解酵素である卵白リゾチーム（卵白リゾチームＦＧ、長瀬産業製）を０．０１ｐｈｒ添加し、５０℃で２時間ホールドした。次いで、３０％水酸化ナトリウムを用いて、ｐＨを８．５±０．２に調整した。その後、タンパク質分解酵素であるアルカラーゼ（Ｎｏｖｏｚｙｍｅｓ社製）を添加し、５０℃で３０％水酸化ナトリウムにてｐＨ８．５にコントロールしながら、２時間以上ホールドした。 (Enzyme treatment)
To the inactivated culture solution obtained above, 0.01 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.), which is a degrading enzyme of peptidoglycan, was added and held at 50 ° C. for 2 hours. The pH was then adjusted to 8.5 ± 0.2 with 30% sodium hydroxide. Then, alcalase (manufactured by Novozymes), which is a proteolytic enzyme, was added, and the mixture was held for 2 hours or more while controlling the pH to 8.5 with 30% sodium hydroxide at 50 ° C.
（溶菌、濃縮）
上記で得られた酵素処理液に対してアルカリ水溶液を添加し、ｐＨを１１．５以上に調整し、４５℃以上で４時間以上保持した。その後、０．６～１．０ｗｔ％のドデシル硫酸ナトリウム（花王製）を添加し、かつ、ｐＨが１２前後になるように３０％水酸化ナトリウムを添加するとともに、アルカリ水溶液（ｐＨ１０～１１．５）で３倍に希釈した。これを遠心分離した後、上清を除去して２倍濃縮した。この濃縮したＰＨＢ共重合体の水性懸濁液に、除去した上清と同量の水酸化ナトリウム水溶液（ｐＨ１１）を添加して遠心分離し、上清を除去した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を繰り返した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。その後、ＰＨＢ共重合体水性懸濁液をブフナー漏斗でのろ別により固形分濃度５０％まで濃縮したが、濡れた粉状になっており、流動性もなく、噴霧乾燥ができない状態であった。 (Lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above, the pH was adjusted to 11.5 or higher, and the pH was maintained at 45 ° C. or higher for 4 hours or longer. Then, 0.6 to 1.0 wt% sodium dodecyl sulfate (manufactured by Kao) is added, and 30% sodium hydroxide is added so that the pH becomes around 12, and an alkaline aqueous solution (pH 10 to 11.5) is added. ) Was diluted 3-fold. After centrifuging this, the supernatant was removed and concentrated twice. To the aqueous suspension of the concentrated PHB copolymer, the same amount of sodium hydroxide aqueous solution (pH 11) as the removed supernatant was added and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm. After that, the aqueous suspension of the PHB copolymer was concentrated to a solid content concentration of 50% by filtration through a Büchner funnel, but it was in the form of a wet powder, had no fluidity, and could not be spray-dried. ..
上記で得られた酵素処理液に対してアルカリ水溶液を添加し、ｐＨを１１．５以上に調整し、４５℃以上で４時間以上保持した。その後、０．６～１．０ｗｔ％のドデシル硫酸ナトリウム（花王製）を添加し、かつ、ｐＨが１２前後になるように３０％水酸化ナトリウムを添加するとともに、アルカリ水溶液（ｐＨ１０～１１．５）で３倍に希釈した。これを遠心分離した後、上清を除去して２倍濃縮した。この濃縮したＰＨＢ共重合体の水性懸濁液に、除去した上清と同量の水酸化ナトリウム水溶液（ｐＨ１１）を添加して遠心分離し、上清を除去した。水酸化ナトリウム水溶液の添加、遠心分離、および上清除去の操作を繰り返した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。その後、ＰＨＢ共重合体水性懸濁液をブフナー漏斗でのろ別により固形分濃度５０％まで濃縮したが、濡れた粉状になっており、流動性もなく、噴霧乾燥ができない状態であった。 (Lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above, the pH was adjusted to 11.5 or higher, and the pH was maintained at 45 ° C. or higher for 4 hours or longer. Then, 0.6 to 1.0 wt% sodium dodecyl sulfate (manufactured by Kao) is added, and 30% sodium hydroxide is added so that the pH becomes around 12, and an alkaline aqueous solution (pH 10 to 11.5) is added. ) Was diluted 3-fold. After centrifuging this, the supernatant was removed and concentrated twice. To the aqueous suspension of the concentrated PHB copolymer, the same amount of sodium hydroxide aqueous solution (pH 11) as the removed supernatant was added and centrifuged to remove the supernatant. The operations of adding the aqueous sodium hydroxide solution, centrifuging, and removing the supernatant were repeated. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm. After that, the aqueous suspension of the PHB copolymer was concentrated to a solid content concentration of 50% by filtration through a Büchner funnel, but it was in the form of a wet powder, had no fluidity, and could not be spray-dried. ..
〔比較例２〕
（溶菌、濃縮）の遠心分離操作までは、比較例１と同様の方法でＰＨＢ共重合体の水性懸濁液を得た。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。 [Comparative Example 2]
An aqueous suspension of the PHB copolymer was obtained in the same manner as in Comparative Example 1 until the centrifugation operation (lysis and concentration). The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm.
（溶菌、濃縮）の遠心分離操作までは、比較例１と同様の方法でＰＨＢ共重合体の水性懸濁液を得た。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。 [Comparative Example 2]
An aqueous suspension of the PHB copolymer was obtained in the same manner as in Comparative Example 1 until the centrifugation operation (lysis and concentration). The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm.
（造粒）
上記で得られたＰＨＢ共重合体水性懸濁液（固形分濃度２０％）に、エチレンオキサイド／プロピレンオキサイド共重合体非イオン性分散剤（ポリエチレンオキサイド分子量８０００、ポリプロピレンオキサイド分子量２０００、商品名プロノン２０８）を０．９５ｐｈｒ添加した。次いで、メチルセルロース（商品名ＳＭ-１５）を０．３ｐｈｒ添加した。この液を撹拌し、液温度を６０℃まで上昇させ、ｐＨが４．０になるように１０％硫酸を添加し、１２０分間撹拌を継続し、ＰＨＢ共重合体水性懸濁液を得た。その後、前記混合物の固形分濃度を、水を加えることにより１５質量％に調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。さらに、前記ＰＨＢ共重合体水性懸濁液のせん断粘度をＡｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて測定したところ、０．１Ｐａ・ｓであった。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１１５℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、ＰＨＢ共重合体粉体を得た。得られたＰＨＢ共重合体粉体に含まれるペプチドグリカン量は０．０１ｐｈｒ、残タンパク質量は０．５ｐｈｒ、ＰＨＢ共重合体粉体の嵩密度は０．２０ｇ／ｍＬ、メジアン粒子径は７１μｍ、純度は９８％であった。 (Granulation)
In the PHB copolymer aqueous suspension (solid content concentration 20%) obtained above, an ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronone 208) was added. ) Was added 0.95 phr. Then, 0.3 phr of methyl cellulose (trade name SM-15) was added. This liquid was stirred, the liquid temperature was raised to 60 ° C., 10% sulfuric acid was added so that the pH became 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. Then, the solid content concentration of the mixture was adjusted to 15% by mass by adding water. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa · s. The obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder was obtained. The amount of peptidoglycan contained in the obtained PHB copolymer powder is 0.01 phr, the amount of residual protein is 0.5 phr, the bulk density of the PHB copolymer powder is 0.20 g / mL, the median particle size is 71 μm, and the purity. Was 98%.
上記で得られたＰＨＢ共重合体水性懸濁液（固形分濃度２０％）に、エチレンオキサイド／プロピレンオキサイド共重合体非イオン性分散剤（ポリエチレンオキサイド分子量８０００、ポリプロピレンオキサイド分子量２０００、商品名プロノン２０８）を０．９５ｐｈｒ添加した。次いで、メチルセルロース（商品名ＳＭ-１５）を０．３ｐｈｒ添加した。この液を撹拌し、液温度を６０℃まで上昇させ、ｐＨが４．０になるように１０％硫酸を添加し、１２０分間撹拌を継続し、ＰＨＢ共重合体水性懸濁液を得た。その後、前記混合物の固形分濃度を、水を加えることにより１５質量％に調整した。このＰＨＢ共重合体水性懸濁液中の体積メジアン径をＨＯＲＩＢＡ製レーザ回折／散乱式粒子径分布測定装置ＬＡ－９５０を用いて測定したところ、６２μｍであった。さらに、前記ＰＨＢ共重合体水性懸濁液のせん断粘度をＡｎｔｏｎ Ｐａａｒ社製ＭＣＲ３０２を用いて測定したところ、０．１Ｐａ・ｓであった。得られたＰＨＢ共重合体水性懸濁液を、ロータリーアトマイザー型噴霧乾燥機（ＯＣ－１６、大川原社製）にて噴霧乾燥を実施し（熱風温度：１１５℃、排風温度：７５℃、ロータリーアトマイザー回転速度：１１０００ｒｐｍ）、ＰＨＢ共重合体粉体を得た。得られたＰＨＢ共重合体粉体に含まれるペプチドグリカン量は０．０１ｐｈｒ、残タンパク質量は０．５ｐｈｒ、ＰＨＢ共重合体粉体の嵩密度は０．２０ｇ／ｍＬ、メジアン粒子径は７１μｍ、純度は９８％であった。 (Granulation)
In the PHB copolymer aqueous suspension (solid content concentration 20%) obtained above, an ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronone 208) was added. ) Was added 0.95 phr. Then, 0.3 phr of methyl cellulose (trade name SM-15) was added. This liquid was stirred, the liquid temperature was raised to 60 ° C., 10% sulfuric acid was added so that the pH became 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. Then, the solid content concentration of the mixture was adjusted to 15% by mass by adding water. The volume median diameter in this PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA and found to be 62 μm. Further, the shear viscosity of the aqueous suspension of the PHB copolymer was measured using MCR302 manufactured by Antonio Par Co., Ltd. and found to be 0.1 Pa · s. The obtained PHB copolymer aqueous suspension was spray-dried with a rotary atomizer type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115 ° C., exhaust air temperature: 75 ° C., rotary). Atomizer rotation speed: 11000 rpm), PHB copolymer powder was obtained. The amount of peptidoglycan contained in the obtained PHB copolymer powder is 0.01 phr, the amount of residual protein is 0.5 phr, the bulk density of the PHB copolymer powder is 0.20 g / mL, the median particle size is 71 μm, and the purity. Was 98%.
表１より、本製造方法によると、嵩密度が高い、３ＨＢ単位以外のヒドロキシアルカノエート単位の比率が高いＰＨＢ共重合体を製造できることが分かった。
From Table 1, it was found that according to this production method, a PHB copolymer having a high bulk density and a high ratio of hydroxyalkanoate units other than 3HB units can be produced.
本発明によれば、３ＨＢ単位以外のヒドロキシアルカノエート単位の組成比が高いＰＨＢ共重合体を製造することができる。また、本発明の製造方法により得られたＰＨＢ共重合体は嵩密度が高いため、農業、漁業、林業、園芸、医学、衛生品、衣料、非衣料、包装、自動車、建材、その他の分野に好適に利用することができる。
According to the present invention, it is possible to produce a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units. Further, since the PHB copolymer obtained by the production method of the present invention has a high bulk density, it can be used in agriculture, fisheries, forestry, horticulture, medicine, sanitary goods, clothing, non-clothing, packaging, automobiles, building materials, and other fields. It can be suitably used.
Claims (13)
- ポリヒドロキシ酪酸共重合体を製造する方法であって、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
（ａ）前記ポリヒドロキシ酪酸共重合体を含有する菌体を含む培養液に、アルカリ性タンパク質分解酵素を添加して、前記菌体を酵素処理する工程、
（ｂ）前記工程（ａ）で得られた培養液にアルカリ水溶液を添加してｐＨを１０．０～１２．０に調整し、該調整前、該調整と同時、または該調整後のいずれかにおいて界面活性剤を添加する工程、
（ｃ）前記工程（ｂ）で得られた水性懸濁液から、ｐＨが７．０以下であり、かつ、せん断粘度が０．００５Ｐａ・ｓ以上、０．５Ｐａ・ｓ以下の水性懸濁液を調製する工程、および
（ｄ）前記工程（ｃ）で調製した水性懸濁液を噴霧乾燥する工程、
を含む、ポリヒドロキシ酪酸共重合体の製造方法。 A method for producing a polyhydroxybutyrate copolymer, which is a method for producing a polyhydroxybutyrate copolymer.
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
(A) A step of adding an alkaline proteolytic enzyme to a culture solution containing cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the cells.
(B) An alkaline aqueous solution is added to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and either before the adjustment, at the same time as the adjustment, or after the adjustment. In the process of adding a surfactant,
(C) From the aqueous suspension obtained in the step (b), an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa · s or more and 0.5 Pa · s or less. And (d) the step of spray-drying the aqueous suspension prepared in the above step (c),
A method for producing a polyhydroxybutyric acid copolymer. - 前記工程（ｄ）の後に、
（ｅ）前記工程（ｄ）で得られたポリヒドロキシ酪酸共重合体粉体を洗浄する工程、および
（ｆ）前記工程（ｅ）で得られたポリヒドロキシ酪酸共重合体粉体を脱水および／または乾燥する工程
をさらに含む、請求項１に記載のポリヒドロキシ酪酸共重合体の製造方法。 After the step (d),
(E) The step of washing the polyhydroxybutyrate copolymer powder obtained in the step (d), and (f) the polyhydroxybutyrate copolymer powder obtained in the step (e) of dehydration and / /. The method for producing a polyhydroxybutyrate copolymer according to claim 1, further comprising a step of drying. - 前記３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位が、３－ヒドロキシヘキサノエート単位である、請求項１または２に記載のポリヒドロキシ酪酸共重合体の製造方法。 The method for producing a polyhydroxybutyric acid copolymer according to claim 1 or 2, wherein the hydroxyalkanoate unit other than the 3-hydroxybutyrate unit is a 3-hydroxyhexanoate unit.
- 前記工程（ｂ）の界面活性剤がドデシル硫酸ナトリウムである、請求項１～３のいずれか１項に記載のポリヒドロキシ酪酸共重合体の製造方法。 The method for producing a polyhydroxybutyric acid copolymer according to any one of claims 1 to 3, wherein the surfactant in the step (b) is sodium dodecyl sulfate.
- 前記水性懸濁液が、分散剤をさらに含む、請求項１～４のいずれか１項に記載のポリヒドロキシ酪酸共重合体の製造方法。 The method for producing a polyhydroxybutyric acid copolymer according to any one of claims 1 to 4, wherein the aqueous suspension further contains a dispersant.
- 前記分散剤が、アルキレンオキサイド系分散剤、セルロース系分散剤およびポリビニルアルコールからなる群より選択される少なくとも１種である、請求項５に記載のポリヒドロキシ酪酸共重合体の製造方法。 The method for producing a polyhydroxybutyric acid copolymer according to claim 5, wherein the dispersant is at least one selected from the group consisting of an alkylene oxide-based dispersant, a cellulosic-based dispersant, and polyvinyl alcohol.
- 前記工程（ｃ）で調製する水性懸濁液におけるポリヒドロキシ酪酸共重合体の濃度が、３０～６５重量％である、請求項１～６のいずれか１項に記載のポリヒドロキシ酪酸共重合体の製造方法。 The polyhydroxybutyric acid copolymer according to any one of claims 1 to 6, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight. Manufacturing method.
- 前記工程（ｅ）の洗浄液がアルカリ性タンパク質分解酵素および／または溶菌酵素を含む、請求項２に記載のポリヒドロキシ酪酸共重合体の製造方法。 The method for producing a polyhydroxybutyric acid copolymer according to claim 2, wherein the cleaning solution of the step (e) contains an alkaline proteolytic enzyme and / or a lytic enzyme.
- ポリヒドロキシ酪酸共重合体と、ペプチドグリカンと、分散剤と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。 Containing a polyhydroxybutyrate copolymer, peptidoglycan, and a dispersant,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm. - 前記ペプチドグリカン含有量が０．１～１．５ｐｈｒである、請求項９に記載のポリヒドロキシ酪酸共重合体粉体。 The polyhydroxybutyric acid copolymer powder according to claim 9, wherein the peptidoglycan content is 0.1 to 1.5 phr.
- 残タンパク質量が０．１～１．０ｐｈｒである、請求項９または１０に記載のポリヒドロキシ酪酸共重合体粉体。 The polyhydroxybutyric acid copolymer powder according to claim 9 or 10, wherein the amount of residual protein is 0.1 to 1.0 phr.
- ポリヒドロキシ酪酸共重合体と、窒素化合物と、を含み、
前記ポリヒドロキシ酪酸共重合体は、３－ヒドロキシブチレート単位／３－ヒドロキシブチレート単位以外のヒドロキシアルカノエート単位の組成比が８０／２０～８８／１２（ｍｏｌ／ｍｏｌ）であり、
嵩密度が０．４５ｇ／ｍＬ以上であり、メジアン粒子径が８０～２００μｍである、ポリヒドロキシ酪酸共重合体粉体。 Containing a polyhydroxybutyrate copolymer and a nitrogen compound,
The polyhydroxybutyric acid copolymer has a composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate unit / 3-hydroxybutyrate unit of 80/20 to 88/12 (mol / mol).
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm. - 前記ポリヒドロキシ酪酸共重合体粉体中の全窒素量が０．０１０～０．０７５ｐｈｒである、請求項１２に記載のポリヒドロキシ酪酸共重合体粉体。
The polyhydroxybutyrate copolymer powder according to claim 12, wherein the total amount of nitrogen in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr.
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