WO2024166803A1 - ポリヒドロキシアルカン酸の製造方法 - Google Patents

ポリヒドロキシアルカン酸の製造方法 Download PDF

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WO2024166803A1
WO2024166803A1 PCT/JP2024/003416 JP2024003416W WO2024166803A1 WO 2024166803 A1 WO2024166803 A1 WO 2024166803A1 JP 2024003416 W JP2024003416 W JP 2024003416W WO 2024166803 A1 WO2024166803 A1 WO 2024166803A1
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pha
aqueous suspension
producing
culture solution
polyhydroxyalkanoic acid
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French (fr)
Japanese (ja)
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郁弥 迫
智哉 西中
竜輝 安成
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Kaneka Corp
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Kaneka Corp
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    • 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
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable
    • 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

Definitions

  • the present invention relates to a method for producing polyhydroxyalkanoic acid.
  • PHA Polyhydroxyalkanoic acid
  • PHA Polyhydroxyalkanoic acid
  • Patent Documents 1 and 2 leave room for improvement in terms of productivity, because components derived from the bacterial cells clog the membrane during membrane separation, reducing the membrane's permeability.
  • one aspect of the present invention aims to provide a simple method for producing PHA that can suppress the decrease in membrane permeability caused by components derived from bacterial cells.
  • this production method a method for producing PHA (hereinafter referred to as "this production method"), comprising the steps of: (a) maintaining a culture solution containing PHA-containing bacteria at 40 to 80°C; (b) adding an enzyme to the culture solution obtained in step (a) to enzymatically treat the bacteria; (c) adding an oxidizing agent to the culture solution obtained in step (b); (e) centrifuging the culture solution obtained in step (c); and (f) filtering the suspension obtained in step (e) through a membrane.
  • a method for producing PHA can be provided that can easily suppress the decrease in membrane permeability caused by components derived from bacterial cells.
  • FIG. 1 is a graph plotting the measurement results of average permeation flux in Example 1, Comparative Example 1, and Comparative Example 2.
  • 1 is a graph showing the results of measuring the molecular sizes of impurities contained in the supernatants of Example 1 and Comparative Example 1.
  • FIG. 2 is a schematic diagram of a filtration device used in the examples.
  • a method of performing membrane separation is known as a method of separating and purifying PHA from the living body of a microorganism.
  • a method of preventing membrane clogging there is a method of reducing the content of impurities other than PHA by sufficiently performing centrifugation and washing before performing membrane separation to increase the degree of purification.
  • the number of work steps but also the amount of waste washing water is increased, which is problematic from an economic point of view.
  • Patent Document 1 is an excellent technology that can obtain high-purity 3-hydroxyalkanoic acid in high yields, but because an oxidizing agent is added to the 3-hydroxyalkanoic acid separated from the microorganism, it is difficult to sufficiently prevent membrane blockage due to components derived from the bacteria.
  • Patent Document 2 involves a bleaching process in which an oxidizing agent is added after filtration, so it is similarly difficult to sufficiently prevent membrane blockage during filtration.
  • the inventors conducted extensive research to solve the above problems, and discovered for the first time that in a PHA manufacturing method, by adding an oxidizing agent to an aqueous PHA suspension prior to membrane filtration, the components derived from the bacteria are broken down into smaller molecules, preventing membrane blockage and easily suppressing a decrease in membrane permeability.
  • this manufacturing method even if an aqueous PHA suspension with a low degree of purification is used, the impurities in the aqueous PHA suspension are broken down into smaller molecules, making it difficult for membrane blockage to occur.
  • the above-mentioned configuration can reduce the amount of wastewater used to clean the membranes used for filtration, which can contribute to the achievement of the Sustainable Development Goals (SDGs), such as Goal 14 "Conserve and sustainably use the oceans and marine resources for sustainable development.”
  • SDGs Sustainable Development Goals
  • the PHA produced by this manufacturing method may be in the form of either an aqueous suspension or a powder.
  • this manufacturing method may be "a method for producing an aqueous PHA suspension including the above steps (a)-(c) and (e)-(f)” or "a method for producing a PHA powder including the above steps (a)-(c) and (e)-(f)."
  • Step (a) in this production method is a step of maintaining a culture solution containing PHA-containing bacterial cells at 40 to 80° C.
  • Step (a) can inactivate the PHA-producing bacteria. Furthermore, by previously inactivating the bacterial cells in the culture solution by subjecting them to an inactivation temperature in step (a), a purification step, etc., which will be described later, can be easily carried out.
  • the term “inactivation temperature” refers to a temperature at which the microorganisms in the culture solution can be killed.
  • the term “inactivation temperature” refers to a temperature at which catalase, an enzyme contained in the microorganisms, can be inactivated.
  • the inactivation temperature is 40°C to 80°C, which is the temperature at which the microorganisms are killed and the catalase is inactivated, and is preferably 50°C to 80°C, and more preferably 60°C to 80°C.
  • PHA is a general term for polymers having hydroxyalkanoic acid as a monomer unit.
  • Hydroxyalkanoic acids constituting PHA are not particularly limited, but examples thereof include 3-hydroxybutanoic acid, 4-hydroxybutanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, and 3-hydroxyoctanoic acid.
  • These polymers may be homopolymers or copolymers containing two or more types of monomer units.
  • examples of PHA include 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 preferred because they are easy to produce
  • composition ratio of the repeating units by changing the composition ratio of the repeating units, it is possible to change the melting point and degree of crystallinity, and as a result, physical properties such as Young's modulus and heat resistance can be changed. It is also possible to impart physical properties between those of polypropylene and polyethylene, and as described above, it is easy to produce industrially and is a physically useful plastic. From this viewpoint, P3HB3HH, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferable.
  • the composition ratio of the repeating units of P3HB3HH is preferably 80/20 to 99.9/0.1 (mol/mol), more preferably 85/15 to 97/3 (mol/mol), in terms of the balance between flexibility and strength.
  • the composition ratio of 3-hydroxybutyrate units/3-hydroxyhexanoate units is 99.9/0.01 (mol/mol) or less, sufficient flexibility is obtained, and when it is 80/20 (mol/mol) or more, sufficient hardness is obtained.
  • the weight average molecular weight (hereinafter sometimes referred to as "Mw") of the PHA obtained in step (a) is preferably 1.5 million to 2.5 million, more preferably 1.6 million to 2.4 million, and even more preferably 1.7 million to 2.3 million. If the weight average molecular weight of the PHA is within the above range, excellent culture productivity will be achieved.
  • the weight average molecular weight of the PHA can be determined as the molecular weight in terms of polystyrene by gel permeation chromatography (GPC) (Shodex GPC-101 manufactured by Showa Denko) using a polystyrene gel (Shodex K-804 manufactured by Showa Denko) in a column and chloroform as the mobile phase.
  • GPC gel permeation chromatography
  • the solids 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 30 to 40% by weight. If the solids concentration of the culture solution obtained in step (a) is within the above range, a sufficient amount of PHA can be obtained.
  • the bacterial cells used in step (a) are not particularly limited as long as they are microorganisms capable of producing PHA within the cells.
  • microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants that can be prepared from them can be used.
  • the first bacterial cell to produce P3HB an example of PHA, was Bacillus megaterium, discovered in 1925, and other natural microorganisms include Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. It is known that PHA accumulates within the bacterial cells of these microorganisms.
  • bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB.
  • Aeromonas caviae which produces P3HB3HV and P3HB3HH
  • Alcaligenes eutrophus which produces P3HB4HB.
  • P3HB3HH Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, p4821-4830 (1997)) into which genes of a group of PHA synthases have been introduced is more preferred.
  • the bacterial cells may be genetically modified microorganisms into which various PHA synthesis-related genes have been introduced depending on the PHA to be produced.
  • PHA-producing bacteria the microorganisms capable of producing PHA within the cells described above may also be referred to as "PHA-producing bacteria.”
  • Step (b) In the step (b) of this production method, an enzyme is added to the culture solution obtained in the step (a) to enzymatically treat the cells.
  • an enzyme is added to the culture solution obtained in the step (a) to enzymatically treat the cells.
  • impurities derived from the cells are destroyed and removed, so that PHA can be efficiently recovered from the cells.
  • the enzyme treatment in step (b) may be a lytic enzyme treatment and/or an alkaline protease treatment. It is preferable that the lytic enzyme treatment and the alkaline protease treatment are each performed at least once, and if necessary, the lytic enzyme treatment and/or the alkaline protease treatment may be performed two or more times.
  • the order in which the lytic enzyme treatment and/or alkaline proteolytic enzyme treatment are performed is not particularly limited.
  • step (b) When performing the enzyme treatment (e.g., lytic enzyme treatment and alkaline protease treatment) in step (b), it is preferable to adjust the pH and temperature of the culture solution to the optimal pH and temperature of the enzyme used.
  • the enzyme treatment e.g., lytic enzyme treatment and alkaline protease treatment
  • the method for adjusting the pH and temperature of the culture solution there are no particular limitations on the method for adjusting the pH and temperature of the culture solution, and any known method can be used.
  • Step (b) may include a step of adding a surfactant.
  • the surfactant may be added before, simultaneously with, or after the addition of the alkaline protease.
  • the surfactant is added after the addition of the alkaline protease.
  • the lytic enzyme treatment is a step of adding a lytic enzyme to the culture solution to enzymatically treat the bacterial cells.
  • lytic enzyme refers to an enzyme that has the activity of degrading (lysing) the cell wall (e.g., peptidoglycan) of a bacterial cell.
  • the lytic enzyme is not particularly limited, and examples thereof include lysozyme, labia, ⁇ -N-acetylglucosaminidase, endolysin, autolysin, and the like. From the viewpoint of economical advantage, lysozyme is preferred. One of these may be used alone, or two or more may be used in combination.
  • lytic enzymes such as "lysozyme” and “achromopeptidase” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • the optimal pH of the lytic enzyme is not particularly limited as long as the lytic enzyme has cell wall decomposition activity, but is, for example, 5.0 to 11.0, preferably 6.0 to 9.0, and more preferably 6.0 to 8.0.
  • the optimum temperature of the lytic enzyme is not particularly limited, but from the viewpoint of not requiring excessive heating and being able to prevent thermal changes (thermal decomposition) of the PHA, it is preferably 60°C or less, and more preferably 50°C or less.
  • the lower limit of the optimum temperature is not particularly limited, but from the viewpoint of not requiring excessive cooling operations and being economical, it is preferably room temperature (e.g., 25°C) or higher.
  • alkaline protease refers to a protease that has the activity of decomposing proteins in an alkaline environment (for example, in a solution of pH 8.5).
  • alkaline protease treatment refers to the process of adding the alkaline protease to the culture solution and enzymatically treating the bacterial cells.
  • the alkaline protease is not particularly limited as long as it has the activity of decomposing proteins in an alkaline environment, and examples thereof include serine-specific proteases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteases (e.g., papain, bromelain, cathepsin), aspartic acid-specific proteases (e.g., pepsin, cathepsin D, HIV protease), etc. From the viewpoint of economic advantage, serine-specific proteases, particularly subtilisin (e.g., alcalase), are preferred. These may be used alone or in combination of two or more.
  • serine-specific proteases e.g., subtilisin, chymotrypsin, trypsin
  • cysteine-specific proteases e.g., papain, bromelain, cathepsin
  • aspartic acid-specific proteases e.g., pepsin,
  • Alkaline protease may be a commercially available product, such as "Alcalase 2.5L” 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 Nippon Chemical Industry Co., Ltd.; “Delborase” manufactured by DSM Japan Co., Ltd.; “Bioprase OP", “Bioprase SP-20FG” and “Bioprase SP-4FG” manufactured by Nagase ChemteX Corporation; “Orientase 22BF” manufactured by HBI Corporation; “Aroase XA-10” manufactured by Yakult Pharmaceutical Co., Ltd.; and “Esperase” manufactured by Novozyme.
  • Alkaline protease may be a commercially available product, such as "Alcalase 2.5L” manufactured
  • the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease has activity in an alkaline environment, but is, for example, 8.0 to 14.0, preferably 8.0 to 12.0, more preferably 8.0 to 10.0, even more preferably 8.0 to 9.0, and most preferably 8.5.
  • the optimum temperature of the alkaline protease is not particularly limited, but is preferably 60°C or less, and more preferably 50°C or less, from the viewpoint of not requiring excessive heating and being able to prevent thermal changes (thermal decomposition) of P3HA.
  • the lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25°C) or higher, from the viewpoint of not requiring excessive cooling operations and being economical.
  • the enzymatic treatment in step (b) can be carried out using a combination of lysozyme and alcalase.
  • Step (c) in this production method is a step of adding an oxidizing agent to the culture solution obtained in step (b).
  • low molecular weight means that "80% by weight or more of the impurities contained in the supernatant are 3 kDa or less" when measured by the method described in the Examples.
  • the aqueous suspension containing PHA obtained in step (c) and the subsequent steps may be abbreviated as “PHA aqueous suspension” or "aqueous suspension”.
  • step (c) the bacterial impurities contained in the culture solution can be broken down into smaller molecular weight impurities.
  • clogging of the membrane can be suppressed in step (f) described below, improving the productivity of PHA.
  • step (f) described below, improving the productivity of PHA.
  • step (f) by adjusting the weight average molecular weight of PHA, it is possible to produce PHA with the desired weight average molecular weight.
  • the oxidizing agent is not particularly limited, but examples thereof include hydrogen peroxide (H 2 O 2 ), ozone; other inorganic peroxides such as sodium peroxide (Na 2 O 2 ), sodium perborate (Na 2 H 4 B 2 O 8 ), sodium percarbonate (Na 2 H 3 CO 6 ), and sodium persulfate (Na 2 S 2 O 8 ); similar halogen compounds such as chlorite, chlorate, metachloroperbenzoic acid (C 7 H 5 ClO 3 ), perchlorate, perchloric acid (ClO 4 ), and chlorine dioxide (ClO 2 ); peracids such as performic acid (CH 2 O 3 ) and peracetic acid (CH 3 CO 3 H); permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO 3 ); sodium bismuthate; and cerium (IV) compounds such as ammonium cerium nitrate and cerium sulfate.
  • H 2 O 2
  • the concentration of hydrogen peroxide in the culture solution is, for example, 0.2 to 30% by weight, preferably 0.2 to 15% by weight, and more preferably 0.2 to 10% by weight. If the concentration is 0.2% by weight or more, the bacterial impurities contained in the culture solution can be reduced in molecular weight. Furthermore, if the concentration is 30% by weight or less, the cost of PHA production can be reduced.
  • hydrogen peroxide can be added so as to achieve the above concentration.
  • the amount of ozone added is, for example, 0.01 to 0.1 g, preferably 0.02 to 0.08 g, and more preferably 0.02 to 0.07 g per 1 g of biomass containing PHA. If the amount is 0.01 g or more, the viscosity of the culture solution can be reduced. If the amount is 0.1 g or less, the amount of remaining ozone is small.
  • NaHCO 3 when hydrogen peroxide is added, NaHCO 3 may be added together with the hydrogen peroxide. It is expected that NaHCO 3 will assist the function of hydrogen peroxide.
  • a chelating agent may be added together with the hydrogen peroxide.
  • the 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, etc.
  • the pH of the culture liquid is preferably 10.0 to 11.0, more preferably 10.2 to 10.8, and even more preferably 10.4 to 10.6. If the pH is 10.0 or higher, the bacterial cell-derived impurities can be efficiently reduced in molecular weight. Furthermore, if the pH is 11.0 or lower, the decrease in the weight average molecular weight of the PHA can be suppressed.
  • the pH may be adjusted, for example, by adding an alkaline aqueous solution.
  • the alkaline aqueous solution may be an aqueous solution containing the same type of basic compound as in step (a') described below.
  • step (c) the order in which the oxidizing agent is added and the pH is adjusted is not particularly limited, but from the viewpoint of efficiently lowering the pH, it is preferable to add the oxidizing agent and then adjust the pH.
  • the temperature of the culture medium is 30°C to 75°C, preferably 35°C to 70°C, and more preferably 40°C to 60°C.
  • the PHA aqueous suspension obtained in step (c) is preferably a Newtonian fluid. If the PHA aqueous suspension is a Newtonian fluid, the viscosity has low shear rate dependency, so the PHA aqueous suspension is more likely to become uniform in the stirring device, improving handleability.
  • step (c) includes a step of maintaining the adjusted pH for preferably 0.1 to 30 hours, more preferably 0.25 to 24 hours, and even more preferably 0.5 to 15 hours.
  • step (c) includes a step of maintaining the adjusted pH for preferably 0.1 to 30 hours, more preferably 0.25 to 24 hours, and even more preferably 0.5 to 15 hours.
  • the method for maintaining the adjusted pH is not particularly limited, and is carried out, for example, by adding an alkaline aqueous solution.
  • the alkaline aqueous solution is not particularly limited, and 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, and preferably 40 to 60°C.
  • Step (e) is a step of centrifuging the PHA aqueous suspension obtained in step (c) and removing the supernatant to obtain a PHA aqueous suspension in which PHA is concentrated. That is, it is a step of concentrating and purifying the PHA aqueous suspension separated from the bacterial cells.
  • 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.
  • the PHA aqueous suspension in step (e), may be concentrated up to 2 times, 2.5 times, 3 times, 3.3 times, or 4 times. By concentrating the PHA aqueous suspension, the degree of purification of the PHA aqueous suspension can be increased.
  • step (e) the method for centrifuging the PHA aqueous suspension is not particularly limited, and any known method can be used.
  • step (e) the PHA aqueous suspension may be centrifuged, the supernatant removed, and then a solution may be added to the sediment, followed by another centrifugation and supernatant removal step.
  • This operation can increase the degree of purification of the PHA aqueous suspension.
  • the degree of purification of the PHA aqueous suspension can be determined, for example, by measuring the absorbance at 280 nm or 254 nm.
  • the solution added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the PHA aqueous suspension.
  • the solution is preferably the same as the alkaline aqueous solution used in step (d).
  • the amount of impurities remaining in the final product is largely determined by step (e). It is preferable to reduce these impurities as much as possible. Naturally, depending on the application, impurities may be present as long as they do not impair the physical properties of the final product, but in cases where a high purity PHA is required, such as for medical applications, it is preferable to reduce the impurities as much as possible.
  • An example of the degree of purification is 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 that can achieve the amount of residual protein in the PHA powder.
  • the protein amount is preferably 3000 ppm or less, more preferably 2500 ppm or less, and even more preferably 2000 ppm or less per weight of PHA in the PHA aqueous suspension.
  • the solvent ("solvent” is also referred to as "aqueous medium" constituting the PHA aqueous suspension is not particularly limited, and may be water or a mixed solvent of water and an organic solvent.
  • 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, and examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethyl sulfoxide, pyridine, and piperidine.
  • alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, pentanol, hexanol, and heptanol
  • ketones such as acetone and methyl ethyl ketone
  • methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easy to remove.
  • methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily 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, even 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 bacteria, compounds generated during purification, etc., as long as this does not impair the essence of the present invention.
  • Step (f) is a step of filtering the aqueous suspension obtained in step (e) through a membrane, whereby bacterial cell-derived impurities are removed from the aqueous PHA suspension through membrane filtration to obtain an aqueous PHA suspension.
  • the pH of the PHA aqueous suspension in step (f) is preferably 8.0 to 12.0, more preferably 9.0 to 11.5, and even more preferably 10.0 to 11.0. If the pH is 8.0 to 12.0, the PHA can be concentrated to a higher concentration while suppressing a decrease in the molecular weight of the PHA.
  • the filtration method carried out in step (f) is not particularly limited, and may be cross-flow filtration, dead-end filtration, etc. Among these, cross-flow filtration is preferred from the viewpoint of suppressing fouling on the membrane surface.
  • the average pore diameter of the filtration membrane used in step (f) is not particularly limited, but is, for example, 0.05 to 0.5 ⁇ m, and more preferably 0.1 to 0.3 ⁇ m. If the average pore diameter is within the above range, the permeation flux is increased and the required membrane area can be reduced, thereby improving productivity.
  • the average pore diameter of the filtration membrane can be determined by the bubble point method.
  • the inner diameter of the filtration membrane used in step (f) is not particularly limited, but is, for example, 4 to 10 mm, and preferably 4 to 7 mm. If the inner diameter is within the above range, filtration can be carried out efficiently.
  • the "inner diameter of the filtration membrane” means the inner diameter of the circular hollow cross section of the filtration membrane.
  • the material of the filtration membrane is not particularly limited, but examples include polypropylene, fluorine-based resins (e.g., polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl fluoride, tetrafluoroethylene-hexafluoropropylene copolymer, ethylene-tetrafluoroethylene copolymer, etc.), cellulose esters (e.g., cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, etc.), polysulfone-based resins (e.g., polysulfone, polyethersulfone, etc.), polyacrylonitrile, polyimide, and other resins, inorganic materials such as porous ceramics such as alumina, mullite, zirconia, cordierite, and porous sintered metals such as stainless steel, and the like.
  • fluorine-based resins e.g., polytetrafluoroethylene, poly
  • the transmembrane pressure difference in step (f) is not particularly limited, but is preferably 0.001 to 0.1 MPa, and more preferably 0.01 to 0.1 MPa. If the transmembrane pressure difference is within the above range, filtration is performed sufficiently and membrane deterioration can be suppressed.
  • the linear velocity of the PHA aqueous suspension in step (f) is not particularly limited, but is preferably 1 to 5 m/s, more preferably 1.5 to 4 m/s, and even more preferably 2 to 4 m/s. If the linear velocity is within the above range, filtration can be performed sufficiently and deterioration of the membrane can be suppressed. Furthermore, if the filtration membrane is made of a resin such as polypropylene, it is preferable to set the linear velocity to 3 m/s or less from the viewpoint of the durability of the filtration membrane.
  • the production method may include a step (a') of adjusting the molecular weight of the PHA in the bacterial cells by adding an alkaline aqueous solution to the culture solution obtained in the step (a). That is, the step (a') is a step of adjusting the molecular weight of the PHA in the bacterial cells to an appropriate size by adding an alkaline aqueous solution to the culture solution in which the bacterial cells have been inactivated in the step (a). In other words, the step (a') can be said to be an alkali treatment step.
  • the step (a') has the advantage of improving the processability of the PHA powder.
  • the step (a') also has the advantage of solubilizing the protein in the bacterial 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, but examples thereof include 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 or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, and potassium hydrogen phosphate.
  • 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 compounds may be used alone or in combination of two or more kinds.
  • step (a') the pH of the culture liquid is preferably adjusted to 8.0 to 12.0 by adding an alkaline aqueous solution, more preferably to 8.2 to 11.5, and even more preferably to 8.4 to 11.5.
  • the pH to 8.0 or higher and maintaining the pH at a constant value any constant value between pH 8.0 and 12.0
  • the reaction time in step (a') is, for example, 4 hours to 30 hours, and preferably 8 hours to 20 hours. If the reaction time in step (a') is within the above range, the molecular weight of the PHA in the bacterial cells can be adjusted to a suitable range and excessive reduction in molecular weight of the PHA can be prevented.
  • the temperature in step (a') is preferably less than 100°C, and more preferably less than 80°C. There is no particular lower limit, but it is preferably, for example, 50°C or higher.
  • the weight-average molecular weight of the PHA in the culture solution obtained in step (a') is preferably 100,000 to 800,000, more preferably 200,000 to 800,000, and even more preferably 400,000 to 800,000. If the weight-average molecular weight is 100,000 or more, sufficient mechanical properties are obtained when the PHA is made into a powder, and if it is 800,000 or less, a sufficient crystallization rate is obtained and good moldability is achieved.
  • the production method preferably includes a step (d) of adding a surfactant and an aqueous alkaline solution to adjust the pH to 10.0 to 12.0 before the step (e).
  • the 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) is a step of adding an alkaline aqueous solution to the PHA aqueous suspension obtained in step (c) to adjust the pH to 10.0 to 12.0.
  • impurities derived from the bacterial cells are dispersed and dissolved, and a high-purity PHA can be separated from the bacterial cells.
  • step (d1) the description of the alkaline aqueous solution in step (a') applies.
  • step (d1) the pH is preferably adjusted to 10.0 to 12.0 by adding an alkaline aqueous solution, more preferably to 10.2 to 11.8, and even more preferably to 10.4 to 11.6. Adjusting the pH to 10.0 or higher has the advantage of allowing the decomposition and dissolution of bacterial components. In addition, adjusting the pH to 12.0 or lower can prevent unintended damage to the bacterial cells.
  • the temperature in step (d1) is preferably less than 100°C, and more preferably less than 80°C. There is no particular lower limit, but it is preferably, for example, 40°C or higher.
  • Step (d2) is a step of adding a surfactant to the culture solution, which can efficiently treat impurities contained in the cells, particularly cell membranes, and can remove a larger amount of impurities derived from the cells, allowing a higher purity PHA to be separated from the cells.
  • the surfactant is not particularly limited, but examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, etc.
  • anionic surfactants are preferred from the viewpoint of their high cell membrane removal ability.
  • One of these surfactants may be used alone, or two or more of them may be used in combination.
  • anionic surfactants include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfate ester salts, alkenyl sulfate ester salts, alkyl ether sulfate ester salts, alkenyl ether sulfate ester salts, ⁇ -olefin sulfonates, ⁇ -sulfofatty acid salts, esters of ⁇ -sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acyl amino acid surfactants.
  • alkyl sulfate ester salts are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred from the viewpoints of its high cell membrane removal ability and low cost.
  • SDS sodium dodecyl sulfate
  • the anionic surfactants may be used alone or in combination of two or more of these.
  • the amount of surfactant added is not particularly limited, and is, for example, 0.1 to 5.0% by weight, preferably 0.3 to 2.5% by weight, relative to the culture medium.
  • Step (d2) may be carried out before, simultaneously with, or after step (d1). That is, in step (d), the surfactant and the alkaline aqueous solution may be added by adding an alkaline aqueous solution to adjust the pH and then adding the surfactant, or by adding the surfactant and the alkaline aqueous solution simultaneously to adjust the pH, or by adding the surfactant and then adding the alkaline aqueous solution to adjust the pH.
  • the present production method preferably includes a step (g) of drying the PHA aqueous suspension obtained in the step (f).
  • the step (g) is a step of removing water from the PHA aqueous suspension obtained in the step (f).
  • a PHA powder having a reduced water content can be obtained from the PHA aqueous suspension.
  • the drying method may be, for example, a method in which the PHA aqueous suspension is supplied in the form of fine droplets into a dryer and dried in the dryer while being exposed to hot air (spray drying), vacuum drying, etc.
  • spray drying is used.
  • step (g) can be referred to as a drying step.
  • the method of supplying the PHA aqueous suspension into the dryer in the form of fine droplets is not particularly limited, and examples of such methods include known methods using a rotating disk and a nozzle.
  • the method of contacting the droplets with the hot air in the dryer is not particularly limited, and examples of such methods include a parallel flow method, a counter flow method, and a method that combines these.
  • the drying temperature when spray drying is performed in step (g) may be any temperature capable of removing most of the aqueous medium from the droplets of the PHA aqueous suspension, and may be appropriately set under conditions that allow drying to the desired moisture content and minimize the occurrence of quality deterioration (reduction in molecular weight, loss of color, etc.), melting, etc.
  • the temperature of the hot air blown into the spray dryer may be appropriately selected within the range of 100 to 300°C.
  • the volume of hot air in the dryer may also be appropriately set depending on, for example, the size of the dryer, etc.
  • the method may include a step of further drying the obtained PHA (e.g., PHA powder) after step (g) (e.g., a step of subjecting the PHA to reduced pressure drying, etc.).
  • the present manufacturing method may include other steps (e.g., a step of adding various additives to the PHA aqueous suspension, etc.).
  • the present invention includes the following configurations. ⁇ 1> (a) maintaining a culture solution containing a bacterial cell containing polyhydroxyalkanoic acid at 40 to 80°C; (b) adding an enzyme to the culture solution obtained in the step (a) to enzymatically treat the cells; (c) adding an oxidizing agent to the culture solution obtained in the step (b); (e) centrifuging the aqueous suspension obtained in step (c); (f) subjecting the aqueous suspension obtained in step (e) to membrane filtration;
  • a method for producing a polyhydroxyalkanoic acid comprising: ⁇ 2> The method for producing a polyhydroxyalkanoic acid according to ⁇ 1>, further comprising, prior to the step (e), (d) adding a surfactant and an aqueous alkaline solution to the culture solution obtained in the step (c) to adjust the pH to 10.0 to 12.0.
  • ⁇ 3> The method for producing a polyhydroxyalkanoic acid according to ⁇ 1> or ⁇ 2>, wherein the enzyme in the step (b) is a lytic enzyme and/or an alkaline protease.
  • the enzyme in the step (b) is a lytic enzyme and/or an alkaline protease.
  • ⁇ 4> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 3>, wherein the oxidizing agent in the step (c) is hydrogen peroxide or ozone.
  • ⁇ 5> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 4>, wherein the membrane filtration in the step (f) is cross-flow filtration.
  • ⁇ 6> The method for producing a polyhydroxyalkanoic acid powder according to any one of ⁇ 1> to ⁇ 5>, further comprising: (g) a step of drying the aqueous suspension obtained in the step (f).
  • ⁇ 7> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 6>, wherein in the step (c), the aqueous suspension has a pH of 10.0 to 11.0.
  • ⁇ 8> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 7>, wherein in the step (e), centrifugation is performed until the amount of protein contained in the aqueous suspension becomes 3000 ppm or less.
  • ⁇ 9> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 8>, wherein the pH of the aqueous suspension in the step (f) is 8.0 to 12.0.
  • ⁇ 10> The method for producing a polyhydroxyalkanoic acid according to any one of ⁇ 1> to ⁇ 9>, wherein the temperature of the culture solution in the step (c) is 30°C to 75°C.
  • the filtrate obtained in 3.2 was filtered for a certain period of time using a centrifugal filter device with a pore size of 0.2 ⁇ m (product name: Nanosep, manufactured by Nippon Pall Corporation), and the filtrate and concentrated liquid were collected.
  • the filtrate obtained in 4.3 was filtered for a certain period of time using a centrifugal filtration device with a molecular weight cutoff of 300 kDa (product name: Nanosep, manufactured by Nippon Pall Corporation), and the filtrate and concentrated liquid were collected.
  • the filtrate obtained in 5.4 was filtered for a certain period of time using a centrifugal filter device with a molecular weight cutoff of 30 kDa (product name: Nanosep, manufactured by Nippon Pall Corporation), and the filtrate and concentrated liquid were collected.
  • the filtrate obtained in 6.5 was filtered for a certain period of time using a centrifugal filtration device with a molecular weight cutoff of 3 kDa (product name: Nanosep, manufactured by Nippon Pall Corporation), and the filtrate and concentrated liquid were collected. 7. Pure water was added to the concentrated liquids obtained in 3 to 5 and the filtrate obtained in 6 so that all the liquid amounts were the same. 8.
  • the absorbance of each of the solutions obtained in 7 was measured at a wavelength of 254 nm (measuring device: spectrophotometer (U-5100, Hitachi High-Technologies Corporation)).
  • Average permeation flux (L/m 2 h) total permeated liquid volume (L)/(membrane area (m 2 ) ⁇ filtration time (h)) (Protein content)
  • the amount of protein attached to the PHA surface was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 20 to 50 mg (amount containing about 10 mg of P3HB3HH particles in the liquid) of the PHA aqueous suspension immediately before step (c') was placed in a 15 mL Falcon tube, 2 mL of the reagent of the kit was added, and the mixture was shaken at 60°C for 30 minutes.
  • Example 1 (culture) 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 fungal culture solution containing fungal cells containing PHA.
  • Ralstonia eutropha is currently classified as Capriavidus necator.
  • the composition ratio of the repeating units of PHA was 94/6 (mol/mol).
  • Step (a): Inactivation The bacterial 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 molecular weight was 1.8 million, and the solid content of the inactivated culture solution was 30% by weight.
  • the pH of the inactivated culture solution was adjusted to 9.0 ⁇ 0.2 using 30% sodium hydroxide, and the molecular weight adjustment of PHA was carried out for 12 hours at an internal temperature of 50 ⁇ 2° C.
  • the molecular weight of PHA after step (a′) was 500,000.
  • Step (b): Enzyme treatment Lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that degrades sugar chains (peptidoglycans) in cell walls, was added to the inactivated culture solution to a liquid concentration of 10 ppm, and the solution was maintained at 50° C. for 2 hours. Thereafter, Alcalase 2.5L (manufactured by Novozyme, Inc.), a protease, was added to a liquid concentration of 300 ppm, and then 30% sodium hydroxide was added at 50° C., and the solution was maintained for 2 hours while adjusting the pH to 8.5, to obtain an enzyme-treated culture solution.
  • Alcalase 2.5L manufactured by Novozyme, Inc.
  • Hydrogen peroxide manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • 30% aqueous sodium hydroxide solution was added to adjust the pH to 10.5.
  • 30% aqueous sodium hydroxide solution was added continuously to maintain the pH at 10.5 for 4 hours, and an aqueous PHA suspension was obtained.
  • Step (d): Surfactant and Alkali Treatment A surfactant, sodium dodecyl sulfate (SDS, manufactured by Kao Corporation), was added to the aqueous PHA suspension after the hydrogen peroxide treatment so as to give a concentration of 0.6 to 1.0% by weight. Then, an aqueous sodium hydroxide solution was added to the aqueous PHA suspension to which the surfactant had been added, to adjust the pH to 11.0 ⁇ 0.2, thereby obtaining an aqueous PHA suspension that had been alkali-treated.
  • SDS sodium dodecyl sulfate
  • Step (e): Centrifugation An equal amount of distilled water was added to the alkali-treated PHA aqueous suspension obtained above. After centrifuging, the supernatant was removed and the suspension was concentrated twice. The concentrated aqueous suspension of PHA was added with an aqueous sodium hydroxide solution (pH 11.0) in the same amount as the removed supernatant, centrifuged, and the supernatant was removed. Water was added again to the suspension to suspend it, and 0.2% by weight of sodium dodecyl sulfate and 1/100 weight of protease (Novozyme, Esperase) of PHA were added, and the suspension was stirred for 2 hours while maintaining pH 10.0 and 50°C.
  • aqueous sodium hydroxide solution pH 11.0
  • the supernatant was then removed by centrifugation to concentrate the suspension 2.5 times, 3 times, 3.3 times, 4.7 times, and 4 times.
  • an aqueous PHA suspension was obtained in which the median diameter (D50) of the PHA particles was 2 ⁇ m and the solid content concentration was 26% by weight (PHA particle content: 260 g/L).
  • the protein amounts were (1) 1630 ppm, (2) 1430 ppm, (3) 1400 ppm, (4) 1220 ppm, and (5) 1200 ppm, respectively.
  • Step (f): Membrane filtration In the filtration apparatus shown in Fig. 3, which used a tubular membrane (MEMBRALOX (registered trademark) 1T1-70, manufactured by PALL, material: alumina ceramic) as the filtration membrane, the PHA aqueous suspension was circulated and fed to the tubular membrane, and filtration was performed until the solid content concentration reached 55% by weight.
  • the linear velocity during filtration (linear velocity at which the liquid passed through the tubular membrane) was 3 to 4 m/s, and the transmembrane pressure difference was 80 kPa.
  • Comparative Example 2 Except for carrying out the oxidizing agent treatment after centrifugation, a PHA aqueous suspension was obtained in the same manner as in Example 1. Comparative Example 2 corresponds to the method described in Patent Document 1. The concentration ratios were 3 times, 3.5 times, and 5 times, respectively.
  • Example 1 in which oxidizing agent treatment was performed before centrifugation, had a higher average permeation flux of the PHA aqueous suspension at the same absorbance (i.e., the same degree of purification) than Comparative Example 1, in which oxidizing agent treatment was not performed, and Comparative Example 2, in which hydrogen peroxide treatment was performed after centrifugation.
  • a higher average permeation flux of the PHA aqueous suspension during membrane filtration indicates that more of the PHA aqueous suspension has permeated (filtered) through the membrane. From the above, it was demonstrated that this production method can easily suppress a decrease in membrane permeability caused by components derived from the bacterial cells.
  • Figure 2 shows the results of confirming the reduction in molecular weight of the 4-fold concentrated supernatant of Example 1 and the 4-fold concentrated supernatant of Comparative Example 1.
  • the supernatant of Example 1 contained more molecules of 3 kDa or less in size than the supernatant of Comparative Example 1.
  • SDS-PAGE polyacrylamide electrophoresis
  • the present invention can produce PHA with high yield.
  • the present invention can be suitably used in the fields of agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08503131A (ja) * 1992-11-12 1996-04-09 ゼネカ・リミテッド 微生物からの固体物質の分離方法
JPH08508881A (ja) * 1993-04-14 1996-09-24 ゼネカ・リミテッド 微生物からのプラスチック材料の製造
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
WO2023027953A1 (en) * 2021-08-23 2023-03-02 Meredian, Inc. Method for recovering phas from a biomass

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08503131A (ja) * 1992-11-12 1996-04-09 ゼネカ・リミテッド 微生物からの固体物質の分離方法
JPH08508881A (ja) * 1993-04-14 1996-09-24 ゼネカ・リミテッド 微生物からのプラスチック材料の製造
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
WO2023027953A1 (en) * 2021-08-23 2023-03-02 Meredian, Inc. Method for recovering phas from a biomass

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