WO2022091685A1 - Procédé de production de copolymères de polyhydroxybutyrate et leur utilisation - Google Patents

Procédé de production de copolymères de polyhydroxybutyrate et leur utilisation Download PDF

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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
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phb copolymer
copolymer
polyhydroxybutyric acid
aqueous suspension
acid copolymer
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PCT/JP2021/036049
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English (en)
Japanese (ja)
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直樹 出口
翔悟 廣田
優 平野
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株式会社カネカ
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Priority to JP2022558940A priority Critical patent/JPWO2022091685A1/ja
Priority to CN202180063699.4A priority patent/CN116323643A/zh
Publication of WO2022091685A1 publication Critical patent/WO2022091685A1/fr

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    • 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

La présente invention a pour but de fournir un copolymère de PHB permettant d'inhiber l'agrégation des particules de copolymère de PHB pendant le processus de production et présentant une densité apparente élevée. L'invention concerne également un procédé de production dudit copolymère.　La solution selon l'invention consiste à fournir un procédé de production de copolymère de PHB comprenant les étapes suivantes : (a) réalisation d'un traitement enzymatique utilisant une enzyme spécifique sur un micro-organisme contenant un copolymère de PHB ayant un rapport de composition constitué d'une unité 3HB/unité hydroxyalcanoate autre qu'une unité 3HB et se trouvant dans une plage prescrite ; (b) ajout d'un agent tensioactif après ajustement du pH dans une plage prescrite par ajout d'une solution aqueuse alcaline ; (c) préparation d'une suspension aqueuse ayant un pH d'au maximum 7,0 et ayant une viscosité de cisaillement dans une plage prescrite ; et (d) séchage par pulvérisation.
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JP2019097518A (ja) * 2017-12-06 2019-06-24 株式会社カネカ ポリヒドロキシアルカノエート分散液の製造方法
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WO2018186278A1 (fr) * 2017-04-05 2018-10-11 株式会社カネカ Particules de polyhydroxyalcanoate et dispersion aqueuse de celles-ci
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