Method for producing polyhydroxybutyric acid copolymer and its use

Abstract

The purpose of the present invention is to provide a PHB copolymer with which aggregation of PHB copolymer particles during the production process is inhibited and which has high bulk density. The purpose is also to provide a production method for said copolymer.　The aforementioned purpose is achieved by providing a PHB copolymer production method comprising: (a) a step for performing an enzymatic treatment using a specific enzyme on a microorganism containing a PHB copolymer having a composition ratio that is a 3HB unit / a hydroxyalkanoate unit other than a 3HB unit and is in a prescribed range; (b) a step for adding a surfactant after the pH is adjusted to within a prescribed range by adding an alkaline aqueous solution; (c) a step for preparing an aqueous suspension having a pH that is at most 7.0 and having a shear viscosity in a prescribed range; and (d) a step for spray drying.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a polyhydroxybutyric acid copolymer and its use. [Background technology]

[0002] Polyhydroxybutyric acid copolymers (hereinafter sometimes referred to as "PHB copolymers") are known to be biodegradable.

[0003] PHB copolymers produced by microorganisms accumulate within the microbial cells. Therefore, in order to use PHB copolymers as plastics, a process of separating and purifying the PHB copolymers from the microbial cells is required. In the process of separating and purifying the PHB copolymers, biologically derived components other than the PHB copolymers are solubilized, and then the PHB copolymers are extracted from the resulting aqueous suspension. Separation procedures such as centrifugation, filtration, and drying are performed. For drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer, or the like is used, but a spray dryer is preferably used because of its simple operation.

[0004] To date, the present inventors have developed a technique for preventing aggregation of PHB copolymer in an aqueous suspension at a pH of 7.0 or less by adding polyvinyl alcohol (PVA) as a dispersant before adjusting the pH of the aqueous suspension to 7.0 or less, and then spray-drying the resulting aqueous suspension at a pH of 7.0 or less (Patent Document 1). The present inventors have also developed a technique for producing a PHB copolymer with a high composition ratio of 3-hydroxyhexanoate (hereinafter sometimes referred to as "3HH") units, which includes a step of disrupting bacterial cells containing the PHB copolymer by high-pressure disruption (Patent Documents 2 and 3).

[0005] A known polyhydroxybutyrate copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter, sometimes referred to as "PHBH"). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 070492 [Patent Document 2] International Publication No. 2018 / 186278 [Patent Document 3] Japanese Patent Application Publication No. 2019-97518 Summary of the Invention [Problem to be solved by the invention]

[0007] Although the above-mentioned technology is excellent, there is room for further improvement in terms of ease of handling in the manufacturing process.

[0008] Therefore, an object of the present invention is to provide a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles during the production process, particularly a PHB copolymer having a high bulk density and a specific composition ratio of 3-hydroxybutyrate (hereinafter, sometimes referred to as "3HB") units / hydroxyalkanoate units other than 3HB units, and a method for producing the same. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have discovered for the first time that a PHB copolymer having a high bulk density can be obtained while suppressing aggregation of PHB copolymer particles during the production process by including a step of enzymatically treating, with a specific enzyme, bacterial cells containing a polyhydroxybutyric acid copolymer having a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units, in which the composition ratio of hydroxyalkanoate units other than 3HB units is high. This discovery led to the completion of the present invention.

[0010] Therefore, one aspect of the present invention is a method for producing a PHB copolymer, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), the method comprising: (a) adding an alkaline protease to a culture solution containing bacterial cells containing the PHB copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing, from the aqueous suspension obtained in step (b), an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less; and (d) spray-drying the aqueous suspension prepared in step (c).

[0011] Another aspect of the present invention is a PHB copolymer powder comprising a PHB copolymer, peptidoglycan, and a dispersant, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 μm.

[0012] Furthermore, one aspect of the present invention is a PHB copolymer powder comprising a PHB copolymer and a nitrogen compound, wherein the PHB copolymer has a composition ratio of 3-hydroxybutyrate units / hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 μm. [Effects of the Invention]

[0013] According to one aspect of the present invention, it is possible to provide a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles during the production process, particularly a PHB copolymer having a high bulk density and a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units. DETAILED DESCRIPTION OF THE INVENTION

[0014] An embodiment of the present invention will be described in detail below. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more and B or less." In addition, all documents described in this specification are incorporated herein by reference.

[0015] 1. Overview of the Invention A method for producing a PHB copolymer according to one embodiment of the present invention (hereinafter referred to as "this production method") is characterized in that the PHB copolymer has a composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), and includes the steps of: (a) adding an alkaline protease to a culture solution containing bacterial cells containing the PHB copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing an aqueous suspension from the aqueous suspension obtained in step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less; and (d) spray-drying the aqueous suspension prepared in step (c).

[0016] The present inventors have discovered a new problem in that, in a conventional method (Patent Document 1), a high-concentration slurry (PHB copolymer aqueous suspension) is used, but PHB copolymer particles having a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units aggregate, resulting in an increase in the viscosity of the PHB copolymer aqueous suspension, which can make it difficult to transport and spray-dry. Furthermore, they have found that reducing the concentration of the PHB copolymer aqueous suspension to solve the above problem requires a lot of energy for spray-drying, which is undesirable in terms of energy efficiency, and also creates other problems, such as a low bulk density of the resulting powder and poor powder transportability.

[0017] On the other hand, the methods described in Patent Documents 2 and 3 involve physically disrupting bacterial cells by high-pressure disruption, which causes the process to become complicated when scaled up. Furthermore, the method described in Patent Document 3 also has the problem of the production process itself being long.

[0018] Therefore, the present inventors have conducted extensive research to solve the above problems, and have found for the first time that a PHB copolymer having a high bulk density can be obtained while suppressing aggregation of PHB copolymer particles during the production process, even when spray-drying, by including a step of enzymatically treating, with a specific enzyme, bacterial cells containing a PHB copolymer having a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units. The present inventors speculate that this is because the above step causes the aqueous PHB copolymer suspension to contain peptidoglycan derived from bacterial cells, thereby preventing aggregation of PHB copolymer particles.

[0019] Furthermore, the present inventors have further investigated the PHB copolymer (PHB copolymer powder (intermediate)) obtained above from the viewpoint of increasing the purity, and as a result have found for the first time that a highly pure PHB copolymer having a high bulk density can be obtained by washing the PHB copolymer (PHB copolymer powder (intermediate)) with a specific enzyme (here, "high purity" means that the content of nitrogen compounds, which are impurities, is low).

[0020] Therefore, this production method can produce a PHB copolymer (e.g., PHB copolymer powder) having a high bulk density and a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units. This production method can also produce a PHB copolymer (e.g., PHB copolymer powder) having a high bulk density and a specific composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units, with a low content of nitrogen compounds as impurities. Furthermore, this production method can produce the PHB copolymer without using a physical crushing step (e.g., high-pressure crushing), which is advantageous from the perspective of scale-up.

[0021] Furthermore, the above-described configuration can reduce the amount of plastic waste generated, thereby contributing to the achievement of Sustainable Development Goals (SDGs) such as Goal 12 "Ensure sustainable consumption and production patterns" and Goal 14 "Conserve and sustainably use the oceans and marine resources for sustainable development." The configuration of this manufacturing method will be described in detail below.

[0022] 2. Method for producing PHB copolymer This production method includes the following steps (a) to (d) as essential steps: In addition, the PHB copolymer in this production method has a composition ratio of 3HB units / hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol). Step (a): adding an alkaline protease to a culture solution containing bacterial cells containing a PHB copolymer to enzymatically treat the bacterial cells. Step (b): adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment. Step (c): preparing an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa·s or more and 0.5 Pa·s or less from the aqueous suspension obtained in step (b). Step (d): spray-drying the aqueous suspension prepared in step (c). In step (c) of this production method, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa·s or more and 0.5 Pa·s or less is prepared from the aqueous suspension obtained in step (b). In this aqueous suspension, the PHB copolymer is present in a dispersed state in the aqueous medium. In this specification, an aqueous suspension containing at least the PHB copolymer may be abbreviated as "PHB copolymer aqueous suspension."

[0023] <Process (a)> Step (a) is a step of adding an alkaline protease to a culture solution containing bacterial cells containing a PHB copolymer to enzymatically treat the bacterial cells.

[0024] (PHB copolymer) The PHB copolymer in this production method is a copolymer of 3HB and a hydroxyalkanoate other than 3HB.

[0025] Examples of hydroxyalkanoates other than 3HB include 3-hydroxyhexanoate (3HH), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), 3-hydroxyoctadecanoate (3HOD), and 3-hydroxydecanoate (3HD).

[0026] A preferred example of the PHB copolymer is, but is not limited to, PHBH, and for the sake of convenience, the following description will be mainly based on PHBH as a representative example.

[0027] By changing the composition ratio of the 3HB and 3HH repeating units, the melting point and crystallinity of PHBH can be altered, which in turn changes physical properties such as Young's modulus and heat resistance, making it possible to impart physical properties between those of polypropylene and polyethylene.

[0028] The PHB copolymer produced by this method has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), preferably 81 / 19 to 87 / 13 (mol / mol), and more preferably 82 / 18 to 86 / 14 (mol / mol). When the composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units is 88 / 12 (mol / mol) or less, sufficient hardness is obtained, and when it is 80 / 20 (mol / mol) or more, sufficient flexibility is obtained.

[0029] In one embodiment of the present invention, the weight-average molecular weight (hereinafter sometimes referred to as "Mw") of the PHB copolymer is not particularly limited, but is preferably 150,000 to 800,000, more preferably 200,000 to 700,000, and even more preferably 250,000 to 600,000. A weight-average molecular weight of 150,000 or more provides sufficient mechanical properties, while a weight-average molecular weight of 800,000 or less provides a sufficient crystallization rate and achieves good moldability. The weight-average molecular weight of the P3HB resin can be determined as the molecular weight converted into polystyrene by gel permeation chromatography (GPC) (Shodex GPC-101 manufactured by Showa Denko) using a polystyrene gel (Shodex K-804 manufactured by Showa Denko) as a column and chloroform as the mobile phase.

[0030] (fungal body) The bacterial cells used in step (a) are not particularly limited as long as they are microorganisms capable of producing PHB copolymer intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for bacterial strains (e.g., IFO, ATCC, etc.), or mutants and transformants prepared from such microorganisms, can be used. For example, the first bacterial cell to produce P3HB, an example of a PHB copolymer, was Bacillus megaterium, discovered in 1925. Other examples include naturally occurring microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. It is known that PHB copolymer accumulates intracellularly in these microorganisms.

[0031] Examples of bacteria that produce copolymers of hydroxybutyrate and other hydroxyalkanoates, which are examples of PHB copolymers, include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with regard to P3HB3HH, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, pp. 4821-4830 (1997)), into which genes encoding PHB copolymer synthases have been introduced, are preferred for increasing P3HB3HH productivity. In addition to the above, the bacteria may also be genetically modified microorganisms into which various PHB copolymer synthesis-related genes have been introduced depending on the PHB copolymer to be produced.

[0032] The PHB copolymer can also be produced by the method described in, for example, WO 2010 / 013483.

[0033] (alkaline protease) As used herein, the term "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).

[0034] In one embodiment of the present invention, the alkaline protease is not particularly limited as long as it has the activity of degrading proteins in an alkaline environment, and examples thereof include serine-specific proteases (e.g., subtilisin and chymotrypsin) and cysteine-specific proteases (e.g., papain and bromelain). From the viewpoints of versatility and economy, serine-specific proteases, particularly alcalase, including subtilisin, are preferred. These may be used alone or in combination of two or more.

[0035] Commercially available alkaline protease enzymes can be used, such as "Alcalase" and "Esperase" manufactured by Novozyme; "Protin SD-AY10" and "Protease P "Amano" 3SD" manufactured by Amano Enzyme Inc.; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd.; "Sumiteam MP" manufactured by Shin-Nihon Chemical Industry Co., Ltd.; "Delvolase" 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 Inc.; and "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd.

[0036] In step (a), when the bacterial cells are treated with an alkaline protease, the pH and temperature of the culture medium are preferably adjusted to match the optimum pH and temperature of the alkaline protease used. Furthermore, the pH in step (a) is preferably lower than the pH adjusted by adding an aqueous alkaline solution in step (b). The method for adjusting the pH and temperature of the culture medium is not particularly limited, and known methods can be used.

[0037] In one embodiment of the present invention, 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 12.0, preferably 8.0 to 11.0, more preferably 8.0 to 10.0, even more preferably 8.0 to 9.0, and most preferably 8.5.

[0038] In one embodiment of the present invention, the optimum temperature of the alkaline protease is not particularly limited, but is preferably 70°C or lower, more preferably 60°C or lower, from the viewpoint of not requiring excessive heating and being able to prevent thermal changes (thermal decomposition) of the PHB copolymer. 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 and being economical.

[0039] In one embodiment of the present invention, the amount of alkaline protease added is not particularly limited, but is, for example, 0.05 to 1.0 phr, preferably 0.1 to 0.5 phr, and more preferably 0.15 to 0.3 phr. If the amount of alkaline protease added is within the above range, bacterial cells can be decomposed appropriately.

[0040] In step (a), it is preferable that substantially no lytic enzyme is added simultaneously with the alkaline protease. As used herein, the term "lytic enzyme" refers to an enzyme that has the activity of degrading (lysing) bacterial cell walls (e.g., peptidoglycan). Furthermore, as used herein, "substantially no lytic enzyme is added" refers to adding a lytic enzyme at 0.0005 phr or less, which may be 0 phr. The lytic enzyme is not particularly limited as long as it falls within the scope of the above definition, and examples thereof include lysozyme, rabia, β-N-acetylglucosaminidase, endolysin, and autolysin. By substantially not adding a lytic enzyme in step (a), peptidoglycan remains in the PHB copolymer aqueous suspension, thereby achieving the effects of the present invention.

[0041] In step (a), the bacterial cells containing the PHB copolymer are preferably inactivated. The inactivation method is not particularly limited, but an example thereof is a method in which a culture solution containing bacterial cells containing the PHB copolymer is heated and stirred at 60 to 70°C for 7 hours, as described in the Examples. After the heating and stirring treatment, the culture solution is preferably further cooled to a temperature suitable for step (a).

[0042] <Process (b)> Step (b) is a step of adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment.

[0043] Step (b) includes the following steps (b1) and (b2). Step (b1): adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0. Step (b2): Adding a surfactant (Process (b1)) As described above, step (b1) is a step of adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0. This step disperses and dissolves bacterial cell-derived impurities (nucleic acids, proteins, etc.), thereby enabling separation of a highly pure PHB copolymer from the bacterial cells.

[0044] In one embodiment of the present invention, the alkaline aqueous solution is an aqueous solution containing a basic compound. The basic compound contained in the alkaline aqueous solution is not particularly limited, but examples thereof include alkali metal or alkaline earth metal hydroxides 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.

[0045] In one embodiment of the present invention, the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, more preferably sodium hydroxide. The basic compounds may be used alone or in combination of two or more.

[0046] In step (b1), 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 enabling decomposition and dissolution of bacterial components. Furthermore, adjusting the pH to 12.0 or lower can prevent unintended damage to the bacterial cells.

[0047] The temperature in step (b1) is preferably less than 100° C., more preferably less than 80° C. There is no particular lower limit to the temperature, but it is preferably, for example, 40° C. or higher.

[0048] (Process (b2)) Step (b2) is a step of adding a surfactant to the culture solution obtained in step (a). This step can efficiently treat cell membranes in particular and remove a larger amount of impurities derived from the bacterial cells, thereby enabling the isolation of a more pure PHB copolymer from the bacterial cells.

[0049] In one embodiment of the present invention, the surfactant is not particularly limited, but examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Among these, anionic surfactants are preferred from the viewpoint of their high ability to remove cell membranes. These surfactants may be used alone or in combination of two or more.

[0050] Examples of 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, α-olefin sulfonates, α-sulfofatty acid salts, esters of α-sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acylamino acid surfactants. Among these, 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. These surfactants may be used alone or in combination of two or more.

[0051] In step (b2), the amount of surfactant to be 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.

[0052] Step (b2) may be carried out before, simultaneously with, or after step (b1), and preferably, step (b2) is carried out after step (b1).

[0053] <Process (b')> In one embodiment of the present invention, the production method may further comprise step (b').

[0054] Step (b') is a step of centrifuging the culture solution obtained in step (b) and removing the supernatant to obtain an aqueous suspension of concentrated PHB copolymer, i.e., a step of removing impurities from the PHB copolymer separated from the bacterial cells, followed by concentration and purification.

[0055] In step (b'), the method for centrifuging the culture medium is not particularly limited, and any known method can be used.

[0056] In step (b'), it is preferable to repeat the steps of centrifuging the culture solution, removing the supernatant, adding a solution to the precipitate, and then centrifuging again and removing the supernatant. This procedure allows for a more concentrated and purified PHB copolymer aqueous suspension to be obtained. Here, the solution added after removing the supernatant is preferably an alkaline aqueous solution adjusted to the same pH as the culture solution.

[0057] Since the amount of impurities remaining in the final product is largely determined by step (b'), 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. However, when a highly pure PHB copolymer is required, such as for medical applications, it is preferable to reduce the impurities as much as possible. An example of an index of the degree of purification is the amount of protein in the PHB copolymer aqueous suspension. The amount of protein in the PHB copolymer aqueous suspension is not particularly limited, as long as it achieves the residual protein amount of the PHB copolymer powder described below. The amount of protein is preferably 10,000 ppm or less, more preferably 5,000 ppm or less, and even more preferably 3,000 ppm or less, per weight of PHB copolymer in the PHB copolymer aqueous suspension.

[0058] In step (b'), the solvent ("solvent" is also referred to as "aqueous medium") constituting the PHB copolymer 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 in the mixed 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, isobutanol, 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. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easily removed. Furthermore, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily available. Furthermore, methanol, ethanol, and acetone are particularly preferred.

[0059] The water content in the aqueous medium constituting the PHB copolymer 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.

[0060] The aqueous suspension of PHB copolymer in step (b') may contain other solvents, components derived from bacterial cells, compounds generated during purification, etc., as long as the essence of the present invention is not impaired.

[0061] <Process (c)> In step (c) of this production method, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa·s or more and 0.5 Pa·s or less is prepared from the aqueous suspension obtained in step (b).

[0062] The PHB copolymer aqueous suspension before being subjected to step (c) of the present production method usually has a pH greater than 7.0 due to step (b). Therefore, the pH of the PHB copolymer aqueous suspension is adjusted to 7.0 or less in step (c) of the present production method. The adjustment method is not particularly limited, and examples thereof include a method of adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid, regardless of whether it is volatile. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0063] The upper limit of the pH of the PHB copolymer aqueous suspension adjusted in the above adjustment step is 7.0 or less, preferably 5.0 or less, and more preferably 4.0 or less, from the viewpoint of reducing discoloration when the PHB copolymer is heat-melted and ensuring molecular weight stability during heating and / or drying. The lower limit of the pH is preferably 1 or more, more preferably 2.0 or more, and even more preferably 3.0 or more, from the viewpoint of acid resistance of the container. By adjusting the pH of the PHB copolymer aqueous suspension to 7.0 or less, discoloration during heat-melting is reduced and a PHB copolymer with suppressed molecular weight reduction during heating and / or drying is obtained.

[0064] The concentration of the PHB copolymer in the aqueous suspension prepared in step (c) of this production method is preferably 30% by weight or more, more preferably 40% by weight or more, and even more preferably 50% by weight or more. A PHB copolymer concentration of 30% by weight or more is economically advantageous in terms of drying utility, improves productivity, and increases the bulk density of the powder obtained in the subsequent step (d), thereby improving transportability. The upper limit of the PHB copolymer concentration is preferably 65% ​​by weight or less, more preferably 60% by weight or less, because close packing may occur and sufficient fluidity may not be ensured. The method for adjusting the PHB copolymer concentration is not particularly limited, and examples include adding an aqueous medium and removing a portion of the aqueous medium (e.g., by centrifuging and then removing the supernatant). The PHB copolymer concentration may be adjusted in either step (c) or step (b).

[0065] In one embodiment of the present invention, the concentration of the PHB copolymer in the aqueous suspension prepared in step (c) is preferably 30 to 65% by weight.

[0066] In step (c), the shear viscosity of the PHB copolymer aqueous suspension is 0.005 to 0.5 Pa·s, preferably 0.007 to 0.4 Pa·s, and more preferably 0.009 to 0.3 Pa·s, from the viewpoint of ease of feeding into a spray dryer. The shear viscosity of the PHB copolymer aqueous suspension refers to the shear viscosity (Pa·s) at 20°C when a shear rate of 10 (1 / s) is applied. The shear viscosity of the PHB copolymer aqueous suspension is measured by the method described in the Examples.

[0067] <Dispersant> The aqueous suspension in one embodiment of the present invention may contain a dispersant. That is, the step (c) in one embodiment of the present invention may be a step of preparing an aqueous suspension further containing a dispersant in addition to the PHB copolymer. In one embodiment of the present invention, when the dispersant is used, it is preferable to add the dispersant to the PHB copolymer aqueous suspension before adjusting the pH to 7.0 or less. By including a dispersant in the PHB copolymer aqueous suspension in one embodiment of the present invention, the productivity and thermal stability of the PHB copolymer can be suitably improved.

[0068] The dispersant is not particularly limited, but examples thereof include alkylene oxide-based dispersants, cellulose-based dispersants, polyvinyl alcohol (PVA), and sorbitan alkylate-based dispersants. By using these dispersants, it is possible to obtain PHB copolymers with large particle sizes at lower hot air temperatures. The dispersant may be one type or two or more types.

[0069] In one embodiment of the present invention, the dispersant is at least one selected from the group consisting of alkylene oxide-based dispersants, cellulose-based dispersants, and polyvinyl alcohols.

[0070] In one embodiment of the present invention, the dispersant is preferably an alkylene oxide-based dispersant, from the viewpoint of suitably preventing aggregation of the PHB copolymer when adjusting the pH of the PHB copolymer aqueous suspension to 7.0 or less and suitably suppressing adhesion to the extruder shaft during powder processing. Furthermore, using a cellulose-based dispersant in combination with an alkylene oxide-based dispersant is more preferred, since it is expected to further suppress aggregation of the PHB copolymer and also function as a binder that makes the powder less brittle after drying.

[0071] In one embodiment of the present invention, the alkylene oxide dispersant is not particularly limited as long as it exhibits the above-mentioned effects, but it is preferably composed of a poly(ethylene oxide) (PEO) block and a poly(propylene oxide) (PPO) block, and is in the form of PEO-PPO-PEO.

[0072] As used herein, the term "poly(ethylene oxide) (PEO) block" refers to a polymer portion formed by polymerization of ethylene oxide (EO) in the structure of an alkylene oxide dispersant.

[0073] In this specification, the term "poly(propylene oxide) (PPO) block" refers to a polymer portion formed by polymerization of propylene oxide (PO) in the structure of an alkylene oxide-based dispersant.

[0074] In one embodiment of the present invention, by setting the PEO molecular weight and the PEO molecular weight / PPO molecular weight ratio in the alkylene oxide-based dispersant within specific ranges, the viscosity of the aqueous suspension can be kept low, and a PHB copolymer (e.g., a PHB copolymer powder) can be produced with high productivity.

[0075] In one embodiment of the present invention, the range of the PEO molecular weight and the PEO molecular weight / PPO molecular weight in the alkylene oxide dispersant is preferably the following combination.

[0076] In this specification, the "PEO molecular weight" may be referred to as the "EO amount," and the "PPO molecular weight" may be referred to as the "PO amount."

[0077] That is, in one embodiment of the present invention, the molecular weight of the PEO in the alkylene oxide dispersant may be 1500 or more, preferably 1750 or more, and more preferably 2000 or more. Also, in one embodiment of the present invention, the upper limit of the molecular weight of the PEO in the alkylene oxide dispersant is, for example, 30000 or less, preferably 25000 or less, and more preferably 20000 or less.

[0078] In one embodiment of the present invention, the PEO molecular weight / PPO molecular weight ratio in the alkylene oxide dispersant is preferably 0.5 or more, more preferably 0.6 or more, and even more preferably 0.7 or more. The upper limit of the PEO molecular weight / PPO molecular weight ratio is preferably 5.0 or less, more preferably 4.8 or less, and even more preferably 4.5 or less.

[0079] In one embodiment of the present invention, if the PEO molecular weight and the PEO molecular weight / PPO molecular weight in the alkylene oxide dispersant are within the above-mentioned ranges, the alkylene oxide dispersant will be hydrophilic and the number of molecules relative to the added weight of the alkylene oxide dispersant will be large, making it easier to maintain the dispersibility of the aqueous suspension.

[0080] In one embodiment of the present invention, the alkylene oxide dispersant has a PEO molecular weight of 1500 or more, and a PEO molecular weight / PPO molecular weight ratio of 0.5 to 5.0.

[0081] The alkylene oxide dispersant used in step (c) of this production method is not particularly limited, and for example, commercially available products can be used. Examples of commercially available products include Pluronic (registered trademark) 10400 (manufactured by BASF), Pluronic (registered trademark) 10500 (manufactured by BASF), Genapol (registered trademark) PF80 (manufactured by Clariant), Unilube (registered trademark) 70DP-600B (manufactured by NOF Corporation), Unilube (registered trademark) 70DP-950B (manufactured by NOF Corporation), Pronon (registered trademark) 208 (manufactured by NOF Corporation), Epan (registered trademark) U105 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Epan (registered trademark) U108 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Epan (registered trademark) 750 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and the like.

[0082] The amount of the dispersant added to the aqueous suspension in step (c) of the present production method is not particularly limited, but is preferably 0.1 to 20 parts by weight, more preferably 0.5 to 10 parts by weight, and even more preferably 0.75 to 5 parts by weight, relative to a total of 100 parts by weight of the PHB copolymer contained in the aqueous suspension. By setting the amount of the dispersant added within the above range, the dispersion stability of the PHB copolymer in the PHB copolymer aqueous suspension is further improved, allowing for efficient spray drying, and as a result, the productivity and thermal stability of the PHB copolymer tend to be more suitably improved.

[0083] In one embodiment of the present invention, the cellulose-based dispersant is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include methyl cellulose (MC), ethyl cellulose, propyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC), carboxyethyl cellulose, carboxypropyl cellulose, carboxymethyl hydroxyethyl cellulose, acetyl cellulose, cyanoethyl cellulose, sodium cellulose sulfate, etc. Among these, methyl cellulose and hydroxypropyl methyl cellulose are preferred from the viewpoint of the wide range of substitution degree that makes them water-soluble. The cellulose-based dispersant may be used alone or in combination of two or more.

[0084] The cellulose-based dispersant used in step (c) of this production method is not particularly limited, and for example, commercially available products can be used. Examples of commercially available cellulose-based dispersants include MCE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SFE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.), SFE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SE-50 (manufactured by Shin-Etsu Chemical Co., Ltd.), and NE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.).

[0085] The amount of cellulose-based dispersant added to the aqueous suspension of PHB copolymer in step (c) of the present production method is not particularly limited, but is preferably 0.01 to 10 parts by weight, more preferably 0.05 to 5 parts by weight, and even more preferably 0.08 to 3 parts by weight, per 100 parts by weight of the PHB copolymer contained in the aqueous suspension. By setting the amount of cellulose-based dispersant added within the above range, the effects of the present invention can be achieved.

[0086] In one embodiment of the present invention, the dispersant is preferably a biodegradable substance from the viewpoint of environmental issues.

[0087] The volume median diameter of the PHB copolymer in the aqueous suspension of PHB copolymer obtained by step (c) of this production method (hereinafter sometimes simply referred to as the "volume median diameter of the PHB copolymer") is preferably 30 times or less, more preferably 20 times or less, and even more preferably 10 times or less, the volume median diameter of the primary particles of the PHB copolymer (hereinafter referred to as the "primary particle diameter"). When the volume median diameter of the PHB copolymer is 30 times or less than the primary particle diameter, the aqueous suspension of the PHB copolymer exhibits superior fluidity, which allows the subsequent step (d) to be carried out with high efficiency and tends to further improve the productivity of the PHB copolymer. The volume median diameter of the PHB copolymer is measured, for example, using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer.

[0088] The volume median diameter of the PHB copolymer can be used as an indicator of the dispersion state of the PHB copolymer in the aqueous suspension of the PHB copolymer. The method for adjusting the volume median diameter of the PHB copolymer is not particularly limited, and known means (e.g., stirring) can be applied. For example, a PHB copolymer aqueous suspension whose dispersion state has been disrupted due to exposure to acidic conditions can be subjected to physical, chemical, or biological treatment that can be considered by those skilled in the art, thereby restoring the PHB copolymer in the aqueous suspension to a dispersed state (e.g., a state having the above-mentioned volume median diameter of the PHB copolymer).

[0089] <Process (d)> In step (d) of this production method, the PHB copolymer aqueous suspension prepared in step (c) is spray-dried. Examples of spray-drying methods include supplying the PHB copolymer aqueous suspension in the form of fine droplets into a dryer and drying it while contacting it with hot air in the dryer. The method (atomizer) for supplying the PHB copolymer aqueous suspension in the form of fine droplets into the dryer is not particularly limited, and examples include known methods using a rotating disk or a nozzle. The method for contacting the droplets with hot air in the dryer is not particularly limited, and examples include a co-current method, a counter-current method, and a combination of these.

[0090] The drying temperature during spray drying in step (d) may be any temperature capable of removing most of the aqueous medium from the droplets of the PHB copolymer aqueous suspension, and can be appropriately set under conditions that allow drying to the desired moisture content and minimize the occurrence of quality deterioration (reduction in molecular weight, color tone, etc.), melting, etc. For example, the temperature of the hot air blown into the spray dryer can be appropriately selected from the range of 100 to 300°C. The volume of the hot air blown into the dryer can also be appropriately set depending on, for example, the size of the dryer.

[0091] The present production method may further include a step of drying the obtained PHB copolymer (e.g., PHB copolymer powder) after step (d) (e.g., a step of subjecting the PHB copolymer to drying under reduced pressure). The present production method may also include other steps (e.g., a step of adding various additives to the PHB copolymer aqueous suspension).

[0092] The present production method may further include the following steps (e) and (f) after step (d). These steps can reduce the nitrogen content derived from bacterial cell residue contained in the PHB copolymer, thereby producing a highly pure PHB copolymer.

[0093] <Process (e)> Step (e) is a step of washing the PHB copolymer powder obtained in step (d) above. Step (e) preferably includes the following steps (e1) and (e2). Step (e1): A step of dispersing the PHB copolymer powder obtained in the step (e) in pure water to a predetermined concentration to prepare a dispersion slurry, adding an alkaline aqueous solution to adjust the pH of the dispersion slurry, and stirring the slurry together with a washing liquid. Step (e2): A step of adding an aqueous alkaline solution to the dispersed slurry obtained in the step (e1), centrifuging the mixture, and removing the supernatant.

[0094] (Process (e1)) As described above, step (e1) is a step of preparing a dispersion slurry by dispersing the PHB copolymer powder obtained in step (d) in pure water to a predetermined concentration, adding an alkaline aqueous solution to adjust the pH of the dispersion slurry, and stirring the slurry together with a washing liquid. During stirring, the liquid may be heated.

[0095] The predetermined concentration of the dispersed slurry in step (e1) is not particularly limited, but is preferably 20 to 40%, more preferably 30 to 40%, from the viewpoint of minimizing the size of the dispersion tank and ensuring that the slurry has fluidity.

[0096] In step (e1), the alkaline aqueous solution is not particularly limited, but the alkaline aqueous solution described in (step (b1)) can be used. The pH of the dispersion slurry is preferably adjusted to, for example, 5.0 to 14.0.

[0097] The stirring time in the washing in step (e1) is not particularly limited, but may be several hours. From the viewpoint of thoroughly washing the PHB copolymer, the stirring time may be 1 to 12 hours, or may be 2 to 10 hours.

[0098] The dispersed slurry in step (e1) may further contain sodium sulfate.

[0099] The washing solution in step (e1) may contain an alkaline protease and / or a lytic enzyme. The type of alkaline protease is not particularly limited, and those described in the section (Alkaline protease) of this specification may be used. The alkaline protease used in this step may be the same as or different from the alkaline protease used in step (a). The type of lytic enzyme is not particularly limited, and may be the lytic enzyme described in the explanation of step (a).

[0100] (Process (e2)) As described above, the step (e2) is a step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), separating the PHB copolymer powder, and removing the supernatant.

[0101] In step (e2), the aqueous alkaline solution is not particularly limited, and may be the same as or different from the aqueous alkaline solution used in step (e1).

[0102] In step (e2), the method for separating the PHB copolymer powder is not particularly limited, and any known method can be used, such as centrifugation, filter dehydration, or separation using a liquid cyclone.

[0103] In step (e2), it is preferable to repeat the steps of adding an alkaline aqueous solution to the dispersed slurry, centrifuging the slurry, and removing the supernatant, thereby obtaining a more concentrated and purified PHB copolymer.

[0104] <Process (f)> Step (f) is a step of dehydrating and / or drying the polyhydroxyalkanoic acid powder obtained in step (e). For dehydration and / or drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer, etc. are used, but a spray dryer is preferably used because of its simple operation. The spray drying method and drying temperature may be, for example, the method and temperature described in step (d).

[0105] [3.PHB copolymer powder] (Intermediate) A PHB copolymer powder according to one embodiment of the present invention (hereinafter referred to as "the first PHB copolymer powder") comprises a PHB copolymer, peptidoglycan, and a dispersant, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80 / 20 to 88 / 12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 μm. The first PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units and has a high bulk density, making it extremely useful in a variety of fields.

[0106] In this embodiment, the above definitions are used for "PHB copolymer," "peptidoglycan," and "dispersant."

[0107] The first PHB copolymer powder contains a PHB copolymer. The content of the PHB copolymer in the first PHB copolymer powder is not particularly limited, but is, for example, 90 to 99% by weight, preferably 93 to 98% by weight, and more preferably 95 to 97% by weight. By setting the content of the PHB copolymer within the above range, there is an advantage that the physical properties of PHBH, such as fluidity, are not lost.

[0108] The first PHB copolymer powder contains peptidoglycan. The content of peptidoglycan in the first PHB copolymer powder is not particularly limited, but is preferably 0.1 to 1.5 parts by weight (phr), more preferably 0.3 to 1.3 parts by weight (phr), per 100 parts by weight of the PHB copolymer constituting the PPHB copolymer powder. By setting the peptidoglycan content within the above range, the bulk density of the PHB copolymer powder tends to be improved. The content of peptidoglycan in the first PHB copolymer powder is measured by the method described in the Examples.

[0109] The first PHB copolymer powder contains a dispersant. The dispersant is preferably an alkylene oxide-based dispersant. The content of the dispersant in the PHB copolymer powder is not particularly limited, but is preferably 0.1 to 20 parts by weight, more preferably 0.5 to 10 parts by weight, and even more preferably 0.75 to 5 parts by weight, per 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. By setting the amount of dispersant added within the above range, productivity of the PHB copolymer powder tends to improve.

[0110] The bulk density of the first PHB copolymer powder is 0.45 g / mL or more, preferably 0.48 g / mL or more, and more preferably 0.50 g / mL or more, from the viewpoint of improving powder transportability. The upper limit is not particularly limited, but from the viewpoint of powder transportability, it can be, for example, 0.55 g / mL. The bulk density of the first PHB copolymer powder is measured by the method described in the Examples.

[0111] The median particle size of the first PHB copolymer powder is 80 to 200 μm, preferably 100 to 180 μm, and more preferably 105 to 160 μm, from the viewpoint of achieving excellent fluidity. The median particle size of the first PHB copolymer powder is measured by the method described in the Examples.

[0112] The purity of the first PHB copolymer powder is not particularly limited to an upper limit, but is preferably less than 98%. The lower limit is 85% or more, preferably 90% or more, and more preferably 92% or more, from the viewpoint of not impairing the physical properties of the PHB copolymer. The purity of the PHB copolymer powder refers to the content (wt%) of the PHB copolymer in the PHB copolymer powder. The purity of the first PHB copolymer powder is measured by the method described in the Examples.

[0113] In one embodiment of the present invention, the residual protein content of the first PHB copolymer powder is preferably 0.1 to 1.0 phr, more preferably 0.12 to 0.5 phr, and even more preferably 0.15 to 0.3 phr, from the viewpoint of coloration. The residual protein content refers to the amount of protein remaining in the PHB copolymer powder and is expressed as the amount per 100 parts by weight of the PHB copolymer in the PHB copolymer powder. The residual protein content of the first PHB copolymer powder is measured by the method described in the Examples.

[0114] Furthermore, the first PHB copolymer powder may contain various components that are generated or not removed during the production process, as long as the effects of the present invention are achieved.

[0115] In one embodiment of the present invention, the first PHB copolymer powder is an intermediate for the final product described below, and is produced by the above-described production method (for example, steps (a) to (d)).

[0116] (final product) A PHB copolymer powder according to one embodiment of the present invention (hereinafter referred to as "the second PHB copolymer powder") comprises a PHB copolymer and a nitrogen compound, wherein the PHB copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), a bulk density of 0.45 g / mL or more, and a median particle size of 80 to 200 μm. The second PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate units, and has a high bulk density and high purity, making it extremely useful in a variety of fields.

[0117] As used herein, the term "nitrogen compounds" refers to compounds containing at least nitrogen. Nitrogen compounds are decomposition products of proteins remaining in the PHB copolymer powder and peptidoglycan, a component of cell walls.

[0118] In this embodiment, the terms "PHB copolymer" and "nitrogen compound" are defined above. The terms "bulk density" and "median particle size" are defined above in the section (Intermediate).

[0119] The second PHB copolymer powder contains a nitrogen compound. The total nitrogen content in the second PHB copolymer powder is not particularly limited, but is preferably 0.010 to 0.075 parts by weight (phr), and more preferably 0.01 to 0.06 parts by weight (phr), per 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. By setting the total nitrogen content within the above range, the purity of the PHB copolymer powder tends to be improved. The total nitrogen content in the second PHB copolymer powder is measured by the method described in the Examples.

[0120] Furthermore, the second PHB copolymer powder may contain various components that are generated or not removed during the production process, as long as the effects of the present invention are achieved.

[0121] In one embodiment of the present invention, the second PHB copolymer powder is a final product and is produced by the above-described production method (for example, steps (a) to (f)).

[0122] The PHB copolymer powder can be used for a variety of purposes, including paper, films, sheets, tubes, plates, rods, containers (such as bottles), bags, and parts.

[0123] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0124] That is, one embodiment of the present invention is as follows. <1> 1. A method for producing a polyhydroxybutyric acid copolymer, comprising: The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), (a) adding an alkaline protease to a culture solution containing the bacterial cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less from the aqueous suspension obtained in step (b); and (d) spray drying the aqueous suspension prepared in step (c); A method for producing a polyhydroxybutyric acid copolymer, comprising: <2> After the step (d), (e) washing the polyhydroxybutyric acid copolymer powder obtained in the step (d); and (f) a step of dehydrating and / or drying the polyhydroxybutyric acid copolymer powder obtained in the step (e) further comprising: <1> A method for producing the polyhydroxybutyric acid copolymer described in <3> the hydroxyalkanoate units other than 3-hydroxybutyrate units are 3-hydroxyhexanoate units; <1> or <2> A method for producing the polyhydroxybutyric acid copolymer described in <4> The surfactant in step (b) is sodium dodecyl sulfate. <1> ~ <3> 1. A method for producing the polyhydroxybutyric acid copolymer according to any one of the preceding claims. <5> the aqueous suspension further comprising a dispersing agent; <1> ~ <4> 1. A method for producing the polyhydroxybutyric acid copolymer according to any one of the preceding claims. <6> The dispersant is at least one selected from the group consisting of alkylene oxide dispersants, cellulose dispersants, and polyvinyl alcohols. <5> A method for producing the polyhydroxybutyric acid copolymer described in <7> The concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight. <1> ~ <6> 1. A method for producing the polyhydroxybutyric acid copolymer according to any one of the preceding claims. <8> The washing solution in step (e) contains an alkaline protease and / or a lytic enzyme. <2> A method for producing the polyhydroxybutyric acid copolymer described in <9> a polyhydroxybutyrate copolymer, a peptidoglycan, and a dispersing agent; The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm. <10> The peptidoglycan content is 0.1 to 1.5 phr. <9> The polyhydroxybutyric acid copolymer powder according to claim 1. <11> Residual protein content is 0.1 to 1.0 phr. <8> or <9> The polyhydroxybutyric acid copolymer powder according to claim 1. <12> A polyhydroxybutyric acid copolymer and a nitrogen compound are included, The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm. <13> The total nitrogen content in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr. <12> The polyhydroxybutyric acid copolymer powder according to claim 1.

[0125] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0126] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the examples, "PHBH" is used as "PHB copolymer," and "PHB copolymer" can be read as "PHBH."

[0127] [Measurement and evaluation methods] Measurements and evaluations in the examples and comparative examples were carried out by the following methods.

[0128] (Volume Median Diameter) The volume median diameter in the PHB copolymer aqueous suspension was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA.

[0129] (Shear viscosity of PHB copolymer aqueous suspension) The shear viscosity of the PHB copolymer aqueous suspension was measured by the following method. Specifically, shear viscosity was measured in a coaxial double cylinder using an Anton Paar MCR302. The PHB copolymer aqueous suspension was poured into a 20 mL cylinder and cooled at a shear rate of 10 (1 / s) until the liquid temperature reached 15°C. The liquid temperature was then raised to 20°C, and after reaching the target liquid temperature, the viscosity was measured when the change in torque with time became less than 1%.

[0130] (peptidoglycan amount) The amount of peptidoglycan remaining in the PHB copolymer powder was measured using the following method. Specifically, 50 μL of peptidoglycan standard was placed in a microplate. Next, 50 μL of the reagent from the SLP-HS Single Reagent Set (Wako LAL System) was placed in the microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm. The absorbance 3 hours after the start of the measurement was defined as the final absorbance, and the time until the absorbance reached 0.4 times the final absorbance was measured. Similarly, the peptidoglycan standard was diluted 1000-10,000 times with distilled water, and a calibration curve was created using the same procedure as above. Next, the PHB copolymer aqueous suspension prepared in step (c) was diluted 1000-10,000 times with distilled water, and 50 μL of the diluted solution was placed in a microplate. Next, 50 μL of the reagent from the SLP-HS Single Reagent Set (Wako LAL System) was placed in the microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the absorbance 3 hours after the start of measurement was defined as the final absorbance. The amount of peptidoglycan was calculated from the time it took for the absorbance to reach 0.4 times the final absorbance.

[0131] (Residual protein amount) The residual protein content of the PHB copolymer powder was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was placed in a 14 mL Falcon tube, 2 mL of the above reagent was added, and the tube was shaken at 60°C for 30 minutes. After 30 minutes, the tube was cooled, and the absorbance at a wavelength of 562 nm was measured.

[0132] (bulk density) The bulk density of the PHB copolymer powder was measured using a bulk specific gravity measuring device (manufactured by Kuramochi Scientific Instruments) in accordance with JIS K 7365:1999.

[0133] (median particle size) The median particle size of the PHB copolymer powder was measured by the following method. Specifically, the median particle size was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 (HORIBA). 0.05 g of sodium dodecyl sulfate as a surfactant was added to 20 mL of ion-exchanged water to obtain a surfactant aqueous solution. Next, 0.2 g of the PHB copolymer powder to be measured was added to the surfactant aqueous solution, and the PHB copolymer powder was dispersed in the surfactant aqueous solution to obtain a dispersion for measurement. The prepared dispersion was introduced into the laser diffraction / scattering particle size distribution analyzer, and measurement was performed.

[0134] (purity) The purity of the PHB copolymer powder was measured using the following method. Specifically, approximately 10 mg of dried PHBH powder (weight: W) was heated at 10°C / min from 50°C to 500°C under a nitrogen atmosphere using a TG-DTA (2000SE, manufactured by Nietzsch). A graph of temperature vs. sample weight was created. The weights at point A, where a linear line representing the weight loss from 280-285°C intersects with a linear line representing the weight loss from 400-450°C, and point B, where a linear line representing the weight loss from 280-285°C intersects with a linear line representing the weight loss from 150-200°C, were calculated from the graph. The difference in weight between points A and B was used to calculate W', which was the amount of PHBH. W' / W was used as the purity of PHBH.

[0135] (Total nitrogen measurement) The total nitrogen content of the PHB copolymer powder was measured using a trace total nitrogen analyzer TN-2100H (Nitto Seiko Analytech Co., Ltd.).

[0136] Example 1 (Preparation of bacterial culture solution) Ralstonia eutropha, described in International Publication No. WO 2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same publication to obtain a fungal culture medium containing fungal cells containing PHB copolymer. Ralstonia eutropha is currently classified as Capriavidus necator. The repeating unit composition ratio of the PHB copolymer (3HB units / hydroxyalkanoate units other than 3HB units) was 80 / 20 to 88 / 12 (mol / mol).

[0137] (inactivation) The bacterial cell culture solution obtained above was heated and stirred at an internal temperature of 60 to 70°C for 7 hours, sterilized, and then cooled to 55°C to obtain an inactivated culture solution.

[0138] (enzyme treatment) The pH of the inactivated culture solution was adjusted to 8.5±0.2 using 30% sodium hydroxide, followed by the addition of 0.2 phr of Alcalase (Novozymes), a protease, and the mixture was kept at 50°C for at least 2 hours while controlling the pH at 8.5 using 30% sodium hydroxide.

[0139] (Bacterial lysis, concentration) The enzyme-treated solution obtained above was added with an alkaline aqueous solution to adjust the pH to 11.5 or higher and maintained at 45°C or higher for 4 hours or more. Subsequently, 0.6 to 1.0 wt% sodium dodecyl sulfate (Kao Corporation) was added, and 30% sodium hydroxide was added to adjust the pH to approximately 12. The solution was then diluted 2-fold with an alkaline aqueous solution (pH 10 to 11.5). After centrifugation, the supernatant was removed and the solution was concentrated 2-fold. To this concentrated aqueous suspension of PHB copolymer, an equal amount of aqueous sodium hydroxide solution (pH 11) was added, followed by centrifugation and removal of the supernatant. The process of adding aqueous sodium hydroxide, centrifugation, and supernatant removal was repeated 7 times. The aqueous PHB copolymer suspension was concentrated 4-fold to adjust the PHB copolymer concentration to 52 wt% or higher. The volume median diameter of this aqueous PHB copolymer suspension was measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer and found to be 1.9 μm.

[0140] (granulation) To the PHB copolymer aqueous suspension obtained above, 0.95 phr (0.95 parts by weight per 100 parts by weight of the PHB copolymer present in the aqueous suspension) of an ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added. Next, 0.3 phr of methylcellulose (trade name SM-15) was added. The resulting solution was stirred, the temperature was raised to 60°C, 10% sulfuric acid was added to adjust the pH to 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. The solids concentration of the mixture was then adjusted to 50% by mass by adding water. The volume median diameter of this PHB copolymer aqueous suspension was measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer and found to be 1.9 μm. Furthermore, the shear viscosity of the PHB copolymer aqueous suspension was measured using an Anton Paar MCR302 and found to be 0.1 Pa·s. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawahara Co., Ltd.) (hot air temperature: 115°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a pre-washed PHB copolymer powder. The peptidoglycan content of the resulting pre-washed PHB copolymer powder was 1.04 phr, the residual protein content was 0.18 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g / mL, the median particle size was 110 μm, the purity was 97%, and the total nitrogen content was 0.099 phr.

[0141] (Washing 1) The unwashed PHB copolymer powder obtained above was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 1 phr of sodium sulfate was added to the dispersion slurry. The pH of the dispersion slurry was then adjusted to 6 with 30% sodium hydroxide. The dispersion slurry was heated to 50°C, and 0.0050 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.) was added, followed by stirring for 2 hours.

[0142] (Wash 2) 30% sodium hydroxide was added to the dispersion slurry obtained above to adjust the pH of the dispersion slurry to 11. Then, the same weight of sodium hydroxide solution with a pH of 11 as the dispersion slurry was added to the dispersion slurry, the mixture was centrifuged, and the supernatant was removed. The procedure of adding the sodium hydroxide solution, centrifuging, and removing the supernatant was repeated three times.

[0143] (Dry) 10% sulfuric acid was added to the dispersion slurry obtained above to adjust the pH of the dispersion slurry to 4. Water was then added so that the PHB copolymer concentration in the dispersion slurry was 35%. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawahara Co., Ltd.) (hot air temperature: 160°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a PHB copolymer powder. The total nitrogen content of the resulting PHB copolymer powder was 0.059 phr, and the bulk density of the PHB copolymer powder before washing was 0.50 g / mL and the median particle size was 102 μm.

[0144] Example 2 Pre-washed PHB copolymer powder was obtained in the same manner as in Example 1 up to (granulation). The pre-washed PHB copolymer powder was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 1 phr of sodium sulfate was added to the resulting dispersion slurry. The pH of the dispersion slurry was then adjusted to 10.5 with 30% sodium hydroxide. 0.05 phr of Esperase (Novozyme) was added, and the mixture was stirred for 2 hours. The procedures from (Washing 2) onward described in Example 1 were then repeated, and the total nitrogen content of the resulting PHB copolymer powder was 0.070 phr, the bulk density of the PHB copolymer powder was 0.50 g / mL, and the median particle size was 108 μm.

[0145] Example 3 A dispersion slurry of unwashed PHB copolymer powder was obtained using the same method as in Example 1 up to (Washing 1). 30% sodium hydroxide was added to the obtained dispersion slurry to adjust the pH of the dispersion slurry to 10.5. 0.05 phr of Esperase (Novozyme) was added and the mixture was stirred for 2 hours. The procedures from (Washing 2) onward described in Example 1 were then carried out, and the total nitrogen content of the obtained PHB copolymer powder was 0.033 phr, the bulk density of the PHB copolymer powder was 0.50 g / mL, and the median particle size was 115 μm.

[0146] Example 4 Pre-washed PHB copolymer powder was obtained in the same manner as in Example 1 up to (granulation). The pre-washed PHB copolymer powder was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 30% sodium hydroxide was added to adjust the pH to 11, followed by stirring for 2 hours. The operations from (Washing 2) onward described in Example 1 were then carried out, and the total nitrogen content in the obtained PHB copolymer powder was 0.098 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g / mL, and the median particle size was 110 μm.

[0147] Comparative Example 1 An inactivated culture solution was obtained in the same manner as in Example 1 up to the step (inactivation).

[0148] (enzyme treatment) To the inactivated culture medium obtained above, 0.01 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.), a peptidoglycan-degrading enzyme, was added and the mixture was kept at 50°C for 2 hours. Next, the pH was adjusted to 8.5±0.2 using 30% sodium hydroxide. After that, the protease Alcalase (manufactured by Novozymes) was added, and the mixture was kept at 50°C for at least 2 hours while controlling the pH to 8.5 with 30% sodium hydroxide.

[0149] (Bacterial lysis, concentration) The enzyme-treated solution obtained above was added with an alkaline aqueous solution to adjust the pH to 11.5 or higher and maintained at 45°C or higher for 4 hours or more. Subsequently, 0.6 to 1.0 wt% sodium dodecyl sulfate (Kao Corporation) was added, and 30% sodium hydroxide was added to adjust the pH to approximately 12. The solution was then diluted 3-fold with an alkaline aqueous solution (pH 10 to 11.5). After centrifugation, the supernatant was removed and the solution was concentrated 2-fold. To this concentrated aqueous suspension of PHB copolymer, an equal amount of aqueous sodium hydroxide solution (pH 11) was added, followed by centrifugation and removal of the supernatant. The addition of aqueous sodium hydroxide solution, centrifugation, and supernatant removal were repeated. The volume median diameter of this aqueous PHB copolymer suspension was measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer and found to be 62 μm. Thereafter, the PHB copolymer aqueous suspension was concentrated to a solids concentration of 50% by filtration using a Buchner funnel, but it was in the form of a wet powder with no fluidity and was in a state where it could not be spray-dried.

[0150] Comparative Example 2 An aqueous suspension of PHB copolymer was obtained in the same manner as in Comparative Example 1 up to the centrifugation step (lysing and concentration). The volume median diameter of this aqueous suspension of PHB copolymer was measured using a laser diffraction / scattering particle size distribution analyzer LA-950 manufactured by HORIBA and was found to be 62 μm.

[0151] (granulation) To the PHB copolymer aqueous suspension (solids concentration 20%) obtained above, 0.95 phr of an ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added. Next, 0.3 phr of methylcellulose (trade name SM-15) was added. The resulting solution was stirred, the liquid temperature was raised to 60°C, 10% sulfuric acid was added to adjust the pH to 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. The solids concentration of the mixture was then adjusted to 15% by mass by adding water. The volume median diameter of this PHB copolymer aqueous suspension was measured using a HORIBA LA-950 laser diffraction / scattering particle size distribution analyzer and found to be 62 μm. Furthermore, the shear viscosity of the PHB copolymer aqueous suspension was measured using an Anton Paar MCR302 and found to be 0.1 Pa·s. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawahara Co., Ltd.) (hot air temperature: 115°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a PHB copolymer powder. The resulting PHB copolymer powder contained 0.01 phr of peptidoglycan, 0.5 phr of residual protein, a bulk density of 0.20 g / mL, a median particle size of 71 μm, and a purity of 98%.

[0152] [Table 1] 〔result〕 From Table 1, it was found that the present production method can produce a PHB copolymer having a high bulk density and a high ratio of hydroxyalkanoate units other than 3HB units. [Industrial Applicability]

[0153] According to the present invention, it is possible to produce a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units. Furthermore, since the PHB copolymer obtained by the production method of the present invention has a high bulk density, it can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. 1. A method for producing a polyhydroxybutyric acid copolymer powder, comprising: the polyhydroxybutyrate copolymer contained in the polyhydroxybutyrate copolymer powder has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol); (a) adding an alkaline protease to a culture solution containing the bacterial cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less from the aqueous suspension obtained in the step (b); and (d) spray drying the aqueous suspension prepared in step (c); Including, After the step (d), (e) a step of washing the polyhydroxybutyric acid copolymer powder obtained in the step (d); further comprising A method for producing a polyhydroxybutyric acid copolymer powder, wherein the washing liquid in the step (e) contains an alkaline protease and / or a bacteriolytic enzyme.

2. After the step (e), (f) a step of dehydrating and / or drying the polyhydroxybutyric acid copolymer powder obtained in the step (e). The method for producing polyhydroxybutyric acid copolymer powder according to claim 1, further comprising:

3. 3. The method for producing a polyhydroxybutyrate copolymer powder according to claim 1, wherein the hydroxyalkanoate units other than 3-hydroxybutyrate units are 3-hydroxyhexanoate units.

4. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 3, wherein the surfactant in step (b) is sodium dodecyl sulfate.

5. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 4, wherein the aqueous suspension further contains a dispersant.

6. 6. The method for producing polyhydroxybutyric acid copolymer powder according to claim 5, wherein the dispersant is at least one selected from the group consisting of alkylene oxide-based dispersants, cellulose-based dispersants, and polyvinyl alcohol.

7. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 6, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in step (c) is 30 to 65% by weight.

8. A polyhydroxybutyric acid copolymer and a nitrogen compound are included, The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80 / 20 to 88 / 12 (mol / mol), A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g / mL or more and a median particle size of 80 to 200 μm, The polyhydroxybutyric acid copolymer powder has a total nitrogen content of 0.010 to 0.075 phr.

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