Method for producing polyhydroxyalkanoic acid

Abstract

To provide a method for producing PHA which satisfies both suppression of coagulation in an aqueous suspension of PHA and separability of PHA from the aqueous suspension.SOLUTION: A method for producing PHA includes the steps of: adding inorganic peroxide to a culture solution containing PHA; adding organic peroxide; adding alkaline protein degrading enzyme; adjusting pH to a fixed range to obtain an aqueous suspension of PHA; and centrifugally separating the aqueous suspension.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid. [Background technology]

[0002] Polyhydroxyalkanoic acid (hereinafter sometimes referred to as "PHA") is known to be biodegradable, and in recent years, from the viewpoint of environmental considerations, it has been utilized as a biodegradable plastic.

[0003] PHA produced by microorganisms accumulates within the microbial cells, and therefore, in order to use PHA as a plastic, a process for separating and purifying PHA from the microbial cells is required. In the process for separating and purifying PHA, biologically derived components other than PHA are solubilized, and then PHA is recovered from the resulting aqueous suspension by separation procedures such as centrifugation, filtration, and drying. For example, Patent Documents 1 and 2 disclose techniques for recovering PHA from microbial cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2023 / 021878 [Patent Document 2] International Publication No. 2023 / 140097 Summary of the Invention [Problem to be solved by the invention]

[0005] However, although the above-mentioned conventional techniques are excellent, there is room for improvement in terms of achieving both the suppression of aggregation in the aqueous suspension of PHA and the separability of PHA from the aqueous suspension.

[0006] In view of the above situation, one aspect of the present invention aims to provide a method for producing PHA that can simultaneously suppress aggregation of PHA in an aqueous suspension and ensure the separability of PHA from the aqueous suspension. [Means for solving the problem]

[0007] As a result of extensive research to solve the above problems, the present inventors have discovered for the first time that it is possible to suppress aggregation of PHA in an aqueous suspension while also ensuring separability of PHA from the aqueous suspension by treating a culture solution containing PHA-containing bacterial cells with an inorganic peroxide, an organic peroxide, and an alkaline protease in this order during the PHA production process, and have thus completed the present invention.

[0008] That is, one aspect of the present invention includes the following configuration. [1] A method for producing polyhydroxyalkanoic acid, comprising: (a) adding an inorganic peroxide to a culture solution containing a bacterial cell containing polyhydroxyalkanoic acid; (b) adding an organic peroxide to the culture solution obtained in the step (a) and maintaining the temperature at a temperature of 0 to 100°C that is equal to or lower than the one-minute half-life temperature of the organic peroxide; (c) adding an alkaline protease to the culture solution obtained in the step (b) to perform an enzymatic treatment; (d) adjusting the pH of the culture solution obtained in the step (c) to 10.0 to 12.0; and (e) centrifuging the aqueous suspension of polyhydroxyalkanoic acid obtained in the step (d). [2] The method for producing polyhydroxyalkanoic acid according to [1], wherein the amount of the organic peroxide added in the step (b) is 0.1 to 5 parts by weight per 100 parts by weight of the polyhydroxyalkanoic acid contained in the culture solution obtained in the step (a). [3] The method for producing a polyhydroxyalkanoic acid according to [1] or [2], wherein the organic peroxide is a peroxydicarbonate or peroxyester having a one-minute half-life temperature of 140°C or less. [4] The method for producing a polyhydroxyalkanoic acid according to any one of [1] to [3], wherein the step (a) comprises maintaining a culture solution containing the bacterial cells containing the polyhydroxyalkanoic acid to which the inorganic peroxide has been added at 30 to 75°C. [5] The method for producing a polyhydroxyalkanoic acid according to any one of [1] to [4], wherein in the step (d), a surfactant is further added to the culture solution obtained in the step (c). [6] The method for producing a polyhydroxyalkanoic acid according to any one of [1] to [5], wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyrate repeating units and other hydroxyalkanoate units, and the composition ratio of 3-hydroxybutyrate units / other alkanoate units in the copolymer is 70 / 30 to 88 / 12 (mol / mol). [7] The method for producing a polyhydroxyalkanoic acid according to any one of [1] to [6], wherein the inorganic oxide is hydrogen peroxide or ozone. [Effects of the Invention]

[0009] According to one aspect of the present invention, it is possible to provide a method for producing PHA that can simultaneously suppress aggregation of PHA in an aqueous suspension and ensure separability of PHA from the aqueous suspension. DETAILED DESCRIPTION OF THE INVENTION

[0010] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, the term "A to B" representing a numerical range means "greater than or equal to A (including and greater than A) and less than or equal to B (including and less than B)."

[0011] 1. Overview of the Invention Since PHA accumulates within the cells of microorganisms (PHA-producing bacteria), the process of separating and purifying PHA from the microbial cells is necessary to use PHA as plastic. To separate and purify PHA, the microbial cells are first disrupted, and an aqueous suspension of PHA is prepared in which the PHA within the cells is dispersed in an aqueous solvent. The aqueous suspension is then centrifuged to separate components derived from the microorganisms other than PHA (proteins, etc.), and the PHA is then purified.

[0012] In the separation and purification of PHA, the handling of the aqueous PHA suspension is important because the suspension is transferred by pump and concentrated by centrifugation. However, with conventional aqueous PHA suspensions, the viscosity of the suspension increases excessively, which makes it difficult to handle.

[0013] As a means for solving this problem, Patent Document 1 discloses that an increase in viscosity of a culture solution containing PHA-producing bacteria can be suppressed by adding an inorganic peroxide to the culture solution containing PHA-producing bacteria. However, although this technology can suppress an increase in viscosity of the resulting aqueous suspension of PHA, there is room for improvement in terms of the aggregation properties of PHA, such as the aggregation of PHA in the aqueous suspension, the PHA settling, or adhesion to scale. The problem of aggregation properties becomes particularly pronounced when shear forces are applied to the aqueous suspension of PHA, such as when the aqueous suspension of PHA is sent to a dryer.

[0014] Furthermore, in order to solve the problem of increased viscosity of the suspension, the present inventors have also investigated a method of adding an organic peroxide to a culture solution containing a PHA-producing bacterium. However, the aqueous suspension of PHA obtained by this method has the problem that the PHA is excessively dispersed in the aqueous suspension, making it difficult to sufficiently separate the PHA from the aqueous suspension, resulting in a poor PHA yield. Therefore, this method leaves room for improvement in terms of PHA separability.

[0015] To improve the handleability of aqueous PHA suspensions and stably produce PHA at high yields, it is necessary to both inhibit aggregation of PHA in the aqueous suspension and facilitate separability of PHA from the aqueous suspension. However, as described above, conventional techniques have had problems with either aggregation or separability of PHA, and no method for simultaneously inhibiting aggregation of PHA in aqueous suspension and enabling separability of PHA from the aqueous suspension has been known. Furthermore, this problem is particularly pronounced when the PHA is a copolymer having a composition ratio of 3-hydroxybutyrate units / other alkanoate units of 70 / 30 to 88 / 12 (mol / mol).

[0016] In light of these circumstances, the present inventors conducted extensive research to provide a method for producing PHA that can both inhibit aggregation in an aqueous suspension of PHA and allow the PHA to be easily separated from the aqueous suspension. As a result, they discovered for the first time that an aqueous PHA suspension obtained by subjecting a culture medium containing PHA-containing bacterial cells to inorganic peroxide treatment, organic peroxide treatment, and alkaline protease treatment in this order during the PHA production process can both inhibit aggregation of PHA in the aqueous suspension and allow the PHA to be easily separated from the aqueous suspension, thereby completing the present invention. Furthermore, the present inventors also discovered that an aqueous PHA suspension obtained by the above method can both inhibit aggregation of PHA in the aqueous suspension and allow the PHA to be easily separated from the aqueous suspension, even when the PHA in the aqueous suspension is a copolymer having a composition ratio of 3-hydroxybutyrate units / other alkanoate units of 70 / 30 to 88 / 12 (mol / mol), which has significant problems with aggregation or separability.

[0017] This production method can be produced using renewable biomass resources as raw materials and is rapidly decomposed by microorganisms in the natural environment, enabling stable and high-yield production of PHA, an environmentally friendly biodegradable plastic that can address issues such as plastic waste. Therefore, this production method contributes to the widespread use of PHA, a biodegradable plastic, and through the widespread use of PHA, can contribute to solving the problems of soil and / or marine pollution caused by waste plastic. Therefore, this production method is expected to contribute to the achievement of Sustainable Development Goals (SDGs), such as Goal 12, "Ensure sustainable consumption and production patterns," and / or Goal 14, "Conserve and sustainably use the oceans, seas, and marine resources for sustainable development."

[0018] 2. Method for producing polyhydroxyalkanoic acid A method for producing PHA according to one embodiment of the present invention (hereinafter, sometimes referred to as "this production method") includes: (a) a step of adding an inorganic peroxide to a culture solution containing PHA-containing bacterial cells; (b) a step of adding an organic peroxide to the culture solution obtained in the step (a) and maintaining the temperature at a temperature equal to or lower than the one-minute half-life temperature of the organic peroxide and at a temperature of 0 to 100°C; (c) a step of adding an alkaline protease to the culture solution obtained in the step (b) to perform an enzymatic treatment; (d) a step of adjusting the pH of the PHA culture solution obtained in the step (c) to 10.0 to 12.0; and (e) a step of centrifuging the aqueous suspension of PHA obtained in the step (d). The method for producing PHA includes the steps of:

[0019] This production method can suppress aggregation of PHA in an aqueous suspension while also allowing for the separation of PHA from the aqueous suspension, making it possible to stably produce PHA in high yields.

[0020] Each step that may be included in this production method will be described in detail below.

[0021] <Process (a)> This production method includes, as step (a), a step of adding an inorganic peroxide to a culture solution containing PHA-containing bacterial cells. In this section, the mere mention of "culture solution" means "a culture solution containing PHA-containing bacterial cells," and particularly "a culture solution containing PHA-containing bacterial cells to be subjected to step (a)."

[0022] (PHA) As used herein, "PHA" is a generic term for polymers containing hydroxyalkanoic acid as a monomer unit (monomer repeating unit), and is generally biodegradable. PHA is an aliphatic polyester, preferably a polyester not containing an aromatic ring. As used herein, "PHA" refers to a polymer containing hydroxyalkanoic acid repeating units in an amount of 50 mol% or more of all monomer repeating units (100 mol%). PHA preferably contains hydroxyalkanoate repeating units in an amount of 60 mol% or more, more preferably 70 mol% or more, of all monomer repeating units (100 mol%).

[0023] The PHA produced by this production method is the PHA contained in the bacterial cells in step (a). Examples of PHA produced by this production method include poly(3-hydroxyalkanoate) resins (hereinafter sometimes referred to as "P3HA") and poly(4-hydroxyalkanoate) resins. P3HA is preferred as the PHA produced by this production method because it is highly biodegradable and effective in reducing environmental impact.

[0024] P3HA is a 3-hydroxyalkanoate repeating unit of the formula: [—CHR—CH—CO—O—] (wherein R is C n H 2n+1 where n is an integer of 1 to 15.) as an essential repeating unit. In this specification, "P3HA" refers to a resin containing 50 mol % or more of the 3-hydroxyalkanoate repeating units out of all monomer repeating units (100 mol %).

[0025] The P3HA produced by this production method preferably contains 3-hydroxyalkanoate repeating units in an amount of 60 mol % or more, and more preferably 70 mol % or more, of all monomer repeating units (100 mol %).

[0026] The P3HA produced by this production method is not particularly limited and may be a homopolymer containing the repeating unit described above, or a copolymer containing the repeating unit described above. Examples of the copolymer include copolymers of 3-hydroxybutanoic acid (hereinafter sometimes referred to as "3HB") and one or more monomers selected from the group consisting of 3-hydroxypropionic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytridecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, and 3-hydroxyoctadecanoic acid. Another example of the copolymer is a copolymer of 3HB and one or more monomers selected from the group consisting of 4-hydroxybutanoic acid, 4-hydroxypentanoic acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxynonanoic acid, 4-hydroxydecanoic acid, 4-hydroxyundecanoic acid, 4-hydroxydodecanoic acid, 4-hydroxytridecanoic acid, 4-hydroxytetradecanoic acid, 4-hydroxyhexadecanoic acid, and 4-hydroxyoctadecanoic acid.

[0027] Examples of P3HA produced by this production method include poly(3-hydroxybutyrate) (hereinafter sometimes referred to as "P3HB"), which is a homopolymer of 3HB, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate), and combinations thereof. As used herein, "poly(X-co-Y)" refers to a copolymer containing X repeating units and Y repeating units, and is intended to mean a copolymer obtained by copolymerizing a monomer from which the X repeating unit is derived and a monomer from which the Y repeating unit is derived. Furthermore, during the production of P3HA by microorganisms, a small amount (less than 1 mol%) of a monomer may be copolymerized. However, if this does not significantly affect the physical properties of the resulting P3HA, the monomer is considered to be uncopolymerized, and the product will be referred to by a name that does not include that monomer.

[0028] The P3HA produced by this production method preferably contains 3-hydroxybutyrate (3HB) repeating units, and more preferably contains 3HB repeating units and 3-hydroxyhexanoate (3HH) repeating units. When the P3HA produced by this production method contains 3HB repeating units, i.e., when the P3HA produced by this production method is a copolymer containing 3HB repeating units and other (non-3HB) hydroxyalkanoate repeating units (preferably 3HH repeating units), from the viewpoint of the balance between flexibility and strength of the resulting resin, the composition ratio of 3-hydroxybutyrate units / other alkanoate units in the copolymer is preferably 70 / 30 to 99 / 1 (mol / mol), more preferably 70 / 30 to 90 / 10 (mol / mol), even more preferably 70 / 30 to 88 / 12, and even more preferably 80 / 20 to 88 / 12. When P3HA is a copolymer containing 3HB repeating units and other hydroxyalkanoate repeating units, the handleability of an aqueous suspension containing the P3HA tends to deteriorate as the proportion of the other hydroxyalkanoate repeating units (particularly 3HH repeating units) increases, particularly when the composition ratio exceeds 12 mol / l (i.e., when the composition ratio of 3-hydroxybutyrate units / other alkanoate units in the copolymer is 70 / 30 to 88 / 12 (mol / mol)). Therefore, with conventional methods, this handleability issue has made it difficult to efficiently produce P3HA with a high proportion of other hydroxyalkanoate repeating units. However, with the present production method, even for P3HA with a high proportion of other hydroxyalkanoate repeating units, it is possible to both suppress aggregation of the P3HA in the aqueous suspension and facilitate separability of the P3HA from the aqueous suspension, thereby resolving the handleability issue. Therefore, this production method is particularly suitable for producing P3HA having a large proportion of other hydroxyalkanoate repeating units as described above. The monomer composition ratio of P3HA can be measured by gas chromatography or the like (see, for example, WO 2014 / 020838).

[0029] The weight-average molecular weight (hereinafter sometimes referred to as "Mw") of the PHA produced by this production method is not particularly limited, but is preferably 150,000 to 1,300,000, more preferably 200,000 to 800,000, and even more preferably 250,000 to 600,000. A PHA weight-average molecular weight of 150,000 or more provides sufficient mechanical properties, while a PHA weight-average molecular weight of 1,300,000 or less provides a sufficient crystallization rate and achieves good moldability. In this specification, the weight-average molecular weight of a PHA refers to the value 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 the column and chloroform as the mobile phase.

[0030] (fungal body) The term "bacterial cells" in step (a) refers to "bacterial cells of a PHA-producing microorganism." A PHA-producing microorganism refers to a microorganism that can produce PHA from an appropriate carbon source and accumulate it within its cells.

[0031] The PHA-producing microorganism in this production method is not particularly limited as long as it is a microorganism capable of producing the above-mentioned PHA, preferably P3HA. Examples of PHA-producing microorganisms include bacteria of the genera Cupriavidus, Alcaligenes, Ralstonia, Pseudomonas, Bacillus, Azotobacter, Nocardia, and Aeromonas. Among these, microorganisms belonging to the genera Aeromonas, Alcaligenes, Ralstonia, and Cupriavidus are preferred. In particular, strains such as Alcaligenes lipolytica, Alcaligenes latus, Aeromonas caviae, Aeromonas hydrophila, and C. necator are more preferred, with C. necator being the most preferred. In addition to the above, genetically modified microorganisms into which various PHA synthesis-related genes have been introduced may also be used depending on the desired PHA.

[0032] The bacterial cells used in this production method can be those of a PHA-producing microorganism cultured according to a known method. Examples of known methods for culturing PHA-producing microorganisms include the method described in International Publication No. WO2019 / 142717.

[0033] The bacterial cells used in step (a) are preferably inactivated. The inactivation method is not particularly limited when inactivated bacterial cells are used, but examples thereof include a method in which a culture solution containing a PHA-producing microorganism is heated and stirred at 40 to 80°C, preferably 60 to 70°C, for 112 hours, preferably 5 to 10 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). The inactivation treatment of bacterial cells can also be considered a sterilization treatment.

[0034] (Inorganic peroxide) The inorganic peroxide used in step (a) is not particularly limited, but examples thereof include hydrogen peroxide (H2O2), ozone; sodium peroxide (Na2O2), sodium perborate (Na2H4B2O8), sodium percarbonate (Na2H3CO6), sodium persulfate (Na2S2O8), and the like.

[0035] Among the above, hydrogen peroxide or ozone is preferred as the inorganic peroxide from the viewpoint of easy availability.

[0036] The amount of inorganic peroxide added in step (a) is not particularly limited, but is preferably 0.1 to 10 parts by weight, more preferably 0.1 to 5 parts by weight, and even more preferably 0.5 to 3 parts by weight, relative to 100 parts by weight of polyhydroxyalkanoic acid contained in the culture medium. By adding an amount of inorganic peroxide of 0.1 part by weight or more, it is possible to sufficiently suppress an increase in viscosity of the culture medium and the resulting aqueous suspension of PHA. Furthermore, by adding an amount of inorganic peroxide of 10 parts by weight or less, it is possible to suppress excessive aggregation in the aqueous suspension of PHA and to suppress excessive use of inorganic peroxide, which is economically advantageous.

[0037] When hydrogen peroxide is added as the inorganic peroxide in step (a), sodium hydroxide and / or sodium bicarbonate may be added to the culture solution together with the hydrogen peroxide. The addition of sodium hydroxide and / or sodium bicarbonate is expected to enhance the activity of hydrogen peroxide.

[0038] In addition, when hydrogen peroxide is added as the inorganic peroxide in step (a), a chelating agent may be added to the culture solution together with the hydrogen peroxide. The addition of a chelating agent can stabilize the hydrogen peroxide solution. Chelating agents that can be used in step (a) include, but are not limited to, sodium silicate, EDTA, trans-1,2-cyclohexanediaminetetraacetic acid monohydrate, etc.

[0039] In step (a), the efficiency of viscosity suppression by the inorganic peroxide can be improved, and further, when hydrogen peroxide or ozone is used as the inorganic peroxide, there is an advantage in that volatilization of these can be suppressed, so that the temperature of the culture solution to which the inorganic peroxide has been added is preferably adjusted to 30 to 75° C., more preferably 50 to 75° C. In other words, in step (a), it is preferable to add the inorganic peroxide to the culture solution adjusted to the above temperature.

[0040] In step (a), from the viewpoint of controlling the viscosity of the resulting aqueous suspension of PHA and the weight-average molecular weight of the PHA within a suitable range, it is preferable to add inorganic peroxide when the pH of the culture medium is 10.5 to 13.0. The pH of the culture medium is more preferably 10.6 to 12.9, and even more preferably 10.7 to 12.8. The pH of the culture medium can be adjusted, for example, by adding an alkaline aqueous solution.

[0041] In this specification, the term "alkaline aqueous solution" refers to an aqueous solution containing a basic compound. The basic compound contained in the alkaline aqueous solution is not particularly limited, but examples include alkali metal or alkaline earth metal hydroxides such as sodium hydroxide and potassium hydroxide; metal carbonates such as sodium carbonate and potassium carbonate; and metal phosphates or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, and potassium hydrogen phosphate. Among these, the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or alkaline earth metal hydroxide, and more preferably sodium hydroxide. The alkaline aqueous solution may contain one or more of these basic compounds.

[0042] In step (a), after adding inorganic peroxide to the culture medium, it is preferable to maintain the culture medium at a predetermined temperature and pH for a certain period of time. The time for maintaining the temperature and / or pH of the culture medium to which inorganic peroxide has been added at a predetermined value in step (a) is not particularly limited, but from the viewpoint of efficiently suppressing viscosity by inorganic peroxide, it is preferably 0.1 to 30 hours, more preferably 0.25 to 24 hours, and even more preferably 0.5 to 15 hours. Furthermore, in step (a), the pH of the culture medium may gradually decrease as the inorganic peroxide reacts. Therefore, in step (a), it is preferable to maintain the pH of the culture medium containing the PHA-producing microorganism within the above-mentioned range by, for example, adding an alkaline aqueous solution to the culture medium.

[0043] The "predetermined temperature and pH" of the culture medium to which inorganic peroxide has been added, which is maintained in step (a), can be set to the values described above as the preferred temperature and pH. In other words, the present production method preferably includes, in step (a), a step of maintaining the pH of the culture medium to which inorganic peroxide has been added at 10.5 to 13.0 and / or a step of maintaining the temperature of the culture medium to which inorganic peroxide has been added at 30 to 75°C.

[0044] <Process (b)> This production method includes step (b), which is a step of adding an organic peroxide to the culture solution obtained in step (a) and maintaining the temperature at a temperature equal to or lower than the one-minute half-life temperature of the organic peroxide and at 0 to 100° C. In this section, the mere mention of a "culture solution" means a "culture solution containing PHA-containing bacterial cells," and particularly, a "culture solution obtained in the above step (a) that is to be subjected to step (b) (i.e., a culture solution containing PHA-containing bacterial cells that has been treated with an inorganic peroxide)."

[0045] (organic peroxide) The organic peroxide used in step (b) is not particularly limited, but examples thereof include diacyl peroxides, peroxy esters, dialkyl peroxides, hydroperoxides, peroxy ketals, peroxy monocarbonates, and peroxy dicarbonates.

[0046] More specifically, the organic peroxides used in step (b) include, as diacyl peroxides, dioctanoyl peroxide, dilauroyl peroxide, disuccinic peroxide, a mixture of toluoyl peroxide and benzoyl peroxide, dibenzoyl peroxide, and di(4-methylbenzoyl) peroxide; and as peroxyesters, t-butyl peroxyacetate, t-butyl peroxyisobutyrate, t-butyl peroxypivalate, and t-hexyl peroxide. peroxypivalate, t-butylperoxy 2-ethylhexanoate, t-hexylperoxy-2-ethylhexanoate, 1,1,3,3-tetramethylbutylperoxy-2-ethylhexanoate, t-butylperoxy-3,5,5-trimethylhexanoate, t-amylperoxy, 3,5,5-trimethylhexanoate, t-butylperoxyneodecanoate, t-hexylperoxyneodecanoate, cumylperoxyneodecanoate, t-butylperoxylaurate, t-butylperoxy Oxybenzoates, 2,5-dimethyl-2,5-bis(2-ethylhexanoylperoxy)hexane, 2,5-dimethyl-2,5-di(benzoylperoxy)hexane, dialkyl peroxides such as diisobutyl peroxide, di-t-hexyl peroxide, and dicumyl peroxide, and peroxyketals such as 2,2-di-t-butylperoxybutane, 1,1-bis(t-butylperoxy)cyclohexane, 1,1-bis(t-butylperoxy)-2-methylcyclohexane, and 1, 1-bis(t-butylperoxy)-3,3,5-trimethylcyclohexane, 2,2-bis(4,4-di-t-butylperoxycyclohexyl)propane, butyl bis(t-butylperoxy)valerate, peroxymonocarbonates such as t-butylperoxymethyl monocarbonate, t-pentylperoxymethyl monocarbonate, t-hexylperoxymethyl monocarbonate, t-heptylperoxymethyl monocarbonate, t-octylperoxymethyl monocarbonate, 1,1,3,3-Tetramethylbutylperoxymethyl monocarbonate, t-butylperoxyethyl monocarbonate, t-pentylperoxyethyl monocarbonate, t-hexylperoxyethyl monocarbonate, t-heptylperoxyethyl monocarbonate, t-octylperoxyethyl monocarbonate, 1,1,3,3-tetramethylbutylperoxyethyl monocarbonate, t-butylperoxy-n-propyl monocarbonate, t-pentylperoxy-n-propyl monocarbonate, t-hexylperoxy-n-propyl Pyroxy n-propyl monocarbonate, t-heptylperoxy n-propyl monocarbonate, t-octylperoxy n-propyl monocarbonate, 1,1,3,3-tetramethylbutylperoxy n-propyl monocarbonate, t-butylperoxy isopropyl monocarbonate, t-pentylperoxy isopropyl monocarbonate, t-hexylperoxy isopropyl monocarbonate, t-heptylperoxy isopropyl monocarbonate, t-octylperoxy isopropyl monocarbonate, 1,1,3,3-tetramethylbutylperoxy n-propyl monocarbonate butylperoxy isopropyl monocarbonate, t-butylperoxy n-butyl monocarbonate, t-pentylperoxy n-butyl monocarbonate, t-hexylperoxy n-butyl monocarbonate, t-heptylperoxy n-butyl monocarbonate, t-octylperoxy n-butyl monocarbonate, 1,1,3,3-tetramethylbutylperoxy n-butyl monocarbonate, t-butylperoxy isobutyl monocarbonate, t-pentylperoxy isobutyl monocarbonate, t-hexylperoxy peroxyisobutyl monocarbonate, t-heptylperoxyisobutyl monocarbonate, t-octylperoxyisobutyl monocarbonate, 1,1,3,3-tetramethylbutylperoxyisobutyl monocarbonate, t-butylperoxysec-butyl monocarbonate, t-pentylperoxysec-butyl monocarbonate, t-hexylperoxysec-butyl monocarbonate, t-heptylperoxysec-butyl monocarbonate, t-octylperoxysec-butyl monocarbonate, 1,1,3,3-Tetramethylbutylperoxy sec-butyl monocarbonate, t-butylperoxy t-butyl monocarbonate, t-pentylperoxy t-butyl monocarbonate, t-hexylperoxy t-butyl monocarbonate, t-heptylperoxy t-butyl monocarbonate, t-octylperoxy t-butyl monocarbonate, 1,1,3,3-tetramethylbutylperoxy t-butyl monocarbonate, t-butylperoxy 2-ethylhexyl monocarbonate, t-pentylperoxy 2-ethylhexyl monocarbonate, t-hexylperoxy 2-ethylhexyl monocarbonate Examples of organic peroxides include peroxydicarbonate, t-heptylperoxy 2-ethylhexyl monocarbonate, t-octylperoxy 2-ethylhexyl monocarbonate, 1,1,3,3-tetramethylbutylperoxy 2-ethylhexyl monocarbonate, and 1,6-bis(t-butylperoxycarbonyloxy)hexane; and examples of peroxydicarbonates include di-n-propylperoxydicarbonate, diisopropylperoxydicarbonate, di-sec-butylperoxydicarbonate, bis(4-t-butylcyclohexyl)peroxydicarbonate, and bis(2-ethylhexyl)peroxydicarbonate. Among these organic peroxides, peroxydicarbonates or peroxyesters are preferred as the organic peroxides used in step (b) because they have a superior effect of inhibiting aggregation in aqueous suspensions of PHA.

[0047] The one-minute half-life temperature of the organic peroxide used in step (b) is not particularly limited, but is preferably 140° C. or less in view of excellent reactivity and ease of handling.

[0048] From the viewpoints of the effect of inhibiting aggregation in the aqueous suspension of PHA, as well as reactivity and ease of handling, the organic peroxide used in step (b) is particularly preferably a peroxydicarbonate or peroxyester having a one-minute half-life temperature of 140° C. or less. More specific examples of such peroxydicarbonates having a one-minute half-life temperature of 140° C. or less include di-n-propyl peroxydicarbonate, diisopropyl peroxydicarbonate, di-sec-butyl peroxydicarbonate, bis(4-t-butylcyclohexyl)peroxydicarbonate, bis(2-ethylhexyl)peroxydicarbonate, etc. Specific examples of peroxyesters having a 1-minute half-life temperature of 140°C or less include t-butylperoxyisobutyrate, t-butylperoxypivalate, t-hexylperoxypivalate, t-butylperoxy 2-ethylhexanoate, t-hexylperoxy 2-ethylhexanoate, t-butylperoxyneodecanoate, and t-hexylperoxyneodecanoate.

[0049] As the organic peroxide used in step (b), one of the above-mentioned organic peroxides may be used alone, or two or more of them may be used in combination.

[0050] In step (b), the temperature of the culture medium to which the organic peroxide has been added is adjusted to a temperature of 0 to 100°C that is equal to or lower than the one-minute half-life temperature of the added organic peroxide, and maintained for a certain period of time. In step (b), the temperature of the culture medium to which the organic peroxide has been added is more preferably maintained at a temperature of 10 to 90°C that is equal to or lower than the one-minute half-life temperature of the added organic peroxide, and even more preferably maintained at a temperature of 20 to 80°C that is equal to or lower than the one-minute half-life temperature of the added organic peroxide.

[0051] In step (b), the time for which the temperature of the culture medium to which the organic peroxide has been added is maintained at the above temperature is preferably 1 to 30 hours, more preferably 3 to 24 hours, and even more preferably 5 to 15 hours, since the organic peroxide is completely decomposed by the reaction, thereby preventing the organic peroxide from remaining in the culture medium.

[0052] In step (b), since unintended decomposition of the organic peroxide can be prevented, it is preferable to add the organic peroxide after adjusting the pH of the culture medium to 5.0 to 10.0. The pH of the culture medium is more preferably 6.0 to 9.5, and even more preferably 7.0 to 9.5. The pH of the culture medium can be adjusted, for example, by adding an acid in addition to the aqueous alkaline solution described in the above <Step (a)>. Examples of acids that can be used to adjust the pH include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0053] When the pH of the culture medium is adjusted to the above range in step (b), it is preferable to maintain the pH of the culture medium within the above range while maintaining the temperature of the culture medium at the above temperature.

[0054] The amount of organic peroxide added in step (b) is not particularly limited, but is preferably 0.1 to 10 parts by weight, more preferably 0.5 to 5 parts by weight, and even more preferably 0.5 to 3 parts by weight, per 100 parts by weight of polyhydroxyalkanoic acid contained in the culture medium, in order to fully exert the effect of inhibiting aggregation in the aqueous suspension of PHA.

[0055] <Process (c)> This production method includes step (c), which is a step of adding an alkaline protease to the culture solution obtained in step (b) to perform an enzymatic treatment. In this section, the term "culture solution" simply refers to a "culture solution containing PHA-containing bacterial cells," and particularly refers to a "culture solution obtained in step (b) above (i.e., a culture solution containing PHA-containing bacterial cells treated with inorganic peroxides and organic peroxides) that is to be subjected to step (c)." By carrying out step (c), impurities (cell walls, proteins, etc.) derived from bacterial cells (microorganisms) can be destroyed and removed.

[0056] (alkaline protease) As used herein, the term "alkaline protease" refers to a protease that has the activity of degrading proteins in an alkaline environment (e.g., in a solution of pH 8.5). The alkaline protease is not particularly limited as long as it has the above-mentioned activity, and examples include serine-specific proteases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteases (e.g., papain, bromelain, cathepsin), and aspartic acid-specific proteases (e.g., pepsin, cathepsin D, HIV protease). From the viewpoint of economical advantage, serine-specific proteases, particularly subtilisins (e.g., alcalase), are preferred. These may be used alone or in combination of two or more.

[0057] Commercially available alkaline protease enzymes can be used, such as "Alcalase 2.5L" 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.; "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd.; and "Esperase" manufactured by Novozyme.

[0058] (lytic enzyme) In step (c), in addition to the treatment with the alkaline protease, a treatment with a lytic enzyme may be carried out. In other words, step (c) may further include a step of adding a lytic enzyme to the culture solution to carry out an enzymatic treatment.

[0059] As used herein, the term "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 as long as it has the above activity, and examples include lysozyme, raviol, β-N-acetylglucosaminidase, endolysin, autolysin, etc. From the viewpoint of economical advantage, lysozyme is preferred. These enzymes may be used alone or in combination of two or more.

[0060] Commercially available lytic enzymes can also be used, such as "Lysozyme" and "Achromopeptidase" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0061] In step (c), when a treatment with a lytic enzyme is performed in addition to a treatment with an alkaline protease, each treatment needs to be performed at least once, but if necessary, the treatment with the lytic enzyme and / or the treatment with the alkaline protease may be performed two or more times. Furthermore, the order in which the treatment with the lytic enzyme and the treatment with the alkaline protease are performed in the enzyme treatment step is not particularly limited.

[0062] In step (c), when performing an enzyme treatment (e.g., treatment with an alkaline protease), it is preferable to adjust the pH and temperature of the culture medium to match the optimal pH and temperature of the enzyme used. The method for adjusting the pH and temperature of the culture medium is not particularly limited, and known methods can be used.

[0063] In this production method, it is preferable to carry out the above steps (a), (b), and (c) in this order. By carrying out step (b) after step (a), reducing substances in the culture medium are removed by inorganic peroxide in step (a), so that the reaction of organic peroxide in the subsequent step (b) is not inhibited by reducing substances and the reaction proceeds efficiently. Furthermore, by carrying out step (c) after step (b), there is no risk of the alkaline protease used in step (c) inhibiting the reaction of organic peroxide in step (b).

[0064] <Process (d)> This production method includes step (d), which is a step of adjusting the pH of the PHA culture solution obtained in step (c) to 10.0 to 12.0. In step (d), by adjusting the pH of the PHA culture solution to the above range, it is possible to disperse and dissolve impurities (nucleic acids, proteins, etc.) derived from the bacterial cells in the aqueous suspension of PHA, and it becomes possible to isolate highly pure PHA.

[0065] In step (d), the pH of the PHA culture medium may be adjusted to 10.0 to 12.0, more preferably 10.2 to 11.8, and even more preferably 10.4 to 11.6.

[0066] In step (d), the method for adjusting the pH of the PHA culture solution to the above range is not particularly limited, but a method of adding an alkaline aqueous solution to the PHA culture solution is preferred. Note that the alkaline aqueous solution that can be used in step (d) is the one described in the above <Step (a)>.

[0067] In step (d), the time for which the PHA culture medium is maintained at the above pH is not particularly limited, but is preferably 0.1 to 30 hours, more preferably 0.25 to 24 hours, and even more preferably 0.5 to 15 hours.

[0068] (Surfactant addition process) In step (d), it is preferable to further add a surfactant to the PHA culture solution obtained in step (c). By further adding a surfactant to the PHA culture solution, impurities derived from the bacterial cells (especially cell membranes) can be efficiently removed, and PHA with higher purity can be isolated.

[0069] The surfactant that can be used in step (d) is not particularly limited, but examples thereof include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, etc. Among the surfactants listed above, anionic surfactants are preferred because of their high ability to remove cell membranes. In the surfactant addition step, one of these surfactants may be used alone, or two or more may be used in combination.

[0070] 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. As the anionic surfactant, these may be used alone or in combination of two or more.

[0071] In step (d), the amount of surfactant added to the PHA culture solution is not particularly limited, but is preferably 0.1 to 10.0 parts by weight, more preferably 0.1 to 5.0 parts by weight, and even more preferably 0.2 to 3.0 parts by weight, per 100 parts by weight of polyhydroxyalkanoic acid contained in the culture solution. By adding a surfactant in an amount of 0.1 part by weight or more, impurities derived from the bacterial cells (especially cell membranes) can be efficiently removed, and by adding a surfactant in an amount of 10.0 parts by weight or less, excessive foaming of the PHA culture solution can be suppressed and wastewater load can also be reduced.

[0072] In step (d), the timing of adding the surfactant to the PHA culture solution is not particularly limited, and it may be before, simultaneously with, or after the pH adjustment operation. However, it is preferable to add the surfactant to the PHA culture solution after the pH adjustment operation, since this allows the surfactant's effects to be exerted more efficiently. Furthermore, if the surfactant is added after the pH adjustment operation, the pH adjustment operation may be carried out again thereafter.

[0073] In step (d), after adding the surfactant to the PHA culture solution, it is preferable to maintain the PHA culture solution in that state for a certain period of time. The period for maintaining the PHA culture solution with the surfactant added thereto is not particularly limited, but is preferably 0.1 to 5 hours, more preferably 0.25 to 4 hours, and even more preferably 0.5 to 3 hours.

[0074] Steps (a) to (d) in this production method can also be considered as steps of treating a culture solution containing PHA-producing microorganism cells, separating the PHA from the cells and components other than PHA contained in the cells, and obtaining an aqueous suspension in which the PHA is dispersed in a solvent. Therefore, in this specification, the culture solution containing the cells that has been subjected to steps (a) to (d) may be referred to as an aqueous suspension of PHA. In addition, in this specification, the aqueous suspension of PHA may also be referred to simply as an "aqueous suspension."

[0075] <Process (e)> This production method includes step (e), which is a step of centrifuging the aqueous suspension of PHA obtained in step (d).

[0076] Step (e) is a step of centrifuging the aqueous PHA suspension obtained in step (d) and removing the supernatant to obtain an aqueous PHA suspension containing concentrated PHA. Therefore, step (e) can also be said to be a step of concentrating and purifying the aqueous PHA suspension separated from the bacterial cells.

[0077] In step (e), the method for centrifuging the aqueous suspension of PHA is not particularly limited, and any known method can be used.

[0078] In step (e), the aqueous PHA suspension may be repeatedly centrifuged. More specifically, after centrifuging the aqueous PHA suspension and removing the supernatant, additional solvent may be added to the precipitate to prepare a new aqueous PHA suspension in which the PHA is dispersed in the solvent (this operation may be referred to as dilution or dilution washing). The prepared aqueous PHA suspension may then be centrifuged again and the supernatant removed. Repeated centrifugation and dilution in step (e) can further increase the degree of purity of the aqueous PHA suspension. The degree of purity of the aqueous PHA suspension can be determined, for example, by measuring the absorbance at 280 nm or 254 nm. The solvent added to the precipitate after removing the supernatant, in other words, the solvent used to dilute the precipitate (mainly PHA), is preferably an alkaline aqueous solution adjusted to the same pH as the aqueous PHA suspension used in step (e).

[0079] 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. However, when a highly pure PHA is required, such as for medical applications, it is preferable to reduce the amount of impurities as much as possible. An example of an index 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 after step (e) is not particularly limited, as long as it is an amount that can achieve the desired amount of residual protein in the PHA. However, from the viewpoint of providing a PHA with a sufficiently low impurity content, the amount of protein 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 after step (e).

[0080] As the aqueous suspension of PHA to be subjected to step (e), the aqueous suspension of PHA that has undergone steps (a) to (d) may be used directly, or the aqueous suspension of PHA may be further dissolved in an appropriate solvent and used.

[0081] In step (e), the solvent used to dissolve the aqueous suspension of PHA and the solvent used to dilute the precipitate after centrifugation are not particularly limited as long as they are aqueous media capable of dispersing PHA, and may be water, an alkaline aqueous solution, or a mixed solvent of water and an organic solvent. The alkaline aqueous solution used in step (e) can be any of those described in the above section <Step (a)>. Organic solvents that can be used in the mixed solvent are 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. Furthermore, the concentration of the organic solvent in the mixed solvent is not particularly limited as long as it is equal to or less than the solubility of the organic solvent used in water.

[0082] In addition to the above steps (a) to (e), the present production method may also include other known steps that can be carried out during the production of PHA, such as a step of adjusting the molecular weight of PHA in a culture medium containing bacterial cells, a step of adding various additives to an aqueous suspension of PHA, a step of adjusting the pH of the aqueous suspension of PHA to an acidic value, and a step of drying the aqueous suspension of PHA after centrifugation. [Example]

[0083] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0084] [Measurement and evaluation methods] Measurements and evaluations in the examples and comparative examples were carried out by the following methods.

[0085] (cohesion) The aqueous suspension prepared in step (d) was adjusted to a pH of 10.5-11.0 and a solids content of 20-22 wt% using an alkaline aqueous solution prepared by diluting sodium hydroxide with deionized water at 40°C. This suspension was centrifuged for 10 minutes at a relative centrifugal force (RCF) of 3901 × g in a laboratory batch centrifuge to obtain a sediment with a solids content of 75-80%. This sediment was diluted with an alkaline aqueous solution at 40°C to a pH of 10.5-11.0, a solids content of 36-38 wt%, and a volume of 5 L, and subjected to shearing at 10,000 rpm for 1 hour using a HJIANGTAI HG-200 homomixer. The aqueous suspension after shearing was again adjusted to a pH of 10.5-11.0 and a solids content of 20-22 wt% using the same alkaline aqueous solution at 40°C. The centrifugation, sediment dilution, and shearing of the aqueous suspension under the same conditions were repeated a total of four times. The aqueous suspension was observed and evaluated for any abnormalities during the shear application during this repeated operation. Specifically, if an abnormality such as the homomixer stopping due to an increase in the viscosity of the aqueous suspension or a large amount of resin adhering to the homomixer rotor occurred during any shear application, the coagulation properties of the aqueous suspension were evaluated as poor. If no abnormalities occurred during any of the four shear applications, the coagulation properties of the aqueous suspension were evaluated as good (coagulation was further suppressed).

[0086] (separability) The aqueous suspension prepared in step (d) was adjusted to a pH of 10.5-11.0 and a solids content of 20-22% by weight using an alkaline aqueous solution prepared by diluting sodium hydroxide with deionized water at 40°C. This suspension was centrifuged for 10 minutes at a relative centrifugal force (RCF) of 3901 × g in a laboratory batch centrifuge to obtain a sediment with a solids content of 75-80%. This sediment was then diluted to a pH of 10.5-11.0 and a solids content of 20-22% by weight using an alkaline aqueous solution at 40°C, and further centrifuged under the above conditions to dilute the sediment in the same manner. This centrifugation and dilution procedure was repeated four times. The appearance of the supernatant after centrifugation and the processing yield were observed and evaluated after each procedure. Specifically, if the supernatant after centrifugation was cloudy, the aqueous suspension was evaluated as having poor separability. If the supernatant was not cloudy after any of the four centrifugation procedures, the aqueous suspension was evaluated as having good separability. The treatment yield was calculated based on the following formula: Treatment yield [%] = (solids weight [g] of aqueous suspension after a total of four treatments / solids weight [g] of aqueous suspension before first centrifugation) × 100. The higher the treatment yield, the better the separability of the aqueous suspension. In particular, a treatment yield of 90% or more was evaluated as having sufficiently good separability.

[0087] Example 1 (Preparation of bacterial culture solution) Capriavidus necator, described in International Publication No. WO 2021 / 206155, was cultured using the method described in paragraphs

[0051] to

[0054] of the same document to obtain a bacterial cell culture broth containing PHA. The obtained PHA was a copolymer consisting of 3HB repeating units and 3HH repeating units (i.e., poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)), and the composition ratio of the repeating units in the PHA (composition ratio of 3HB units / 3HH units) was 85 / 15 (mol / mol).

[0088] (inactivation) The bacterial cell culture solution obtained above was heated and stirred at an internal temperature of 60 to 70°C for 7 hours and sterilized to obtain a culture solution containing inactivated bacterial cells with a solid content of 25% (inactivated culture solution).

[0089] (Step (a)) The inactivated culture solution was stirred for 2 hours or more at 70°C while adjusting the pH to 10.5 to 11 with 30% sodium hydroxide. Then, 35% aqueous hydrogen peroxide, an inorganic peroxide, was added so that the amount of hydrogen peroxide added was 0.7 parts by weight per 100 parts by weight of PHA contained in the culture solution, and the mixture was stirred for 1 hour or more.

[0090] (Step (b)) The pH of the culture solution obtained above after step (a) was adjusted to 9.7 with 10% sulfuric acid at 70° C. Then, an aqueous emulsion of di-sec-butyl peroxydicarbonate ("Luperox 225" manufactured by Arkema Yoshitomi Co., Ltd., 1-minute half-life temperature: 107° C.), which is a peroxydicarbonate organic peroxide, was added to the culture solution in an amount of 2 parts by weight per 100 parts by weight of PHA contained in the culture solution, and the mixture was stirred for 10 hours or more.

[0091] (Process (c)) The culture solution obtained above after steps (a) and (b) was adjusted to pH 9.0 using 30% sodium hydroxide at 50°C. Alcalase (Novozymes), an alkaline protease, was added to this culture solution in an amount of 0.1 parts by weight per 100 parts by weight of PHA contained in the culture solution, and the culture solution was maintained at 50°C for 2 hours or more while adjusting the pH of the culture solution to 8.5 to 9.0 using 30% sodium hydroxide. (Step (d)) The PHA culture solution obtained in step (c) above was adjusted to pH 10.5 with 30% sodium hydroxide and stirred for at least 3 hours. Then, at 40°C, a 28% aqueous solution of sodium dodecyl sulfate (Kao Corporation), a surfactant, was added so that the amount of sodium dodecyl sulfate added was 0.4 parts by weight per 100 parts by weight of PHA contained in the PHA culture solution, and 30% sodium hydroxide was added so that the pH was 10.5 to 11, followed by stirring for at least 1 hour. This procedure yielded an aqueous suspension of PHA.

[0092] (Step (e)) The aqueous suspension of PHA obtained by steps (a) to (d) above was centrifuged under the conditions described in the (Coagulation) and (Separability) sections above, and the coagulation and separability of the aqueous suspension were evaluated. As a result, no abnormalities were observed in the coagulation in any of the four shear applications, and the coagulation of the aqueous suspension of PHA was good (coagulation was sufficiently suppressed). Furthermore, in any of the four centrifugation operations, no turbidity was observed in the supernatant, the processing yield was 93%, and the separability of the aqueous suspension of PHA was also good.

[0093] Comparative Example 1 An aqueous suspension of PHA was prepared in the same manner as in Example 1, except that step (a) was not performed, i.e., treatment with inorganic peroxide was not performed, and the coagulation and separability of the aqueous suspension were evaluated. As a result, with regard to separability, no cloudiness was observed in the appearance of the supernatant, and the processing yield was 93%. However, with regard to coagulation, the homomixer stopped applying an overcurrent 3 minutes after the start of the first shearing cycle, and disassembly and cleaning of the homomixer confirmed that a large amount of coagulated resin had adhered to the rotor blades. Therefore, the coagulation of the aqueous suspension of PHA was evaluated as poor.

[0094] Comparative Example 2 An aqueous suspension of PHA was prepared in the same manner as in Example 1, except that step (b) was not performed, i.e., the treatment with organic peroxide was not performed, and the coagulation and separability of the aqueous suspension were evaluated. As a result, no abnormalities occurred in the coagulation properties after all four shear stress applications, but the separability properties of the aqueous suspension of PHA were evaluated as poor because the supernatant was clearly cloudy after all four centrifugations and the treatment yield was a low value of 76%.

[0095] Comparative Example 3 An aqueous suspension of PHA was prepared in the same manner as in Example 1, except that steps (a) and (b) were not performed, i.e., neither treatment with inorganic peroxide nor treatment with organic peroxide was performed, and the coagulation and separability of the aqueous suspension were evaluated. As a result, no abnormalities occurred in the coagulation properties after a total of four shear stress applications, but the separability of the aqueous suspension of PHA was evaluated as being somewhat poor, since the supernatant was slightly cloudy after the first and second centrifugations and the treatment yield was also a somewhat low value of 85%.

[0096] 〔summary〕 From the above, it has been shown that the present manufacturing method, which includes steps (a) and (b), can achieve both the inhibition of aggregation in an aqueous suspension of PHA and the separability of PHA from the aqueous suspension, which were difficult to achieve with conventional methods. [Industrial Applicability]

[0097] This production method can suppress aggregation of PHA in an aqueous suspension while also allowing for the separation of PHA from the aqueous suspension, and therefore can be suitably used in the production of PHA.

Claims

1. (a) adding an inorganic peroxide to a culture solution containing a bacterial cell containing a polyhydroxyalkanoic acid; (b) adding an organic peroxide to the culture solution obtained in the step (a) and maintaining the temperature at a temperature of 0 to 100°C that is equal to or lower than the one-minute half-life temperature of the organic peroxide; (c) adding an alkaline protease to the culture solution obtained in the step (b) to perform an enzymatic treatment; (d) adjusting the pH of the culture solution obtained in the step (c) to 10.0 to 12.0; and (e) centrifuging the aqueous suspension of polyhydroxyalkanoic acid obtained in step (d); A method for producing a polyhydroxyalkanoic acid, comprising:

2. The method for producing polyhydroxyalkanoic acid according to claim 1, wherein the amount of the organic peroxide added in the step (b) is 0.1 to 5 parts by weight per 100 parts by weight of the polyhydroxyalkanoic acid contained in the culture solution obtained in the step (a).

3. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1, wherein the organic peroxide is a peroxydicarbonate or a peroxyester having a one-minute half-life temperature of 140°C or less.

4. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1 or 2, wherein the step (a) comprises maintaining a culture solution containing the polyhydroxyalkanoic acid-containing bacterial cells to which the inorganic peroxide has been added at 30 to 75°C.

5. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1, wherein in the step (d), a surfactant is further added to the culture solution obtained in the step (c).

6. 3. The method for producing a polyhydroxyalkanoic acid according to claim 1, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyrate repeating units and other hydroxyalkanoate units, and the composition ratio of 3-hydroxybutyrate units / other alkanoate units in the copolymer is 70 / 30 to 88 / 12 (mol / mol).

7. The method for producing a polyhydroxyalkanoic acid according to claim 1 or 2, wherein the inorganic oxide is hydrogen peroxide or ozone.