Method for producing polyhydroxyalkanoates

Abstract

The present invention provides a method for producing PHA that offers excellent separation of PHA from bacterial residue (e.g., residual protein). [Solution] A method for producing PHA, comprising: (a) adding an enzyme to a culture medium containing inactivated bacterial cells that contain PHA, thereby enzymatically treating the bacterial cells; and (b) adding an oxidizing agent having a standard oxidation-reduction potential of 2.00V or higher at 25°C under alkaline conditions to the enzymatically treated solution obtained in step (a), and reacting it under conditions of pH 9.0 to 12.0.

Description

[Technical Field]

[0001] This invention relates to a method for producing polyhydroxyalkanoates. [Background technology]

[0002] Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHA") are known to be biodegradable, and in recent years, their utilization has been promoted from an environmental perspective.

[0003] One of the advantages of PHA is that it can be produced by microorganisms using renewable plant-based raw materials. When using PHA produced by microorganisms, it is necessary to first destroy the cells of the PHA-containing microorganisms or solubilize other biological components, disperse the PHA from the cells in water to obtain an aqueous suspension of PHA, and then purify the PHA by further removing impurities other than PHA (cellular residue) from this aqueous suspension of PHA.

[0004] As such PHA purification techniques, the techniques described in Patent Documents 1 and 2 are known. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Public Gazette WO2023 / 021878 [Patent Document 2] International Public Gazette WO2024 / 166803 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] However, conventional PHA purification techniques had room for improvement in terms of the ability to separate PHA from bacterial residue (e.g., residual proteins).

[0007] Given the circumstances described above, the present invention aims to provide a method for producing PHA that offers excellent separation from bacterial residue (e.g., residual protein). [Means for solving the problem]

[0008] As a result of diligent research to solve the aforementioned problems, the inventors of the present invention have discovered a novel finding that the separation of PHA from bacterial residue (e.g., residual protein) can be improved by enzymatically treating bacterial cells containing PHA and then reacting the resulting enzyme-treated solution with an oxidizing agent having a specific standard redox potential under specific conditions. This led to the completion of the present invention.

[0009] In other words, one aspect of the present invention includes the following configuration. [1] A method for producing polyhydroxyalkanoate, comprising the steps of (a) adding an enzyme to a culture medium containing microbial cells containing polyhydroxyalkanoate to enzymatically treat the microbial cells, and (b) adding an oxidizing agent to the enzyme-treated solution obtained in step (a) and reacting it, wherein the oxidizing agent has a standard oxidation-reduction potential of 2.00V or higher at 25°C under alkaline conditions, and the pH of the enzyme-treated solution in step (b) is 9.0 to 12.0. [2] The method for producing a polyhydroxyalkanoate according to [1], wherein in step (b), the concentration of the oxidizing agent in the enzyme treatment solution is 1 to 20% by weight. [3] The method for producing a polyhydroxyalkanoate according to [1] or [2], wherein the oxidizing agent comprises one or more selected from the group consisting of sodium peroxodisulfate, potassium peroxodisulfate, and ammonium peroxodisulfate. [4] A method for producing a polyhydroxyalkanoate according to any one of [1] to [3], wherein the reaction time in step (b) is 6 hours or more and the reaction temperature is 40 to 60°C. [5] A method for producing a polyhydroxyalkanoate according to any one of [1] to [4], further comprising the step of (c) adding a surfactant to the processing solution obtained in step (b) above. [6] The method for producing a polyhydroxyalkanoate according to [5], wherein the surfactant comprises one or more selected from the group consisting of polyoxyethylene lauryl ether, polyoxyethylene alkylphenyl ether, and polyoxyethylene polyoxypropylene glycol. [7] The method for producing a polyhydroxyalkanoate according to [5] or [6], wherein the surfactant comprises sodium dodecyl sulfate. [8] A method for producing a polyhydroxyalkanoate according to any one of [1] to [7], further comprising the step of treating a culture medium containing (a') polyhydroxyalkanoate-containing inactivated bacterial cells with hydrogen peroxide before step (a). [9] A method for producing a polyhydroxyalkanoate according to any one of [1] to [8], further comprising (d) filtering the processed liquid obtained in step (b) or step (c). [Effects of the Invention]

[0010] According to one aspect of the present invention, a method for producing PHA can be provided that exhibits excellent separation properties from microbial residue (e.g., residual protein). [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows a schematic configuration of the filtration device used to measure the degree of fouling in the example. [Figure 2] This figure shows a schematic configuration of the filtration apparatus used to perform membrane filtration in step (d) of the example. [Modes for carrying out the invention]

[0012] 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 each configuration described below, and various modifications are possible within the scope indicated in the claims. In addition, embodiments or examples obtained by combining technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, by combining the technical means disclosed in each embodiment, new technical features can be formed. All academic and patent documents described in this specification are incorporated herein by reference. Also, unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more (including A and greater than A) and B or less (including B and less than B)".

[0013] [1. Method for producing PHA] A method for producing PHA according to an embodiment of the present invention (hereinafter, the "method for producing PHA according to an embodiment of the present invention" may be referred to as "this production method") includes: (a) adding an enzyme to a culture solution containing cells containing PHA to subject the cells to enzyme treatment; and (b) adding an oxidizing agent to the enzyme-treated solution obtained in step (a) and reacting them. The oxidizing agent has a standard redox potential of 2.00 V or more at 25°C under alkaline conditions, and the pH of the enzyme-treated solution in step (b) is 9.0 to 12.0. This is a method for producing PHA.

[0014] The present inventors conducted intensive studies to solve the problem of the separability between PHA and cell residues in conventional PHA purification techniques. As a result, the molecular weight of cell residues (especially residual proteins) was found as a factor affecting the separability between PHA and cell residues in the PHA purification process. More specifically, in the purification of PHA (i.e., the separation of PHA and cell residues), a new finding was obtained that the separability between PHA and cell residues can be improved by reducing the molecular weight of cell residues (especially residual proteins).

[0015] As a result of further intensive studies based on such new findings, an oxidizing agent having a standard redox potential of 2.00 V or more at 25°C under alkaline conditions is added to a solution containing PHA and cell residues to be subjected to purification treatment, and the reaction is carried out under the conditions of pH 9.0 to 12.0 (that is, by carrying out step (b)), it was found that the cell residues in the above solution can be reduced in molecular weight, and the solution after such oxidizing agent treatment is excellent in the separability of PHA and cell residues during purification treatment, and the present invention has been completed.

[0016] The solution after the oxidizing agent treatment obtained by the above method (that is, after passing through step (b)) is excellent in the separability of PHA and cell residues during purification treatment. Therefore, according to the present production method including step (b), it is possible to provide high-purity PHA with few impurities.

[0017] The present inventors also found that when the solution after the oxidizing agent treatment obtained by the above method (that is, after passing through step (b)) is purified by membrane filtration, there are few cell residues remaining after membrane purification, efficient purification is possible with a small amount of water, the permeation flux during membrane purification is high, excellent in filtration efficiency per area (sufficient filtration can be performed even with a small membrane area), and there is little membrane fouling after membrane purification, and the durability of the filter membrane can be improved. That is, the present production method including step (b) can be particularly preferably used as a production method of PHA including a purification treatment of PHA by membrane filtration.

[0018] Hereinafter, each step that may be included in the present production method will be described in detail.

[0019] <Step (a)> The present production method includes a step of adding an enzyme to a culture solution containing cells containing PHA and subjecting the cells to enzyme treatment (sometimes simply referred to as "step (a)"). Step (a) can also be said to be an enzyme treatment step.

[0020] (Culture solution containing cells containing PHA) The culture medium containing PHA-containing microbial cells used in step (a) is a culture medium obtained by culturing microorganisms capable of producing PHA, and includes the microbial cells of the cultured microorganisms (and the PHA produced within the microbial cells).

[0021] ·PHA "PHA" is a general term for polymers containing hydroxyalkanoates as monomer units (monomer repeating units), and is generally biodegradable. In particular, in this specification, "PHA" refers to a (co)polymer containing hydroxyalkanoate repeating units in an amount of 50 mol% or more of the total monomer repeating units (100 mol%), and a resin made from such a (co)polymer. Specific examples of hydroxyalkanoate repeating units constituting PHA include 3-hydroxybutanoic acid units, 4-hydroxybutanoic acid units, 3-hydroxypropionic acid units, 3-hydroxypentanoic acid units, 3-hydroxyhexanoic acid units, 3-hydroxyheptanoic acid units, 3-hydroxyoctanoic acid units, and 2-hydroxypropionic acid units. In this specification, the term (co)polymer includes both homopolymers consisting of only one type of monomer and copolymers consisting of two or more types of monomers.

[0022] Examples of PHAs provided by this manufacturing method include poly(3-hydroxyalkanoate) (hereinafter sometimes referred to as "P3HA") and poly(4-hydroxyalkanoate). Among these, P3HA is preferred because it is suitable for molded article applications.

[0023] P3HA is a 3-hydroxyalkanoate repeating unit represented by the formula: [-CHR-CH2-CO-O-] (where R is C). n H 2n+1 It is a PHA that contains an alkyl group represented by , where n is an integer between 1 and 15, and ) as an essential repeating unit.

[0024] Specific examples of P3HA include the homopolymers of 3HB: poly(3-hydroxybutyrate) (sometimes referred to as "P3HB"), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (sometimes referred to as "P3HB3HH"), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (sometimes referred to as "P3HB4HB"), poly(3-hydroxybutyrate-co-3-hydroxyvariate), and poly(3-hydroxy Examples include poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate), poly(3-hydroxybutyrate-co-3-hydroxydecanoate), poly(3-hydroxybutyrate-co-3-hydroxyvalate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-2-hydroxypropionate), and poly(3-hydroxybutyrate-co-3-hydroxypropionate). Among these, P3HB, P3HB3HH, and P3HB4HB are preferred, and P3HB3HH and P3HB4HB are more preferred, due to their ease of industrial production using microorganisms.

[0025] In this specification, "poly(X-co-Y)" refers to a copolymer containing X repeating units and Y repeating units, and is intended to be a copolymer formed by copolymerizing the monomer from which the X repeating units originate with the monomer from which the Y repeating units originate. As described above, the name of P3HA is determined by the repeating units contained in the P3HA. However, trace amounts (approximately 1 mol% or less) of monomers contained in the P3HA may not be reflected in the name of the P3HA, provided that they do not significantly affect the physical properties of the P3HA. In other words, P3HA may contain trace amounts of other repeating units in addition to the repeating units corresponding to its name.

[0026] When P3HA contains 3HB repeating units, from the viewpoint of balancing flexibility and strength, the composition ratio (3HB repeating units / other repeating units) of 3HB repeating units to other repeating units (100 mol%) in the total monomer repeating units (100 mol%) of the P3HA is preferably 99 / 1 (mol% / mol%) to 60 / 40 (mol% / mol%), more preferably 97 / 3 (mol% / mol%) to 70 / 30 (mol% / mol%), and even more preferably 95 / 5 (mol% / mol%) to 80 / 20 (mol% / mol%). When the composition ratio of 3HB repeating units in P3HA is 60 mol% or more, there is the advantage of being able to provide a resin product with superior rigidity. On the other hand, when the composition ratio of 3HB repeating units in P3HA is 99 mol% or less, there is the advantage of being able to provide a resin product with superior flexibility. The monomer composition ratio of P3HA can be measured by gas chromatography or other methods (see, for example, International Publication No. 2014 / 020838).

[0027] Microorganisms capable of producing PHA As microorganisms capable of producing PHA, P3HA-producing bacteria can be suitably utilized. Examples of such P3HA-producing bacteria include P3HB-producing bacteria, with Bacillus megaterium, discovered in 1925, being the first. Other known natural microorganisms include Cupriavidus necator (formerly classified as Alcaligenes eutrophus, Ralstonia eutropha) and Alcaligenes latus. In these microorganisms, P3HB accumulates within the bacterial cells.

[0028] Furthermore, known microorganisms that produce P3HA, a copolymer of 3HB and other hydroxyalkanoates, include Aeromonas caviae, which produces P3HB3HH, and Alcaligenes eutrophus, which produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate). In particular, to increase the productivity of P3HB3HH, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T.Fukui, Y.Doi, J.Bacteriol., 179, p4821-4830 (1997)) into which genes for P3HA synthases have been introduced is preferred. In addition to the above, genetically modified microorganisms into which various P3HA synthesis-related genes have been introduced can also be used to match the desired physical properties of the P3HA.

[0029] This manufacturing method may include a step of culturing the above-mentioned various PHA-producing bacteria using a known culture method to obtain a culture medium containing PHA-containing bacterial cells.

[0030] ·Inactivation The culture medium containing PHA-containing microorganisms used in step (a) may be a culture medium in which microorganisms capable of producing PHA have been cultured, or an inactivated culture medium in which the microorganisms in the culture medium have been inactivated may be used. In other words, it is preferable that this manufacturing method includes an inactivation step in which the culture medium containing PHA-containing microorganisms used in step (a) is inactivated.

[0031] In the inactivation step of this manufacturing method, the method for inactivating microorganisms in the culture medium is not particularly limited, but one example is heating the culture medium at an inactivation temperature.

[0032] Here, "inactivation temperature" refers to the temperature at which microorganisms in the culture medium can be killed. Specifically, the inactivation temperature is, for example, 40°C to 80°C, preferably 50°C to 80°C, and more preferably 60°C to 70°C. The heating time at the inactivation temperature is not particularly limited as long as it is sufficient to kill the microorganisms in the culture medium, but it may be, for example, 30 minutes to 12 hours.

[0033] (enzyme) The enzyme used in step (a) is not particularly limited, but lytic enzymes and / or proteolytic enzymes are preferred. That is, in one embodiment of the present invention, step (a) may be a step of adding a lytic enzyme and / or proteolytic enzyme to a culture medium containing PHA-containing bacterial cells, or a step of treating a culture medium containing PHA-containing bacterial cells with a lytic enzyme and / or proteolytic enzyme.

[0034] ·Lytic enzyme In this specification, "lytic enzyme" refers to an enzyme that has the activity to break down (lyse) sugar chains (e.g., peptidoglycans) that make up the cell wall of a microbial cell.

[0035] The lytic enzyme that can be used in step (a) is not particularly limited as long as it has the above activity, but examples include lysozyme, labiases, β-N-acetylglucosaminidase, endolysin, and autolysin. From the viewpoint of economic advantage, lysozyme is preferred. In addition, commercially available lytic enzymes can also be used. Examples of such commercially available lytic enzymes include "Lysozyme" and "Achromopeptidase" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0036] • Treatment with proteolytic enzymes In this specification, "protein-degrading enzyme" refers to an enzyme that has the activity to break down proteins.

[0037] The protease that can be used in step (a) is not particularly limited as long as it has the above activity, but examples include serine-specific proteases (e.g., subtilisin, chymotrypsin, trypsin), cysteine-specific proteases (e.g., papain, bromelain, cathepsin), and aspartate-specific proteases (e.g., pepsin, cathepsin D, HIV protease). From the viewpoint of economic advantage, serine-specific proteases, especially subtilisin, are preferred. Commercially available proteases can also be used. Examples of commercially available proteolytic enzymes include Novozyme's "Alcalase 2.5L," Amano Enzyme Co., Ltd.'s "Protin SD-AY10" and "Protease P 'Amano' 3SD," Danisco Japan Co., Ltd.'s "Multifect PR6L" and "Optimase PR89L," Shin Nippon Chemical Industries, Ltd.'s "Sumizyme MP," DSM Japan Co., Ltd.'s "Delborase," Nagase ChemteX Corporation's "Bioprase OP," "Bioprase SP-20FG," and "Bioprase SP-4FG," HBI Corporation's "Orientase 22BF," Yakult Pharmaceutical Co., Ltd.'s "Aloase XA-10," and Novozyme's "Esperase."

[0038] In step (a), one of the above-mentioned enzymes (and other enzymes) may be used alone, or two or more may be used, but in step (a), it is preferable to use at least a lytic enzyme and a proteolytic enzyme.

[0039] In step (a), when using a lytic enzyme and a proteolytic enzyme, the order in which the two enzymes are added is not particularly limited. However, from the viewpoint of improving the efficiency of enzyme treatment, it is preferable to add the lytic enzyme first, allow it to react sufficiently, and then add the proteolytic enzyme. Alternatively, after allowing the proteolytic enzyme to react sufficiently, if necessary, a lytic enzyme and / or a proteolytic enzyme (or other enzymes) may be added and reacted further.

[0040] In step (a), when adding and reacting the enzyme, it is preferable to adjust the pH and temperature of the culture medium to which the enzyme has been added to match the optimal pH and temperature of the added enzyme. Furthermore, the reaction time for the added enzyme is not particularly limited and may be, for example, 30 minutes to 6 hours, or 1 hour to 3 hours.

[0041] <Process (a')> The present manufacturing method preferably includes a step (a') in which a culture medium containing inactivated bacterial cells containing PHA is treated with hydrogen peroxide (H2O2) before step (a). By performing step (a'), a treatment solution containing more low-molecular-weight bacterial residue can be obtained in the subsequent step (b), thereby improving separation performance. Step (a') can also be described as a step of adding hydrogen peroxide to a culture medium containing inactivated bacterial cells containing PHA and allowing it to react, or as a hydrogen peroxide treatment step.

[0042] In step (a'), the amount of hydrogen peroxide added to the culture medium containing inactivated bacterial cells with PHA is not particularly limited, but it is preferable to add an amount such that the concentration of hydrogen peroxide in the culture medium after the addition of hydrogen peroxide is 0.2 to 30% by weight, more preferably 0.2 to 15% by weight, and even more preferably 0.2 to 10% by weight.

[0043] In step (a'), the pH of the culture medium when reacting with hydrogen peroxide is not particularly limited, but from the viewpoint of improving reaction efficiency, it is preferably 10.0 to 11.0, more preferably 10.2 to 10.8, and even more preferably 10.3 to 10.7.

[0044] In step (a'), the temperature of the culture medium when reacting with hydrogen peroxide (reaction temperature) is not particularly limited, but from the viewpoint of improving reaction efficiency, it is preferably 40 to 60°C, more preferably 45 to 55°C, and even more preferably 47 to 53°C.

[0045] In step (a'), the reaction time for hydrogen peroxide is not particularly limited, but from the viewpoint of obtaining a treatment solution containing more low-molecular-weight bacterial residue, it is preferably 2 hours or more, more preferably 3 hours or more, and even more preferably 4 hours or more. Furthermore, the upper limit of the reaction time is not particularly limited and may be, for example, 10 hours or less.

[0046] <Process (b)> This manufacturing method includes a step (b) in which an oxidizing agent is added to the enzyme-treated solution obtained in step (a) and reacted (also simply referred to as "step (b)"). Step (b) can also be described as an oxidizing agent treatment step. By performing step (b), the bacterial residue in the enzyme-treated solution obtained in step (a) (or further via step (a')) can be reduced to a low molecular weight, improving the separation of PHA from the bacterial residue during the purification process.

[0047] Oxidizing agent The oxidizing agent used in step (b) is an oxidizing agent having a standard redox potential of 2.00V or higher at 25°C under alkaline conditions (in this specification, unless otherwise specified, "oxidizing agent" refers to an oxidizing agent having a standard redox potential of 2.00V or higher at 25°C under alkaline conditions). Here, alkaline conditions refer to conditions where the pH is greater than 7.0. Examples of such oxidizing agents having a standard redox potential of 2.00V or higher at 25°C include peroxodisulfates, and among them, sodium peroxodisulfate (standard redox potential at 25°C under alkaline conditions: 2.01V), potassium peroxodisulfate (standard redox potential at 25°C under alkaline conditions: 2.01V), and ammonium peroxodisulfate (standard redox potential at 25°C under alkaline conditions: 2.01V) are preferred because they have an excellent effect in reducing the molecular weight of bacterial residue. In other words, the oxidizing agent used in step (b) preferably includes one or more selected from the group consisting of sodium peroxodisulfate, potassium peroxodisulfate, and ammonium peroxodisulfate, and more preferably includes one or more selected from the group consisting of sodium peroxodisulfate, potassium peroxodisulfate, and ammonium peroxodisulfate.

[0048] In step (b), the amount of oxidizing agent to be added to the enzyme treatment solution obtained in step (a) is not particularly limited, but it is preferable to add an amount such that the concentration of the oxidizing agent in the enzyme treatment solution is 1 to 20% by weight, more preferably 1.5 to 15% by weight, and even more preferably 3 to 10% by weight. In other words, in step (b), it is preferable to react the oxidizing agent under conditions such that the concentration of the oxidizing agent in the enzyme treatment solution is the above-mentioned concentration.

[0049] In step (b), the pH of the enzyme treatment solution when reacting with the oxidizing agent is 9.0 to 12.0, preferably 9.5 to 11.5, more preferably 10.0 to 11.0, even more preferably 10.2 to 10.8, and even more preferably 10.3 to 10.7, from the viewpoint of improving the reaction efficiency of the oxidizing agent and sufficiently reducing the molecular weight of the microbial residue.

[0050] In step (b), the pH of the enzyme treatment solution may decrease as the oxidizing agent reacts. Therefore, in step (b), it is preferable to continuously add alkali to the enzyme treatment solution while the oxidizing agent is reacting to maintain the pH of the enzyme treatment solution within the above range during the reaction.

[0051] In step (b), the alkali used to maintain the pH is not particularly limited and can be, for example, alkali metal or alkaline earth metal hydroxides such as sodium oxide or potassium hydroxide; metal carbonates such as sodium carbonate or potassium carbonate; or metal phosphates or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, or potassium hydrogen phosphate. These alkalis may be in the form of aqueous solutions of any concentration.

[0052] In step (b), the temperature of the enzyme treatment solution (reaction temperature) when reacting with the oxidizing agent is not particularly limited, but from the viewpoint of improving reaction efficiency and further reducing the molecular weight of the microbial residue, it is preferably 40 to 60°C, more preferably 45 to 55°C, and even more preferably 47 to 53°C.

[0053] In step (b), the reaction time for the oxidizing agent is not particularly limited, but from the viewpoint of obtaining a treatment solution containing more low-molecular-weight microbial residue, it is preferably 4 hours or more, more preferably 6 hours or more, and even more preferably 8 hours or more. Furthermore, there is no particular upper limit to the reaction time, and it may be, for example, 12 hours or less.

[0054] <Process (c)> The present manufacturing method preferably further includes a step of adding a surfactant to the processing solution obtained in step (b) (also simply referred to as "step (c)"). By carrying out step (c), the separation of PHA and bacterial residue in the processing solution obtained in step (b) can be further improved.

[0055] (Surfactants) The surfactant that can be added in step (c) is not particularly limited, and any of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants can be used. Furthermore, one of these may be used alone, or two or more may be used in combination. Among the above types of surfactants, anionic surfactants and / or nonionic surfactants are preferred because they offer superior separation improvement, and a combination of an anionic surfactant and a nonionic surfactant is more preferred.

[0056] Examples of anionic surfactants that can be added in step (c) include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfate esters, alkenyl sulfates, alkyl ether sulfates, alkenyl ether sulfates, α-olefin sulfonates, α-sulfo fatty acid salts, esters of α-sulfo fatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid-type surfactants, and N-acyl amino acid-type surfactants. Among these, alkyl sulfates are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred because it has an excellent effect in improving separation performance and is inexpensive. One of these anionic surfactants may be used alone, or two or more may be used in combination.

[0057] Examples of nonionic surfactants that can be added in step (c) include polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene myristyl ether, polyoxyethylene octyldodecyl ether, and polyoxyethylene tridecyl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene dodecylphenyl ether, and polyoxyethylene β-naphthyl ether; polyoxyethylene polyoxypropylene glycol; polyoxyethylene styrene-phenyl ether and polyoxyalkylene alkyl ethers such as polyoxyalkylene trialkyl ethers having 3 or more carbon atoms in the alkylene group; sorbitan monolaurate, sorbitan Examples include sorbitan fatty acid esters such as monopalmitate, sorbitan monostearate, sorbitan distearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate; polyoxyethylene fatty acid esters such as polyethylene glycol monolaurate, polyethylene glycol monostearate, polyethylene glycol distearate, and polyethylene glycol monooleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, and polyoxyethylene sorbitan monooleate; polyoxyethylene alkylamines; glycerin fatty acid esters such as glycerol monostearate and glycerol monooleate; and oxyethylene-oxypropylene block polymers. As nonionic surfactants, one of these may be used alone, or two or more may be used in combination.

[0058] Among these, the surfactant added in step (c) is particularly preferably one or more selected from the group consisting of polyoxyethylene lauryl ether, polyoxyethylene alkylphenyl ether, and polyoxyethylene polyoxypropylene glycol, as it has the advantage of further improving separation and suppressing the generation of fine particles.

[0059] Examples of cationic surfactants that can be added in step (c) include alkylamine salts such as coconutamine acetate, laurylamine acetate, and stearylamine acetate; alkylbenzyldimethylammonium chlorides such as hexadecylbenzyldimethylammonium chloride and laurylbenzyldimethylammonium chloride; alkyltrimethylammonium chlorides such as lauryltrimethylammonium chloride, hexadecyltrimethylammonium chloride, stearyltrimethylammonium chloride, and cocoyltrimethylammonium chloride; and dialkyldimethyl chlorides such as didecyldimethylammonium and dialkyldimethylammonium. One of these cationic surfactants may be used alone, or two or more may be used in combination.

[0060] Examples of amphoteric surfactants that can be added in step (c) include alkyl betaines such as lauryl betaine, stearyl betaine, and dimethyl lauryl betaine; sodium lauryl diaminoethylglycine; amide betaine; imidazoline; lauryl carboxymethyl hydroxyethyl imidazolinium betaine; and the like. These amphoteric surfactants may be used individually or in combination of two or more.

[0061] In one embodiment of the present invention, from the viewpoint of further improving separation performance, the surfactant added in step (c) is particularly preferably a combination of one or more selected from the group consisting of polyoxyethylene lauryl ether, polyoxyethylene alkylphenyl ether, and polyoxyethylene polyoxypropylene glycol, and sodium dodecyl sulfate (SDS).

[0062] In step (c), the amount of surfactant added to the processing solution obtained in step (b) (the total amount if multiple types of surfactants are added) is not particularly limited. However, it is preferable to add an amount such that the concentration of surfactant in the processing solution after surfactant addition is 0.1 to 3.0% by weight, more preferably 0.3 to 1.5% by weight, and even more preferably 0.5 to 1.2% by weight, in order to improve the separation of PHA from bacterial residue while reducing the amount of surfactant remaining in the PHA after purification and providing higher purity PHA.

[0063] (Alkaline treatment) In step (c), an alkali may be added in addition to the surfactant. That is, in one embodiment of this manufacturing method, step (c) may be a step of surfactant and alkali treatment. Adding an alkali further in step (c) has the advantage of dissolving the low-molecular-weight bacterial residue and improving separation.

[0064] In step (c), the alkali that can be added to the treatment solution obtained in step (b) is not particularly limited, and for example, alkali metal or alkaline earth metal hydroxides such as sodium oxide 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 can be used. These alkalis may be in the form of aqueous solutions of any concentration.

[0065] In step (c), the amount of alkali added is not particularly limited, but it is preferable to add an amount of alkali that results in a pH of 10.5 to 11.5 in the treated solution after alkali addition, and more preferably an amount that results in a pH of 10.7 to 11.3, in order to suppress a significant decrease in the molecular weight of PHA.

[0066] <Processing solution> In one embodiment of the present invention, a processed solution obtained through step (b) (and further through step (c) if the present manufacturing method includes step (c)) is provided. The processed solution according to the present manufacturing method is a solution obtained by enzymatic treatment and oxidizing agent treatment of a culture solution containing inactivated bacterial cells containing PHA, preferably a solution obtained by enzymatic treatment and oxidizing agent treatment of the culture solution followed by the addition of a surfactant, more preferably a solution obtained by hydrogen peroxide treatment, enzymatic treatment and oxidizing agent treatment of the culture solution followed by the addition of a surfactant, and even more preferably a solution obtained by hydrogen peroxide treatment, enzymatic treatment and oxidizing agent treatment of the culture solution followed by the addition of a surfactant and alkali. The processed solution according to the present manufacturing method can also be described as a solution in which PHA (and bacterial cell residue) is dispersed in water, or it can be described as a PHA aqueous suspension.

[0067] (Supernatant protein concentration) The processing solution according to this manufacturing method, having undergone the above-described steps, exhibits excellent separation of PHA from bacterial residue when subjected to purification. In this specification, whether or not the processing solution exhibits excellent separation of PHA from bacterial residue when subjected to purification can be evaluated by the supernatant protein concentration of the processing solution. Specifically, if the supernatant protein concentration of the processing solution to be evaluated is 30,000 ppm or less, the processing solution can be evaluated as having excellent separation of PHA from bacterial residue. The supernatant protein concentration of the processing solution is measured by the method described in the examples.

[0068] From the viewpoint of further improving the separation of PHA from bacterial residue, the lower the supernatant protein concentration of the processing solution in this manufacturing method, the better. Specifically, it is preferable that it be 30,000 ppm or less, more preferably 25,000 ppm or less, even more preferably 20,000 ppm or less, and particularly preferably 15,000 ppm or less.

[0069] <Process (d)> The present manufacturing method preferably includes (d) a step of filtering the processed liquid obtained through each of the above steps, that is, the processed liquid obtained in step (b) or step (c) (also simply referred to as "step (d)"). Step (d) is a step of separating PHA from bacterial residue in the processed liquid obtained in the above steps, and can also be said to be a step of purifying PHA. In the present manufacturing method, since the processed liquid obtained through each of the above steps (especially step (b)) is the target of filtration in step (d), it is possible to separate PHA from bacterial residue in the processed liquid with good separation ability.

[0070] (Filtration process) The method of filtration carried out in step (d) is not particularly limited as long as it can separate the PHA and bacterial residue in the processed liquid obtained in the above step, and known filtration methods can be used, but membrane filtration using a filter membrane is preferred, and membrane filtration by a diafiltration method is more preferred. In other words, step (d) is preferably a step of membrane filtration of the processed liquid obtained in the above step, and more preferably a step of membrane filtration by a diafiltration method.

[0071] The material of the filter film that can be used in step (d) is not particularly limited and can be selected from a variety of materials such as ceramic, paper, filter cloth (woven or nonwoven), screen, bisque, polymer film, perforated metal, and wedge wire, but ceramic is preferred because it allows the use of a variety of cleaning agents.

[0072] The pore size of the filter membrane used in step (d) is not particularly limited as long as it can separate the PHA and bacterial residue in the processing solution obtained in the above step, but it is preferably 0.1 to 0.5 μm, and more preferably 0.1 to 0.3 μm, in order to improve the yield of PHA.

[0073] (Resin residual protein concentration) This manufacturing method makes it possible to provide high-purity PHA for filtering a processing solution that exhibits excellent separation of PHA from microbial residue. In this specification, the purity of the obtained PHA can be evaluated by the resin residual protein concentration of the PHA. Specifically, if the resin residual protein concentration of the PHA to be evaluated is 10,000 ppm or less, the PHA can be evaluated as high-purity PHA from which microbial residue has been sufficiently separated and removed. The resin residual protein concentration of the PHA is measured by the method described in the examples.

[0074] From the viewpoint of providing higher purity PHA, it is preferable that the resin residual protein concentration of the PHA obtained through step (d) be as low as possible, specifically, preferably 10,000 ppm or less, and more preferably 5,000 ppm or less.

[0075] (transmission flux) This manufacturing method provides excellent filtration efficiency per unit area of ​​membrane surface area during the filtration operation, in order to filter the treatment solution which offers superior separation of PHA and bacterial residue (in other words, sufficient filtration can be performed even with a small membrane surface area). In this specification, filtration efficiency per unit area of ​​membrane surface area can be evaluated by the average permeate flux during the filtration operation, specifically, when the average permeate flux is 100 L / m 2 If the value is above h, it can be evaluated as having excellent filtration efficiency per unit area. The average permeate flux is measured by the method described in the examples.

[0076] From the viewpoint of improving filtration efficiency per unit area of ​​membrane, a higher permeate flux in step (d) is preferable, specifically 100 L / m³. 2 It is preferable that the rate is 150 L / m² or higher. 2It is more preferable that it be h or greater.

[0077] (Fouling progression) This manufacturing method improves the durability of the filter membrane in order to filter the treatment solution, which has excellent separation properties between PHA and bacterial residue. As a result, the same filter membrane can be used for a longer period, reducing the frequency of filter membrane replacement and resulting in improved production costs and efficiency. In this specification, the durability of the filter membrane can be evaluated by the degree of fouling progression. Specifically, if the degree of fouling progression is 80% or less, the filter membrane can be evaluated as having excellent durability. The degree of fouling progression is measured by the method described in the examples.

[0078] From the viewpoint of improving the durability of the filter film, a lower degree of fouling in step (d) is preferable, specifically, 80% or less is preferable, and 75% or less is more preferable.

[0079] <Process (d')> The present manufacturing method preferably includes (d') a step of centrifuging the processed liquid obtained through each of the above steps, that is, the processed liquid obtained in step (b) or step (c) (also simply referred to as "step (d')"). Step (d'), like step (d), is a step of separating PHA from bacterial residue in the processed liquid obtained in the above steps, and can also be said to be a step of purifying PHA. In the present manufacturing method, in step (d') as well, the processed liquid obtained through each of the above steps (especially step (b)) is the target of centrifugation, so it is possible to separate PHA from bacterial residue in the processed liquid with good separation ability.

[0080] (Centrifugal separation operation) The method of centrifugation performed in step (d') is not particularly limited as long as it can separate the PHA and bacterial residue in the processing liquid obtained in the above step, and any known centrifugation method can be used. Such known centrifugation methods include, for example, centrifugation using a centrifugal sedimentation machine or a centrifugal dehydrator.

[0081] Examples of centrifugal sedimentation machines that can be used in the centrifugal separation process include separation plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.) or cylindrical type centrifugal sedimentation machines. In addition, both palindromic and continuous type centrifugal sedimentation machines can be used. Similarly, either palindromic or continuous type centrifugal dehydrators can be used.

[0082] The number of centrifugations in step (d') is not particularly limited and can be one or any number of two or more. Also, the conditions for centrifugation in step (d') (speed of rotation, rotation time) are not particularly limited as long as the PHA and bacterial residue in the processing liquid can be separated by the desired number of centrifugations. For example, the speed of rotation may be 3000 to 6000 rpm and the rotation time may be 1 to 30 minutes per centrifugation. [Examples]

[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 method] Measurements in the examples and comparative examples were performed using the following method.

[0085] (average permeation flux) The average permeation flux was calculated based on the time required for filtration (filtration time) and the following formula.

[0086] Average permeation flux (L / m 2 ·h)=total permeate volume (L) / (membrane area (m 2 ) × filtration time (h).

[0087] (Supernatant protein concentration) The supernatant protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Specifically, 1 mL of the supernatant obtained by centrifuging the processing solution immediately before step (d) at 10,000 G or higher for 5 minutes was placed in a 15 mL Falcon tube, 2 mL of the reagent from the kit was added, and the mixture was shaken at 60°C for 30 minutes. 30 minutes after the end of shaking, the mixture was cooled to 25°C, and the absorbance at a wavelength of 562 nm was measured. The protein concentration was calculated based on the measured absorbance and was defined as the supernatant protein concentration.

[0088] (Resin residual protein concentration) The residual protein concentration in the PHA resin was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Specifically, 20-50 mg of the aqueous dispersion of PHA obtained in step (d) (an amount resulting in a solid content of 10 mg) was placed in a 15 mL Falcon tube, 2 mL of the reagent from the kit was added, and the mixture was shaken at 60°C for 30 minutes. After 30 minutes from the end of shaking, the mixture was cooled to 25°C, and the absorbance at a wavelength of 562 nm was measured. The protein concentration was calculated based on the measured absorbance and was defined as the residual protein concentration in the resin.

[0089] (Fouling progression) In the filtration apparatus 100 shown in Figure 1, an unused tubular membrane (material: alumina ceramic, nominal pore size 0.2 μm, length 25 cm, diameter 7 mm, membrane area 55 cm²) is used as filter membrane 1. 2 Using the above method, pure water a in tank 3 was circulated to filter membrane 1 by pump 2, and the intermembrane pressure difference was set to 100 kPa. While filtering the filtrate c, the unfiltered liquid b was circulated back to tank 3, and the amount of pure water permeating per minute (permeability) was measured. This was taken as the permeability before membrane filtration. Using the tubular membrane used in membrane filtration in each example and comparative example as filter membrane 1, the amount of pure water permeating per minute (permeability) was measured using the same procedure as above, and this was taken as the permeability after membrane filtration. The degree of fouling progression was calculated based on the following formula: Fouling progression (%) = (1 - Permeable water flow after membrane filtration (L) / Permeable water flow before membrane filtration (L)) × 100.

[0090] [Example 1] (culture) Ralstonia eutropha, as described in International Publication No. WO2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] of the same document to obtain a bacterial culture medium containing PHA-containing cells. Note that Ralstonia eutropha is now classified as Capriavidus nekator. The composition ratio of PHA repeating units (3HB units / 3HH units) was 94 / 6 (mol / mol).

[0091] (inactivation) The bacterial culture medium obtained above was sterilized by heating and stirring at an internal temperature of 60-70°C for 7 hours to obtain an inactivated culture medium.

[0092] (Process (a'): Hydrogen peroxide treatment) Hydrogen peroxide was added to the inactivated culture medium obtained above to a concentration of 1.0% by weight. The pH of this inactivated culture medium was adjusted to 10.5±0.2 using 30% sodium hydroxide, and the internal temperature was maintained at 50±2°C for 4 hours.

[0093] (Process (a): Enzyme treatment process) To the culture solution obtained after hydrogen peroxide treatment in step (a'), lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries), a lytic enzyme, was added to a concentration of 10 ppm, and the mixture was maintained at 50°C for 2 hours. Subsequently, 2.5 L of alcalase (manufactured by Novozyme), a proteolytic enzyme, was added to a concentration of 300 ppm, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5 while maintaining the mixture for 2 hours. Through this procedure, treated solutions treated with lytic enzymes and proteolytic enzymes were obtained.

[0094] (Process (b): Oxidizing agent treatment process) To the treatment liquid obtained in step (a), potassium peroxydisulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a concentration of 3% by weight. Next, a 30% aqueous sodium hydroxide solution was added to adjust the pH to 10.5. While maintaining this solution at 50 °C, a 30% aqueous sodium hydroxide solution was continuously added, and the oxidizing agent was reacted for 8 hours while maintaining the pH at 10.5.

[0095] (Step (c): Surfactant and alkali treatment) To the treatment liquid obtained in step (b), sodium dodecyl sulfate (SDS, manufactured by Kao Corporation), a surfactant, was added to a concentration of 0.6 to 1.0% by weight, and polyoxyethylene lauryl ether (product name: Emulgen 120, manufactured by Kao Corporation) was added to a concentration of 0.1% by weight. Thereafter, an aqueous sodium hydroxide solution was added to the treatment liquid to which the surfactant had been added to adjust the pH to 11.0 ± 0.2. By such an operation, a treatment liquid that had been subjected to surfactant addition and alkali treatment was obtained. For the obtained treatment liquid, the supernatant protein concentration was measured. The results are shown in Table 1.

[0096] (Step (d): Membrane filtration) 1200 mL of the treatment liquid obtained in step (c) (solid content: 25% by weight) was used as a filter membrane 11, and a tubular membrane (material: alumina ceramic, nominal pore size: 0.2 μm, length: 25 cm, diameter: 7 mm, membrane area: 55 cm 2The sample was subjected to a filtration apparatus 101 shown in Figure 2, using a diafiltration type membrane filtration. Specifically, first, the processed liquid obtained in step (c) was stirred in tank 13 with a stirrer 14 and supplied to filter membrane 1 as membrane feed liquid a via pump 12. The processed liquid a was filtered by filter membrane 11, separating it into concentrated liquid b containing PHA and filtrate c containing bacterial residue. Concentrated liquid b was supplied back to tank 13, and filtrate c was removed from the system. Distilled water d of the same weight as the filtrate c removed from the system was supplied to tank 3 (more specifically, 100 mL of distilled water was supplied to tank 13 every time 100 g of filtrate c was permeated). In tank 13, the resupplied concentrated liquid b and distilled water d were stirred and mixed with a stirrer 14, and the mixture was supplied back to filter membrane 11 as membrane feed liquid a and filtered again. This series of operations was repeated until the exchange ratio reached 10 times. Here, the exchange ratio is the value obtained by dividing the weight of filtrate c by the weight of the membrane feed solution a that was initially filtered (i.e., the weight of the 1200 mL of processed solution obtained in step (c)). Filtration was terminated when the exchange ratio reached 10 times, and a purified aqueous dispersion of PHA was obtained. Based on the time required for the above filtration operation, the average permeation flux was calculated, the residual protein concentration of the resin was measured in the obtained aqueous dispersion of PHA, and the degree of fouling progression was calculated using the tubular membrane used as filter membrane 11. The results of each measurement are shown in Table 1.

[0097] [Examples 2-5] Except for changing the concentration or reaction time of the oxidizing agent in step (b) or the type of surfactant in step (c) as shown in Table 1, a treatment solution was obtained in the same manner as in Example 1, and the obtained treatment solution was filtered to obtain an aqueous dispersion of purified PHA. The supernatant protein concentration of the obtained treatment solution was measured, and the physical properties of the obtained aqueous dispersion of PHA were measured or evaluated. The results of each measurement are shown in Table 1.

[0098] [Comparative Example 1] Except for not performing step (b), the treatment solution was obtained in the same manner as in Example 1, and the obtained treatment solution was filtered to obtain a purified aqueous dispersion of PHA. The measurement results of each physical property are shown in Table 1.

[0099] [Comparative Example 2] Except for not performing steps (b) and (a'), the treatment solution was obtained in the same manner as in Example 1, and the obtained treatment solution was filtered to obtain a purified aqueous dispersion of PHA. The measurement results of each physical property are shown in Table 1.

[0100] [Comparative Example 3] Except for changing the pH of the treatment solution during the oxidizing agent treatment in step (b) as shown in Table 1, the treatment solution was obtained in the same manner as in Example 1, and the obtained treatment solution was filtered to obtain an aqueous dispersion of purified PHA. The measurement results of each physical property are shown in Table 1.

[0101] [Comparative Example 4] Except for changing the oxidizing agent in step (b) to hydrogen peroxide (standard oxidation-reduction potential 0.88V), the treatment solution was obtained in the same manner as in Example 1, and the obtained treatment solution was filtered to obtain a purified aqueous dispersion of PHA. The measurement results of each physical property are shown in Table 1.

[0102] [Table 1]

[0103] 〔summary〕 Table 1 shows that the methods of Examples 1 to 5, including steps (a) and (b) of the present manufacturing method, can provide a processed solution with a sufficiently low supernatant protein concentration and excellent separation properties. Furthermore, the results of Example 1 show that purifying the processed solution obtained by this method by filtration results in less residual bacterial residue after purification, providing high-purity PHA, high permeation flux during membrane purification, excellent filtration efficiency per unit area, and reduced membrane clogging after membrane purification, improving the durability of the filter membrane. On the other hand, the methods of Comparative Examples 1 to 4, which either do not perform oxidizing agent treatment, do not perform treatment at the specified pH, or use an oxidizing agent with a standard redox potential of less than 2.00V, result in an excessively high supernatant protein concentration in the obtained processed solution, making it impossible to achieve excellent separation properties. Furthermore, it can be seen that when the processed solution obtained by this method is purified by filtration, a large amount of bacterial residue remains after purification, making it impossible to adequately remove impurities; the permeate flux during membrane purification is low, resulting in poor filtration efficiency per unit area; and there is a problem with the durability of the filter membrane, with frequent membrane clogging after membrane purification. [Industrial applicability]

[0104] This manufacturing method is suitable for use in the production of PHA because it exhibits excellent separation properties between PHA and microbial residue (e.g., residual protein). The PHA produced by this method can be suitably used, for example, as molded products in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing products, packaging, automobiles, building materials, and other fields.

Claims

1. (a) A step of enzymatically treating the bacterial cells by adding an enzyme to a culture medium containing bacterial cells containing polyhydroxyalkanoate, (b) The process includes adding an oxidizing agent to the enzyme treatment solution obtained in step (a) and reacting it, The oxidizing agent has a standard oxidation-reduction potential of 2.00 V or higher at 25°C under alkaline conditions. A method for producing polyhydroxyalkanoate, wherein the pH of the enzyme treatment solution in step (b) is 9.0 to 12.

0.

2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein in step (b), the concentration of the oxidizing agent in the enzyme treatment solution is 1 to 20% by weight.

3. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the oxidizing agent comprises one or more selected from the group consisting of sodium peroxodisulfate, potassium peroxodisulfate, and ammonium peroxodisulfate.

4. A method for producing a polyhydroxyalkanoate according to claim 1, wherein the reaction time in step (b) is 6 hours or more and the reaction temperature is 40 to 60°C.

5. Furthermore, the method for producing a polyhydroxyalkanoate according to claim 1, comprising the step of adding a surfactant to the processing solution obtained in step (b) above.

6. The method for producing a polyhydroxyalkanoate according to claim 5, wherein the surfactant comprises one or more selected from the group consisting of polyoxyethylene lauryl ether, polyoxyethylene alkylphenyl ether, and polyoxyethylene polyoxypropylene glycol.

7. The method for producing a polyhydroxyalkanoate according to claim 5, wherein the surfactant comprises sodium dodecyl sulfate.

8. Furthermore, the method for producing polyhydroxyalkanoate according to claim 1, further comprising the step of treating a culture medium containing (a') polyhydroxyalkanoate-containing inactivated bacterial cells with hydrogen peroxide before step (a).

9. Furthermore, the method for producing a polyhydroxyalkanoate according to claim 1 or 2, comprising the step of filtering the processed liquid obtained in step (b) or step (c).

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