Classifications

• • 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
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• • 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
  • C12P7/625—Polyesters of hydroxy carboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/05—Inorganic components
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W30/00—Technologies for solid waste management
  • Y02W30/50—Reuse, recycling or recovery technologies
  • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

• the present invention relates to a method for producing polyhydroxyalkanoic acid and its use.
• PHA Polyhydroxyalkanoic acid
• PHA produced by microorganisms accumulates within the cells of the microorganisms, so in order to use PHA as a plastic, it is necessary to separate and purify the PHA from the cells of the microorganisms.
• PHA is removed from the obtained aqueous suspension.
• separation operations such as centrifugation, filtration, and drying are performed.
• nucleic acids are released from the cells, increasing the viscosity of the culture solution. There is a problem that it becomes difficult to carry out the purification process. Viscosity-lowering treatments such as heat treatment, addition of hypochlorite, addition of commercially available nuclease, and the like are known to address this problem.
• Patent Document 1 discloses a method of adding a peroxide such as hydrogen peroxide to decompose nucleic acids.
• Patent Document 2 discloses a technique for improving purification efficiency without lowering the molecular weight of 3-hydroxyalkanoic acid by adjusting the pH by adding hydrogen peroxide.
• one aspect of the present invention aims to provide a technique for reducing the viscosity of a culture solution that is easy to industrialize when recovering PHA from PHA-producing bacteria.
• the present inventors added an oxidizing agent to the culture solution containing PHA-producing bacteria after inactivation, adjusted it to a specific pH, and adjusted it to a specific temperature. By maintaining the, it was found for the first time that the increase in the viscosity of the culture solution can be suppressed, and the present invention has been completed.
• the method for producing a polyhydroxyalkanoic acid according to one aspect of the present invention comprises (a) adding 40 and (b) adding an oxidizing agent to the culture solution obtained in step (a) and adjusting the pH to 10.5-13.0, and process to maintain,
• a method for producing a polyhydroxyalkanoic acid comprising:
• an aqueous suspension of polyhydroxyalkanoic acid according to one embodiment of the present invention contains hydrogen peroxide and polyhydroxyalkanoic acid and is It is an aqueous suspension of polyhydroxyalkanoic acid having a shear viscosity at 1/s of 1 to 10 mPa ⁇ s.
• the present inventors have made extensive studies to provide a technique for reducing the viscosity of the culture solution that is easy to industrialize. A new finding was obtained that adding an oxidizing agent to the liquid, adjusting the pH to a specific value, and maintaining the temperature at a specific value can suppress the increase in the viscosity of the culture solution. In addition, the present inventors have found that it is possible to control (promote) the reduction in the molecular weight of PHA by the above method, and that PHA having a desired weight-average molecular weight can be obtained.
• the viscosity of the culture solution is lowered, making it easier to handle and enabling use on an industrial scale.
• the amount of enzymes and surfactants for example, sodium dodecyl sulfate
• the amount of enzymes and surfactants for example, sodium dodecyl sulfate
• the amount of plastic waste generated can be reduced. conservee and sustainably use sea and marine resources.”
• the composition of the present production method and the present aqueous suspension will be described in detail below.
• This production method is a method comprising the following steps (a) to (b): - Step (a): A step of maintaining a culture solution containing bacterial cells containing polyhydroxyalkanoic acid at 40 to 80 ° C. - Step (b): Add an oxidizing agent to the culture solution obtained in the step (a) and adjusting the pH to 10.5-13.0 and maintaining at 30-75°C.
• Step (a) The step (a) in this production method is a step of maintaining a culture solution containing cells containing polyhydroxyalkanoic acid at 40 to 80°C. PHA-producing bacteria can be inactivated by step (a). Further, in the step (a), the cells in the culture medium are inactivated in advance by subjecting them to an inactivation temperature, thereby facilitating the later-described purification step and the like.
• the term "inactivation temperature” means a temperature that can kill microorganisms in a culture solution.
• the “inactivation temperature” means a temperature at which catalase, an enzyme possessed by microorganisms, can be deactivated.
• the inactivation temperature is 40° C. to 80° C., preferably 50° C. to 80° C., which is the temperature at which microorganisms are killed and catalase is inactivated, more preferably 60°C to 80°C.
• PHA is a general term for polymers having hydroxyalkanoic acid as a monomer unit.
• the hydroxyalkanoic acid constituting PHA is not particularly limited, but examples include 3-hydroxybutanoic acid, 4-hydroxybutanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxy heptanoic acid, 3-hydroxyoctanoic acid, and the like.
• These polymers may be homopolymers or copolymers containing two or more monomer units.
• PHA includes, for example, poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HH), poly(3-hydroxybutyrate) -co-3-hydroxyvalerate) (P3HB3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (P3HB3HO), Poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate) (P3HB3HOD), Poly(3-hydroxybutyrate-co-3-hydroxydecanoate) (P3HB3HD), Poly(3 -hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HH) and the like.
• P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferable
• the melting point and crystallinity can be changed.
• a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is used from the viewpoint that it is possible to impart it and that it is a plastic that is easy to produce industrially and has physical properties as described above.
• Some P3HB3HH are more preferred.
• the composition ratio of the repeating units of P3HB3HH is 3-hydroxybutyrate unit/3-hydroxyhexanoate unit composition ratio of 80/20 to 80/20 from the viewpoint of balance between flexibility and strength. It is preferably 99.9/0.1 (mol/mol), more preferably 85/15 to 97/3 (mol/mol).
• the composition ratio of 3-hydroxybutyrate units/3-hydroxyhexanoate units is 99.9/0.01 (mol/mol) or less, sufficient flexibility can be obtained, and 80/20 (mol/ mol) or more, sufficient hardness can be obtained.
• the PHA obtained in the step (a) preferably has a weight average molecular weight (hereinafter sometimes referred to as "Mw") of 1,500,000 to 2,500,000, more preferably. is 1.6 million to 2.4 million, more preferably 1.7 million to 2.3 million. If the weight average molecular weight of PHA is within the above range, the culture productivity is excellent.
• the weight average molecular weight of PHA was determined by gel permeation chromatography (GPC) ("Shodex GPC-101" manufactured by Showa Denko) using polystyrene gel (“Shodex K-804" manufactured by Showa Denko) as a column and chloroform as the mobile phase. , can be obtained as a molecular weight in terms of polystyrene.
• the solid content concentration of the culture solution obtained in step (a) is preferably 20 to 40% by weight, more preferably 25 to 40% by weight, and even more preferably is 30 to 40% by weight. If the solid content concentration of the culture solution obtained in the step (a) is within the above range, a sufficient amount of PHA can be obtained.
• the microbial cells used in step (a) are not particularly limited as long as they are microorganisms capable of intracellularly producing PHA.
• microorganisms that have been deposited in depositories of microorganisms and strains isolated from nature (eg, IFO, ATCC, etc.), or mutants and transformants that can be prepared therefrom can be used.
• Bacillus megaterium which was discovered in 1925, was the first bacterial cell that produces P3HB, which is an example of PHA.
• natural microorganisms such as Ralstonia eutropha and Alcaligenes latus. These microorganisms are known to accumulate PHA in their cells.
• Alcaligenes eutrophus Alcaligenes eutrophus AC32 strain (Alcaligenes eutrophus AC32, FERM BP-6038) (T.Fukui, Y.Doi, J.Bateriol) into which PHA synthase group genes were introduced in order to increase the productivity of P3HB3HH ., 179, p4821-4830 (1997)) and the like are more preferred.
• the microbial cells may be genetically modified microorganisms into which various PHA-synthesis-related genes have been introduced according to the PHA to be produced.
• PHA can also be produced, for example, by the method described in International Publication No. 2010/013483.
• Commercial products of PHA include, for example, Kaneka Corporation "Kaneka Biodegradable Polymer PHBH (registered trademark)" (eg, X131A and 151C used in Examples).
• PHA-producing bacteria the above-mentioned microorganisms capable of producing PHA in cells are sometimes referred to as "PHA-producing bacteria".
• Step (b) The step (b) in this production method includes adding an oxidizing agent to the culture solution obtained in the step (a), adjusting the pH to 10.5 to 13.0, and maintaining it at 30 to 75°C. It is a process.
• viscosity intends "shear viscosity in 50 degreeC 100 1/s” measured by the method as described in an Example.
• PHA aqueous suspension the aqueous suspension containing PHA obtained in step (b) and subsequent steps.
• the step (b) can reduce the viscosity of the obtained culture solution.
• the amount of enzyme used in step (c) described later can be reduced, and the production cost of PHA is reduced.
• the weight average molecular weight of PHA it is possible to produce PHA having a desired weight average molecular weight.
• the oxidizing agent examples include, but are not limited to, hydrogen peroxide (H 2 O 2 ), ozone; sodium peroxide (Na 2 O 2 ), sodium perborate (Na 2 H 4 B 2 O 8 ), Other inorganic peroxides such as sodium percarbonate ( Na2H3CO6 ), sodium persulfate ( Na2S2O8 ) ; chlorite, chlorate , metachloroperbenzoic acid ( C7H5 CIO3 ) similar halogen compounds such as perchlorate , perchloric acid ( CIO4 ), chlorine dioxide ( CIO2 ) ; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate ( KNO3 ); sodium bismuthate; cerium (IV) compounds such as ammonium cerium nitrate and cerium sulfate.
• the oxidizing agent is preferably hydrogen peroxide or ozone from the viewpoint of easy availability.
• the concentration of hydrogen peroxide in the culture medium is, for example, 0.2 to 30% by weight, preferably 0.2 to 15% by weight, more preferably 0.2 to 10% by weight. If it is 0.2% by weight or more, the viscosity of the PHA aqueous suspension can be reduced. Also, if the content is 30% by weight or less, the PHA production cost can be suppressed. Hydrogen peroxide may be added in step (b) to achieve the above concentrations.
• the amount of ozone added is, for example, 0.01 to 0.1 g, preferably 0.02 to 0.08 g, more preferably 0.02 to 0.08 g per 1 g of biomass containing PHA. , 0.02 to 0.07 g. If it is 0.01 g or more, the viscosity of the PHA aqueous suspension can be reduced. Moreover, if it is 0.1 g or less, the amount of residual ozone will be small.
• NaHCO 3 may be added along with the hydrogen peroxide addition. NaHCO 3 is expected to assist the action of hydrogen peroxide.
• a chelating agent may be added together with the addition of hydrogen peroxide. Addition of a chelating agent can stabilize the hydrogen peroxide solution.
• Chelating agents include, but are not limited to, sodium silicate, EDTA, trans-1,2-cyclohexanediaminetetraacetic acid monohydrate, and the like.
• a suitable pH is 10.5 to 13.0, more preferably pH 10.6. ⁇ 12.9, more preferably pH 10.7-12.8. If the pH is 10.5 or more, the viscosity of the PHA aqueous suspension is lowered, and the handleability is improved. Moreover, if the pH is 13.0 or less, sufficient time can be obtained for adjusting the weight average molecular weight of the PHA. Adjustment of pH may be performed, for example, by adding an alkaline aqueous solution.
• 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, but for example, hydroxides of alkali metals or alkaline earth metals such as sodium hydroxide and potassium hydroxide; metal carbonates such as sodium carbonate and potassium carbonate; Metal phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate and potassium hydrogen phosphate, and metal hydrogen phosphates are included.
• the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, more preferably sodium hydroxide.
• a basic compound may be used individually by 1 type, and may use 2 or more types together.
• step (b) the order of adding the oxidizing agent and adjusting the pH is not particularly limited, but from the viewpoint of efficiently lowering the pH, it is preferable to adjust the pH after adding the oxidizing agent.
• the temperature of the culture medium is 30° C. to 75° C., preferably 35° C. to 70° C., in order to control the viscosity of the PHA aqueous suspension and the weight average molecular weight of the PHA, More preferably, it is 40°C to 65°C. If the temperature of the culture solution is 30°C or higher, the time for adjusting the PHA weight average molecular weight does not become longer than necessary, and if the temperature is 80°C or lower, a sufficient adjustment time can be obtained.
• the PHA aqueous suspension obtained in step (b) is preferably a Newtonian fluid. If the PHA aqueous suspension is a Newtonian fluid, the shear rate dependence of the viscosity is low, so that the PHA aqueous suspension tends to become uniform in the stirring device, improving handling properties.
• step (b) the adjusted pH is preferably maintained for 0.1 to 30 hours, more preferably 0.25 to 24 hours, more preferably 0.5 to 15 hours. including the step of maintaining.
• step (b) the pH of the culture solution is gradually lowered as the PHA in the culture solution is decomposed. Therefore, by maintaining the pH of the culture medium in step (b), the viscosity of the resulting PHA aqueous suspension can be further reduced.
• the method for maintaining the adjusted pH is not particularly limited, and for example, it is performed by adding an alkaline aqueous solution.
• the alkaline aqueous solution is not particularly limited, for example, the alkaline aqueous solution described above is used.
• the temperature in the pH maintenance step is not particularly limited, but is, for example, 30 to 80°C, preferably 30 to 75°C.
• the step (b) may include a step of maintaining the pH at a predetermined temperature for a predetermined time and then maintaining the temperature at 30-75°C for 0.5-30 hours.
• the pH of the culture solution gradually decreases due to the decomposition of PHA, so by including the step of maintaining at 30 to 75 ° C. for 0.5 to 30 hours, the neutralization step at a later stage is simplified. be able to.
• the weight average molecular weight of PHA in the PHA aqueous suspension obtained in step (b) is preferably 100,000 to 800,000, more preferably 200,000 to 80,000. 10,000, more preferably 400,000 to 800,000.
• the weight average molecular weight is 100,000 or more, sufficient mechanical properties can be obtained, and when it is 800,000 or less, a sufficient crystallization rate can be obtained and good moldability can be achieved.
• the solid content concentration of the aqueous suspension obtained in step (b) is preferably 20 to 40% by weight, more preferably 25 to 40% by weight, and even more preferably is 30-40% by weight. If the solid content concentration of the aqueous suspension obtained in the step (b) is within the above range, a sufficient amount of PHA can be obtained.
• the production method may further comprise the following steps (c)-(d): - Step (c): A step of adding an enzyme to the aqueous suspension obtained in the step (b) and enzymatically treating the bacterial cells - Step (d): The aqueous solution obtained in the step (c) A step of adding a surfactant and an alkaline aqueous solution to the suspension to adjust the pH to 10.0 to 12.0.
• Step (c) The step (c) in this production method is a step of adding an enzyme to the aqueous suspension obtained in the step (b) to subject the bacterial cells to enzyme treatment.
• an enzyme By destroying and removing impurities (cell walls, proteins, etc.) derived from the microbial cells in step (c), PHA can be efficiently recovered from the microbial cells.
• the enzymatic treatment in step (c) can be lytic enzyme treatment and/or alkaline proteolytic enzyme treatment.
• the bacteriolytic enzyme treatment and the alkaline protease treatment are preferably performed at least once each, and if necessary, the bacteriolytic enzyme treatment and/or the alkaline protease treatment may be performed twice or more.
• the order of lytic enzyme treatment and/or alkaline protease treatment is not particularly limited.
• step (c) when enzymatic treatment (e.g., lytic enzyme treatment and alkaline proteolytic enzyme treatment) is performed, the pH and temperature of the culture solution are adjusted according to the optimum pH and optimum temperature of the enzyme to be used. preferably. Methods for adjusting the pH and temperature of the culture solution are not particularly limited, and known methods can be used.
• the step (c) may include a step of adding a surfactant.
• the addition of the surfactant in step (c) can be performed before, at the same time as, or after the addition of the alkaline protease. Preferably after the addition of alkaline protease.
• the bacteriolytic enzyme treatment is a step of adding a bacteriolytic enzyme to the culture solution and enzymatically treating the bacterial cells.
• lytic enzyme refers to an enzyme that has the activity of degrading (bacteriolysing) the cell wall (eg, peptidoglycan) of bacterial cells.
• the lytic enzyme is not particularly limited, and examples thereof include lysozyme, labyrinthine, ⁇ -N-acetylglucosaminidase, endolysin, autolysin and the like. Lysozyme is preferred from the viewpoint of being economically advantageous. One of these may be used alone, or two or more of them may be used in combination.
• lytic enzymes Commercially available products can also be used as lytic enzymes, and examples include “lysozyme” and “acromopeptidase” manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.
• the optimum pH of the lytic enzyme is not particularly limited as long as the lytic enzyme has cell wall degrading activity, but is, for example, 5.0 to 11.0, preferably 6.0 to 9. .0, more preferably 6.0 to 8.0.
• the optimum temperature of the lytic enzyme is not particularly limited, but from the viewpoint that it does not require excessive heating and can prevent thermal change (thermal decomposition) of PHA, it is 60 ° C. or less. is preferred, and 50°C or lower is more preferred.
• the lower limit of the optimum temperature is not particularly limited, it is preferably room temperature (for example, 25° C.) or higher from the viewpoint of economy without excessive cooling operation.
• alkaline protease means a protease having the activity of degrading proteins in an alkaline environment (for example, in a pH 8.5 solution).
• alkaline protease treatment intends a step of adding the alkaline protease to the culture solution to enzymatically treat the bacterial cells.
• the alkaline protease is not particularly limited as long as it has the activity of degrading protein in an alkaline environment.
• specific protease eg papain, bromelain, cathepsin
• aspartic acid-specific protease eg pepsin, cathepsin D, HIV protease
• serine-specific proteolytic enzymes especially subtilisins (eg, Alcalase) are preferred.
• subtilisins eg, Alcalase
• alkaline protease commercially available products can be used, for example, Novozyme "Alcalase 2.5L”; Amano Enzyme Co., Ltd. “Protin SD-AY10” and “Protease P “Amano” 3SD”; Japan Co., Ltd. “Multifect PR6L” and “Optimase PR89L”; Shinnihon Chemical Industry Co., Ltd. “Sumiteam MP”; DM Japan Co., Ltd. "Delbolase”; Nagase ChemteX Co., Ltd. "Bioprase OP” , “Bioplase SP-20FG” and “Bioplase SP-4FG”; HBI Co., Ltd. "Orientase 22BF”; Yakult Pharmaceutical Co., Ltd. "Alloase XA-10";
• the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease has activity in an alkaline environment, for example 8.0 to 14.0, preferably 8.0 to 12.0, more preferably 8.0 to 10.0, still more preferably 8.0 to 9.0, most preferably 8.5.
• the optimum temperature for the alkaline protease is not particularly limited, but from the viewpoint that it does not require excessive heating and can prevent thermal change (thermal decomposition) of P3HA, 60° C. or lower is preferable, and 50° C. or lower is more preferable.
• the lower limit of the optimum temperature is not particularly limited, but it is preferably room temperature (for example, 25° C.) or higher from the viewpoint of economy without excessive cooling operation.
• the enzymatic treatment of step (c) can be performed with a combination of lysozyme and alcalase.
• Step (d) The step (d) in this production method is a step of adding a surfactant and an alkaline aqueous solution to the aqueous suspension obtained in the step (c) to adjust the pH to 10.0 to 12.0. be.
• Step (d) preferably includes the following steps (d1) and (d2).
• Step (d1) A step of adding an alkaline aqueous solution to the culture solution obtained in the step (c) to adjust the pH to 10.0 to 12.0
• Step (d2) A step of adding a surfactant ⁇ Step (d1)> Step (d1) is, as described above, a step of adding an alkaline aqueous solution to the aqueous PHA suspension obtained in step (c) to adjust the pH to 10.0 to 12.0.
• impurities nucleic acids, proteins, etc.
• step (d1) the description of the section (step (b)) is used for the alkaline aqueous solution.
• step (d1) the pH is preferably adjusted to 10.0 to 12.0, more preferably 10.2 to 11.8, and 10.4 by adding an alkaline aqueous solution. Adjusting to ⁇ 11.6 is even more preferred. Adjusting the pH to 10.0 or higher has the advantage of being able to decompose and dissolve bacterial components. In addition, by adjusting the pH to 12.0 or less, unintended damage to the cells can be prevented.
• the temperature in step (d1) is preferably less than 100°C, more preferably less than 80°C. Although the lower limit is not particularly limited, it is preferably 40° C. or higher, for example.
• Step (d2) is a step of adding a surfactant to the culture solution.
• impurities contained in the microbial cells, particularly cell membranes can be efficiently treated, and more impurities derived from the microbial cells can be removed, so that PHA of higher purity can be separated from the microbial cells. can be done.
• the surfactant is not particularly limited, but examples include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and the like. Of these, anionic surfactants are preferred from the viewpoint of their high ability to remove cell membranes. One of these may be used alone, or two or more of them may be used in combination.
• anionic surfactants include alkyl sulfates, alkylbenzenesulfonates, alkyl sulfates, alkenyl sulfates, alkyl ether sulfates, alkenyl ether sulfates, ⁇ -olefinsulfonates, ⁇ - Examples include sulfo fatty acid salts, esters of ⁇ -sulfo fatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid type surfactants, N-acylamino acid type surfactants, and the like.
• alkyl sulfate ester salts are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred from the viewpoint of its high ability to remove cell membranes and its low cost.
• SDS sodium dodecyl sulfate
• the amount of 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 with respect to the culture medium. preferable.
• Step (d2) may be performed before, at the same time as, or after step (d1). That is, in the step (d), the addition of the surfactant and the aqueous alkali solution may be carried out by adding the aqueous alkali solution to adjust the pH, and then adding the surfactant, or adding the surfactant and the aqueous alkali solution at the same time. After addition of the surfactant, the pH may be adjusted by adding an alkaline aqueous solution.
• the manufacturing method may further include step (e).
• Step (e) is a step of centrifuging the PHA aqueous suspension obtained in step (d) and removing the supernatant to obtain a PHA aqueous suspension in which PHA is concentrated. That is, it is a step of removing impurities from the PHA separated from the cells, and concentrating and purifying the PHA.
• step (e) the method for centrifuging the culture solution is not particularly limited, and a known method can be used.
• step (e) it is preferable to repeat the steps of centrifuging the culture solution, removing the supernatant, adding a solution to the sediment, centrifuging again, and removing the supernatant.
• the solution added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the culture medium.
• said solution is preferably the same as the alkaline aqueous solution used in said step (d).
• step (e) it is preferable to reduce these impurities as much as possible, for which the amount of impurities remaining in the final product is generally determined by step (e).
• impurities may be mixed as long as the physical properties of the final product are not impaired.
• impurities should be reduced as much as possible. preferable.
• An indicator of the degree of purification at that time is, for example, the amount of protein remaining in the PHA aqueous suspension (residual protein amount).
• the amount of protein in the PHA aqueous suspension is not particularly limited as long as it is an amount capable of achieving the residual protein amount of the PHA powder.
• the amount of protein is preferably 3000 ppm or less, more preferably 2500 ppm or less, and still more preferably 2000 ppm or less per PHA weight in the PHA aqueous suspension.
• the solvent constituting the PHA aqueous suspension (“solvent” is also referred to as “aqueous medium”) is not particularly limited, and may be water or a mixed solvent of water and an organic solvent. good too.
• the concentration of the organic solvent is not particularly limited as long as it is equal to or lower than the solubility of the organic solvent used in water.
• the organic solvent is not particularly limited, but for example, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, heptanol; acetone, methyl ethyl ketone ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethylsulfoxide, pyridine, piperidine and the like.
• alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, heptanol
• acetone methyl ethyl ketone ethers
• nitriles such as ace
• 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 readily available.
• methanol, ethanol and acetone are particularly preferred.
• the water content in the aqueous medium constituting the PHA aqueous suspension is preferably 5% by weight or more, more preferably 10% by weight or more, still more preferably 30% by weight or more, and particularly preferably , 50% by weight or more.
• the PHA aqueous suspension in step (e) may contain other solvents, components derived from bacterial cells, compounds generated during purification, etc., as long as they do not impair the essence of the present invention.
• Aqueous suspension of polyhydroxyalkanoic acid contains hydrogen peroxide and polyhydroxyalkanoic acid, and has a shear viscosity of 1 to 10 mPa ⁇ s at 50° C. and 100 1/s. Since the present PHA aqueous suspension has a low viscosity, it is excellent in handleability and allows efficient production of PHA.
• the shear viscosity of the present PHA aqueous suspension at 50°C and 100 1/s is 1 to 10 mPa ⁇ s, preferably 1 to 9 mPa ⁇ s, more preferably 1 to 8 mPa ⁇ s.
• the shear viscosity of the present PHA aqueous suspension at 50° C. 10 1/s is, for example, 1 to 20 mPa ⁇ s, preferably 1 to 18 mPa ⁇ s, more preferably 1 to 15 mPa ⁇ s.
• the shear viscosity of the present PHA aqueous suspension is measured by the method described in Examples.
• the PHA aqueous suspension has a solid content concentration of, for example, 20 to 40% by weight, preferably 25 to 40% by weight.
• the solid content concentration of the aqueous suspension is 20 to 40% by weight, the viscosity of the aqueous suspension is usually high, making the subsequent purification treatment difficult.
• the inclusion of hydrogen peroxide makes it possible to provide an aqueous suspension in which an increase in viscosity is suppressed.
• the solid content concentration of the present PHA aqueous suspension is measured by the method described in Examples.
• the weight average molecular weight of the PHA contained in the present PHA aqueous suspension is, for example, 100,000 to 800,000, preferably 100,000 to 800,000, more preferably 200,000 to 800,000, and even more preferably. is between 400,000 and 800,000.
• weight average molecular weight is 100,000 or more, sufficient mechanical properties can be obtained, and when it is 800,000 or less, a sufficient crystallization rate can be obtained and good moldability can be achieved.
• the concentration of hydrogen peroxide in the present PHA aqueous suspension is, for example, 0.2 to 30% by weight, preferably 0.2 to 15% by weight, more preferably 0.2 to 10% by weight. is. If it is 0.2% by weight or more, the viscosity of the PHA aqueous suspension can be lowered. Also, if the content is 30% by weight or less, the PHA production cost can be suppressed.
• the present PHA aqueous suspension may contain various components generated or not removed during the course of the present production method, as long as the effects of the present invention are exhibited.
• one aspect of the present invention includes the following. ⁇ 1> (a) a step of maintaining a culture medium containing bacterial cells containing polyhydroxyalkanoic acid at 40 to 80° C., and (b) adding an oxidizing agent to the culture medium obtained in step (a) and adjusting the pH to 10.5-13.0 and maintaining at 30-75°C;
• a method for producing a polyhydroxyalkanoic acid comprising: ⁇ 2> The method for producing a polyhydroxyalkanoic acid according to ⁇ 1>, wherein the oxidizing agent is aqueous hydrogen peroxide or ozone.
• ⁇ 3> Furthermore, (c) adding an enzyme to the aqueous suspension obtained in the step (b) to treat the bacterial cells with an enzyme; and (d) adding an enzyme to the aqueous suspension obtained in the step (c).
• the weight-average molecular weight of the polyhydroxyalkanoic acid in the culture medium obtained in the step (a) is 1,500,000 to 2,500,000
• the weight average molecular weight of the polyhydroxyalkanoic acid in the aqueous suspension obtained in the step (b) is 100,000 to 800,000
• ⁇ 7> When hydrogen peroxide is added to the culture medium in the step (b), it is added so that the concentration of hydrogen peroxide in the culture medium is 0.2 to 30% by weight.
• ⁇ 8> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 3> to ⁇ 7>, wherein the enzyme in step (c) is a lytic enzyme and/or an alkaline protease.
• ⁇ 10> An aqueous suspension of polyhydroxyalkanoic acid, containing hydrogen peroxide and polyhydroxyalkanoic acid, and having a shear viscosity of 1 to 10 mPa ⁇ s at 50° C. and 100 1/s.
• ⁇ 11> The aqueous suspension of polyhydroxyalkanoic acid according to ⁇ 10>, which has a solid content concentration of 20 to 40% by weight.
• ⁇ 12> The aqueous suspension of polyhydroxyalkanoic acid according to ⁇ 10> or ⁇ 11>, wherein the polyhydroxyalkanoic acid has a weight average molecular weight of 100,000 to 800,000.
• ⁇ 13> The aqueous suspension of polyhydroxyalkanoic acid according to any one of ⁇ 10> to ⁇ 12>, wherein the concentration of hydrogen peroxide is 0.2 to 30% by weight.
• the viscosity of the PHA aqueous suspension was measured by the following method. Specifically, the viscosity was measured with a coaxial double cylinder using MCR302 manufactured by Anton Paar. The PHA aqueous suspension was put into a 20 mL cylinder, and the liquid temperature was adjusted to 50°C. After reaching the target shear rate, the viscosity was measured when the change in torque with time became less than 1%.
• the residual protein content of the PHA powder was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was put into a 15 mL falcon tube, 2 mL of the reagent of the above kit was added, and then shaken at 60° C. for 30 minutes. After 30 minutes from the end of shaking, the mixture was cooled to 25° C. and absorbance at a wavelength of 562 nm was measured.
• the inactivated PHA-containing culture medium was diluted with distilled water, centrifuged, and then the supernatant was removed. Ethanol was added to the obtained precipitate (PHA), and after dispersing in ethanol, centrifugation was performed. The supernatant was removed and dried in a vacuum dryer for 1 hour or more to completely dry the precipitate to obtain a dry body (PHA powder). After dissolving 10 mg of the obtained PHA powder in 10 mL of chloroform, insoluble matter was removed by filtration.
• Solid content concentration The solid content concentration of the PHA-containing culture solution after inactivation was measured using a heat drying moisture meter ML-50 (manufactured by A&D Co., Ltd.). The culture solution was heated at 130° C. until the weight change rate was less than 0.05%/min, and the solid content concentration was determined from the weight change before and after heating.
• Example 1 (Preparation of cell culture solution)
• Ralstonia eutropha described in International Publication No. WO2019/142717 was cultured by the method described in paragraphs [0041] to [0048] of the same document to obtain a cell culture solution containing cells containing PHA.
• Ralstonia eutropha is now classified as Capriavidus necator.
• the composition ratio of repeating units of PHA was 85/15 to 92/8 (mol/mol).
• Step (a) (Inactivation)
• the cell culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 70° C. for 7 hours to obtain an inactivated culture solution.
• the weight average molecular weight of PHA in the inactivated culture medium was 1.8 million.
• the solid content concentration of the inactivated culture solution was 30% by weight.
• Step (b) (viscosity reduction treatment) Hydrogen peroxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the inactivated culture solution obtained above to obtain the concentrations shown in Table 1. A 30% aqueous sodium hydroxide solution was then added to adjust the pH to 11.0. While maintaining the solution at 60° C., the pH was maintained at 11.0 for the time shown in Table 1 by continuing to add a 30% aqueous sodium hydroxide solution to obtain an aqueous PHA suspension. Table 1 shows the results. The solid content concentration after the viscosity reduction treatment was 28 to 30% by weight.
• the PHA aqueous suspension of Comparative Example 1-1 has a higher viscosity than the PHA aqueous suspensions of Examples 1-1 to 1-6, and the addition of hydrogen peroxide reduces the viscosity. was found to be an important factor in Further, when the pH was 10.0, the PHA aqueous suspensions of Comparative Examples 1-2 to 1-6 had higher viscosities than the PHA aqueous suspensions of Examples 1-1 to 1-6. It has been found that pH is an important factor for viscosity reduction.
• the weight-average molecular weight of the PHA of (Comparative Example 1-1) was 1.8 million, and the weight-average molecular weight of the PHA when reacted for 1 hour at a hydrogen peroxide concentration of 0.99% (Comparative Example 1-4) was 160. was 10,000.
• Example 2 A PHA aqueous suspension was obtained in the same manner as in Example 1, except that the pH, hydrogen peroxide concentration and reaction time were changed as shown in Table 2. Table 2 shows the results.
• Example 3 A PHA aqueous suspension was obtained in the same manner as in Example 1, except that the pH, hydrogen peroxide concentration and reaction time were changed as shown in Table 3. Table 3 shows the results.
• Example 4 A PHA aqueous suspension was obtained in the same manner as in Example 1, except that the hydrogen peroxide concentration, reaction temperature and reaction time were changed as shown in Table 4. Table 4 shows the results.
• Example 5 (Step (c)) (enzyme treatment) 10% sulfuric acid was added to the PHA aqueous suspension of Example 1-3 to adjust the pH to 7.0 ⁇ 0.2. When the solid content concentration of the PHA aqueous suspension to which sulfuric acid was added was measured, it was 30% by weight. After the addition of sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that decomposes sugar chains (peptidoglycan) in cell walls, was added to a liquid concentration of 10 ppm, and the mixture was kept at 50°C for 2 hours.
• lysozyme manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• Alcalase 2.5 L manufactured by Novozyme
• 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5. maintained for 2 hours.
• Step (d) (alkali treatment) Sodium dodecyl sulfate (SDS, manufactured by Kao Corporation) was added to the enzyme-treated solution so as to be 0.6 to 1.0 wt %. After that, an aqueous sodium hydroxide solution was used to adjust the pH to 11.0 ⁇ 0.2. Next, the enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed to obtain a two-fold concentrated PHA aqueous suspension. To the concentrated PHA aqueous suspension, the same amount of sodium hydroxide as the removed supernatant was added, centrifugation was performed again (4500 rpm, 10 minutes), and the supernatant was removed, which was repeated three times. The residual protein concentration in the obtained PHA aqueous suspension was 1217 ppm. Table 5 shows the results.
• Example 6 The treatment was carried out in the same manner as in Example 5 except that the lysozyme concentration was 5 ppm and the alcalase 2.5L concentration was 150 ppm. The residual protein concentration in the obtained PHA aqueous suspension was 1645 ppm. Table 5 shows the results.
• Example 3 The treatment was performed in the same manner as in Example 1 until the inactivation treatment, and the pH was kept at 11.0 for 1.5 hours.
• the viscosity at 50° C. and a shear rate of 10 1/s was 60.17 mPa ⁇ s, indicating poor handleability.
• IW industrial water
• the residual protein concentration in the resulting PHA aqueous suspension was 1675 ppm. Table 5 shows the results.
• Examples 5 and 6 the reaction speed of the enzyme reaction was faster than in Comparative Example 3. This is presumed to be due to the low viscosity of the PHA aqueous suspension.
• Example 7 (Step (c)) (enzyme treatment) Esperase (manufactured by Novozymes), which is an alkaline proteolytic enzyme, was added to the aqueous PHA suspension of Example 1-3 so that the concentration in the liquid was 100 ppm. Then, 30% sodium hydroxide was added at 50° C. and maintained for 2 hours while adjusting the pH to 11.0. Then, after centrifugation (4500 rpm, 10 minutes), the supernatant was removed to obtain a two-fold concentrated PHA aqueous suspension. To the concentrated PHA aqueous suspension, the same amount of sodium hydroxide as the removed supernatant was added to obtain a PHA aqueous suspension.
• Esperase manufactured by Novozymes
• Step (d) (alkali treatment) SDS was added so as to be 0.3 to 1.0 wt % with respect to the enzyme-treated solution. After that, an aqueous sodium hydroxide solution was used to adjust the pH to 11.0 ⁇ 0.2, and the mixture was reacted at 40° C. for 1 hour. Next, the enzyme-treated solution was centrifuged (4500 rpm, 10 minutes), and the supernatant was removed to obtain a two-fold concentrated PHA aqueous suspension. To the concentrated PHA aqueous suspension, the same amount of sodium hydroxide as the removed supernatant was added, centrifugation was performed again (4500 rpm, 10 minutes), and the supernatant was removed, which was repeated three times. The residual protein concentration in the resulting PHA aqueous suspension was 926 ppm.
• Example 8 The treatment was carried out in the same manner as in Example 7, except that esperase was not added, up to the alkali treatment.
• the residual protein concentration in the obtained PHA aqueous suspension was 1244 ppm.
• Example 7 From Table 6, both Examples 7 and 8 were able to obtain a sufficient amount of protein.
• the protein concentration of Example 8 is higher than that of Example 7, presumably due to the inclusion of impurities. Therefore, it was shown that PHA with higher purity can be obtained by performing treatment with esperase.

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