To provide a method for producing a polyhydroxyalkanoate and a method for reducing protein in an aqueous suspension of the polyhydroxyalkanoate.

Abstract

Provided is a method for producing PHA, which can more efficiently reduce the protein content of a PHA aqueous suspension.SOLUTION: A filtration step of subjecting a PHA aqueous suspension liquid to dead-end filtration, in which the PHA aqueous suspension liquid contains at least one organic solvent selected from the group consisting of an alcohol-based solvent, a ketone-based solvent, and an ether-based solvent, and a content of the organic solvent in the PHA aqueous suspension liquid is 0.01% to 1% by weight in 100% by weight of a total amount of the PHA aqueous suspension liquid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoates and a method for reducing protein in aqueous suspensions of polyhydroxyalkanoates. [Background technology]

[0002] Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHAs") are known to be biodegradable, and in recent years, their use has been promoted from the viewpoint of environmental considerations.

[0003] One of the advantages of PHA is that it can be produced by microorganisms using renewable plant materials. To utilize PHA produced by microorganisms, first, the PHA-containing microorganism cells are disrupted or biological components other than PHA are solubilized, and the PHA in the cells is dispersed in water to obtain an aqueous PHA suspension. Then, impurities other than PHA (mainly proteins derived from the microorganisms) are further removed from this PHA aqueous suspension to purify the PHA.

[0004] As such techniques relating to the purification of PHA, for example, the techniques described in Patent Documents 1 to 4 are known. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2023 / 120193 [Patent Document 2] Patent Publication No. 2023-86317 [Patent Document 3] Special table number 2016-524926 [Patent Document 4] Chinese Patent No. 111500650 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the above-mentioned conventional techniques have room for improvement in terms of the efficiency of removing impurities (particularly proteins derived from bacterial cells) from aqueous PHA suspensions.

[0007] In view of the above circumstances, an object of the present invention is to provide a method for producing PHA, which can more efficiently reduce the protein content of a PHA aqueous suspension, and a method for reducing the protein content in a PHA aqueous suspension. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors have discovered the novel finding that proteins in a PHA aqueous suspension can be efficiently reduced (removed) by adding a predetermined amount of organic solvent to the PHA aqueous suspension and subjecting the suspension to dead-end filtration, and have completed the present invention.

[0009] That is, one aspect of the present invention includes the following configuration. [1] A method for producing a polyhydroxyalkanoate, comprising a filtration step of subjecting a polyhydroxyalkanoate aqueous suspension to dead-end filtration, wherein the polyhydroxyalkanoate aqueous suspension contains at least one organic solvent selected from the group consisting of alcohol solvents, ketone solvents, and ether solvents, and the content of the organic solvent in the polyhydroxyalkanoate aqueous suspension is 0.01 to 1 wt % relative to the total amount of the polyhydroxyalkanoate aqueous suspension (100 wt %). [2] The method for producing a polyhydroxyalkanoate according to [1], wherein the polyhydroxyalkanoate aqueous suspension contains at least one organic solvent selected from the group consisting of ethanol, isopropyl alcohol, methyl ethyl ketone, and acetone. [3] The method for producing a polyhydroxyalkanoate according to [1] or [2], wherein the protein content of the aqueous suspension of polyhydroxyalkanoate is 6,000 to 30,000 ppm. [4] The method for producing a polyhydroxyalkanoate according to any one of [1] to [3], which comprises a centrifugation step of centrifuging the unpurified aqueous suspension of polyhydroxyalkanoate prior to the filtration step. [5] The method for producing a polyhydroxyalkanoate according to [4], further comprising a mixing step of mixing the organic solvent with the aqueous suspension of polyhydroxyalkanoate obtained in the centrifugation step after the centrifugation step and before the filtration step. [6] The method for producing a polyhydroxyalkanoate according to any one of [1] to [5], wherein the aqueous suspension of polyhydroxyalkanoate has a shear viscosity of 4 to 15 mPa·s at 40°C and 10 1 / s. [7] In the filtration step, the air permeability is 0.25 cc / cm 2 The method for producing a polyhydroxyalkanoate according to any one of [1] to [6], wherein dead-end filtration is carried out using a filter medium having a flow rate of 1 / sec or less. [8] A method for reducing protein in an aqueous suspension of polyhydroxyalkanoate, comprising: a mixing step of mixing an organic solvent with an aqueous suspension of polyhydroxyalkanoate containing protein; and a protein reduction step of reducing protein in the aqueous suspension of polyhydroxyalkanoate, wherein the organic solvent comprises at least one solvent selected from the group consisting of alcohol solvents, ketone solvents, and ether solvents, and in the mixing step, the organic solvent is mixed so that the content of the organic solvent in the aqueous suspension of polyhydroxyalkanoate after mixing is 0.01 to 1 wt % relative to the total amount (100 wt %) of the aqueous suspension of polyhydroxyalkanoate after mixing. [Effects of the Invention]

[0010] According to one aspect of the present invention, it is possible to provide a method for producing PHA and a method for reducing protein in an aqueous PHA suspension, which can more efficiently reduce the protein content in the aqueous PHA suspension. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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)."

[0012] 1. PHA manufacturing method A method for producing a PHA according to one embodiment of the present invention (hereinafter, "a method for producing a PHA according to one embodiment of the present invention" may be referred to as "this production method") comprises a filtration step of dead-end filtration of an aqueous PHA suspension, wherein the aqueous PHA suspension contains at least one organic solvent selected from the group consisting of alcohol solvents, ketone solvents, and ether solvents, and the content of the organic solvent in the aqueous PHA suspension is 0.01 to 1 wt % relative to the total amount of the aqueous PHA suspension (100 wt %).

[0013] <Technical Concept of the Present Invention> Impurities that may be contained in PHA produced by microorganisms can cause quality degradation in products made from the PHA, such as contamination with foreign matter, deterioration in color, etc. For this reason, when using PHA produced by microorganisms in products, it is first necessary to remove impurities (particularly proteins) contained in the PHA aqueous suspension containing the PHA, and purify the PHA.

[0014] From the viewpoint of providing PHA products of higher quality, it is desirable to remove as many impurities as possible during the PHA purification process. On the other hand, from the viewpoint of PHA production efficiency, it is desirable to purify PHA in a shorter time (with fewer operations). From these two viewpoints, there is a demand for a technology that can remove as many impurities as possible in a shorter time, i.e., a technology that can more efficiently remove impurities (particularly proteins) from aqueous PHA suspensions.

[0015] From the above viewpoint, the present inventors have conducted extensive research to provide a technology for more efficiently removing impurities (particularly proteins) from aqueous PHA suspensions. As a result, they have discovered that when purifying PHA by filtration, by adding a small amount of organic solvent to the aqueous PHA suspension to be filtered, the amount of impurities removed per filtration operation can be increased, making it possible to more efficiently remove impurities from aqueous PHA suspensions, and have thus completed the present invention.

[0016] This production method can also be said to be a method for purifying PHA (PHA purification method) that can more efficiently reduce the protein content of a PHA aqueous suspension.

[0017] Each step included in this manufacturing method will be described in detail below.

[0018] <Filtration process> The present production method includes a filtration step of subjecting the PHA aqueous suspension to dead-end filtration. In this specification, the term "dead-end filtration" refers to "filtration by the dead-end filtration method."

[0019] (filtration operation) The specific mode of the dead-end filtration operation in the filtration step is not particularly limited, and examples thereof include suction filtration, pressure filtration, centrifugal filtration, and gravity filtration.

[0020] The material of the filter medium used in the filtration step is not particularly limited and can be selected from various materials, such as paper, filter cloth (woven or nonwoven), screen, sintered plate, bisque, polymer membrane, punched metal, wedge wire, etc. From the viewpoints of cost and ease of cleaning, filter cloth is preferably used.

[0021] The air permeability of the filter medium used in the filtration step is not particularly limited, but is preferably 0.25 cc / cm 2 / sec or less, and 0.20cc / cm 2 / sec or less. 2 By carrying out filtration using a filter medium with an air permeability of 0.25 cc / cm or less, leakage of PHA into the filtrate can be suppressed, and the yield of PHA can be improved. 2 From the above viewpoint, the lower the air permeability of the filter medium used in the filtration step, the more preferable it is. The lower limit of the air permeability is not particularly limited, but for example, it is 0.01 cc / cm 2 / sec or more.

[0022] In this specification, the air permeability of a filter medium is the air permeability of a unit area (cm ) of the filter medium per second. 2 The air permeability of a filter medium can be measured by the method described in the Examples.

[0023] The filtrate permeation rate in the filtration step is not particularly limited, but is preferably 100 L / m 2 / hr or more is preferable, and 150 L / m 2 / hr or more is more preferable, and 200 L / m 2 / hr or more, and 250L / m 2 The higher the filtrate permeation rate in the filtration step, the faster the filtration rate in the filtration step. In particular, the higher the filtrate permeation rate in the filtration step, the faster the filtration rate in the filtration step. 2 / hr or more, it can be evaluated that a filtration rate sufficient for practical use has been achieved in the filtration step. The filtrate permeation rate in the filtration step can be measured by the method described in the Examples.

[0024] The filtration step may include two or more filtration operations. By performing multiple filtration operations, more impurities can be removed, making it possible to provide a PHA with high purity. Therefore, from the viewpoint of removing more impurities, the more the number of filtration operations performed in the filtration step, the more preferable, preferably one or more, more preferably two or more, and even more preferably three or more. On the other hand, since wastewater is generated during the filtration operation, from the viewpoint of reducing the amount of wastewater generated throughout the present production method and thereby reducing the environmental burden, the fewer the number of filtration operations performed in the filtration step, the more preferable. For example, it is preferably seven or less, more preferably six or less, more preferably five or less, more preferably four or less, more preferably three or less, even more preferably two or less, and particularly preferably one. From the viewpoint of achieving both impurity removal and reduction in the amount of wastewater, the number of filtration operations performed in the filtration step is preferably one to three.

[0025] (PHA aqueous suspension) Next, the PHA aqueous suspension to be subjected to the filtration step will be described. In this specification, the PHA aqueous suspension refers to a solution in which PHA is suspended (dispersed) in water (aqueous medium), and has fluid properties.

[0026] PHA "PHA" is a general term for polymers containing hydroxyalkanoate as a monomer unit (monomer repeat unit) and is generally biodegradable. In particular, "PHA" herein refers to a (co)polymer containing hydroxyalkanoate repeat units at 50 mol% or more of the total monomer repeat units (100 mol%), and a resin composed of such a (co)polymer. Specific examples of hydroxyalkanoate repeat units constituting PHA include 3-hydroxybutanoic acid unit, 4-hydroxybutanoic acid unit, 3-hydroxypropionic acid unit, 3-hydroxypentanoic acid unit, 3-hydroxyhexanoic acid unit, 3-hydroxyheptanoic acid unit, 3-hydroxyoctanoic acid unit, and 2-hydroxypropionic acid unit. In this specification, the term "(co)polymer" encompasses both a homopolymer composed of only one type of monomer and a copolymer composed of two or more types of monomers.

[0027] Examples of PHAs provided by this production method include poly(3-hydroxyalkanoate) (hereinafter sometimes referred to as "P3HA"), poly(4-hydroxyalkanoate), etc. Among these, P3HA is preferred because it is suitable for use in molded articles.

[0028] P3HA is a 3-hydroxyalkanoate repeating unit of the formula: [—CHR—CH—CO—O—] (wherein R is C n H 2n+1 and n is an integer of 1 or more and 15 or less.) as an essential repeating unit.

[0029] Specific examples of P3HA include poly(3-hydroxybutyrate) (hereinafter, sometimes referred to as "P3HB"), which is a homopolymer of 3HB, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter, sometimes referred to as "P3HB3HH"), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (hereinafter, sometimes referred to as "P3HB4HB"), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxy Examples of suitable polyhydroxybutyrates include poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate), poly(3-hydroxybutyrate-co-3-hydroxydecanoate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-2-hydroxypropionate), and poly(3-hydroxybutyrate-co-3-hydroxypropionate). Among these, P3HB, P3HB3HH, and P3HB4HB are preferred, with P3HB3HH and P3HB4HB being more preferred, due to the ease of industrial production using microorganisms.

[0030] In this specification, "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. As described above, the name of a P3HA is determined by the repeating units contained in the P3HA. However, a very small amount (about 1 mol % or less) of a monomer contained in a P3HA may not be reflected in the name of the P3HA, provided that such a monomer does not significantly affect the physical properties of the P3HA. In other words, a P3HA may contain, in addition to the repeating units corresponding to its name, very small amounts of other repeating units.

[0031] When P3HA contains 3HB repeating units, from the viewpoint of the balance between flexibility and strength, the composition ratio of 3HB repeating units to repeating units other than 3HB repeating units (other repeating units) in the total monomer repeating units (100 mol%) in the P3HA (3HB repeating units / other repeating units) 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, it is advantageous in that a resin product with superior rigidity can be provided. On the other hand, when the composition ratio of 3HB repeating units in P3HA is 99 mol% or less, it is advantageous in that a resin product with superior flexibility can be provided. The monomer composition ratio of P3HA can be measured by gas chromatography or the like (see, for example, WO 2014 / 020838).

[0032] P3HA can be produced by microorganisms. Examples of microorganisms capable of producing P3HA include Bacillus megaterium, a P3HB-producing bacterium discovered in 1925, as well as other naturally occurring microorganisms such as Cupriavidus necator (formerly Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. These microorganisms accumulate P3HB within their cells.

[0033] Known microorganisms that produce P3HA, a copolymer of 3HB and other hydroxyalkanoic acids, include Aeromonas caviae, which produces P3HB3HH, and Alcaligenes eutrophus, which produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate). To increase P3HB3HH productivity, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)) is particularly preferred. In addition to the above, genetically modified microorganisms containing various P3HA synthesis-related genes can also be used depending on the desired physical properties of P3HA.

[0034] Organic solvents The PHA aqueous suspension to be subjected to the filtration step contains an organic solvent in addition to PHA and water. By containing a predetermined amount of organic solvent in the PHA aqueous suspension to be subjected to the filtration step, it becomes possible to efficiently remove impurities.

[0035] The organic solvent contained in the PHA aqueous suspension subjected to the filtration step (hereinafter, sometimes referred to as "the organic solvent related to the present production method") contains at least one selected from the group consisting of alcohol solvents, ketone solvents, and ether solvents. The organic solvent related to the present production method may consist of only one of the above-mentioned solvents, or may be a mixed solvent containing two or more of the above-mentioned solvents, or one or more of the above-mentioned solvents and other organic solvents.

[0036] Specific examples of alcohol solvents include methanol, ethanol, isopropyl alcohol, propanol, 1-butanol, 2-butanol, isobutanol, pentanol, and hexanol.

[0037] Specific examples of the ketone solvent include acetone and methyl ethyl ketone.

[0038] Specific examples of the ether solvent include dioxane, diethyl ether, and tetrahydrofuran.

[0039] Among the various solvents mentioned above, ethanol, isopropyl alcohol, methyl ethyl ketone, and acetone are easily soluble in water and can effectively improve the impurity removal efficiency due to the presence of the organic solvent. Therefore, the organic solvent used in the present production method preferably includes at least one selected from the group consisting of ethanol, isopropyl alcohol, methyl ethyl ketone, and acetone.

[0040] The organic solvent used in this production method may include organic solvents other than alcohol solvents, ketone solvents, and ether solvents (other organic solvents), such as nitriles such as acetonitrile and propionitrile, amides such as dimethylformamide and acetamide, dimethyl sulfoxide, pyridine, and piperidine.

[0041] The content of other organic solvents in the organic solvent used in the present production method is not particularly limited, but is preferably 10% by weight or less, and more preferably 5% by weight or less, of the total amount of organic solvents used in the present production method (total amount of each organic solvent) 100% by weight. In other words, the organic solvent used in the present production method preferably contains alcohol solvents, ketone solvents, and ether solvents in a total amount of 90% by weight or more, and more preferably 95% by weight or more, of the total amount of organic solvents used in the present production method (total amount of each organic solvent) 100% by weight.

[0042] The content of the organic solvent according to the present production method in the PHA aqueous suspension subjected to the filtration step is 0.01 to 1 wt % relative to the total amount of the PHA aqueous suspension (total amount of water, PHA, organic solvent, and other components) (100 wt %). By setting the content of the organic solvent according to the present production method to 0.01 wt % or more, the effect of improving impurity removal efficiency due to the presence of the organic solvent can be exerted, and by setting the content to 1 wt % or less, high safety can be ensured. The content of the organic solvent according to the present production method is not particularly limited as long as it is within the above range, but from the viewpoint of improving impurity removal efficiency, it is preferably 0.03 wt % or more, more preferably 0.05 wt % or more, and even more preferably 0.07 wt % or more. On the other hand, from the viewpoint of improving safety, it is preferably 0.8 wt % or less, more preferably 0.5 wt % or less, and even more preferably 0.3 wt % or less.

[0043] Other ingredients The PHA aqueous suspension to be subjected to the filtration step may contain, in addition to the above-mentioned components (water, PHA, and organic solvent), components derived from the PHA-producing microorganism (e.g., cell walls, proteins, etc.) and / or other compounds generated during purification, etc. In other words, the PHA aqueous suspension to be subjected to the filtration step may contain these components in addition to PHA and water.

[0044] ·Physical properties of PHA aqueous suspension The protein content of the PHA aqueous suspension subjected to the filtration step is not particularly limited, but is preferably 6,000 to 30,000 ppm. If the protein content of the PHA aqueous suspension subjected to the filtration step is within the above range, it is possible to improve the filtration rate in the filtration step. From the viewpoint of improving the filtration rate in the filtration step, the lower the protein content of the PHA aqueous suspension subjected to the filtration step, the better. Specifically, the protein content of the PHA aqueous suspension subjected to the filtration step is preferably 25,000 ppm or less, more preferably 15,000 ppm or less, and even more preferably 10,000 ppm or less.

[0045] The pH of the aqueous PHA suspension subjected to the filtration step is not particularly limited, but is preferably 3.0 to 13.0. Furthermore, within the above range, from the viewpoint of further improving the filtration rate in the filtration step, the pH of the aqueous PHA suspension subjected to the filtration step is preferably 3.0 to 5.5. On the other hand, from the viewpoint of improving the efficiency of impurity removal in the filtration step, the pH of the aqueous PHA suspension subjected to the filtration step is preferably greater than 5.5 and 12.5 or less, more preferably 6.2 or more and 12.0 or less, and even more preferably 7.7 or more and 11.0 or less.

[0046] The shear viscosity of the PHA aqueous suspension to be subjected to the filtration step is not particularly limited, but from the viewpoint of improving the filtration rate in the subsequent filtration step, it is preferably 4 to 15 mPa s, and more preferably 5 to 10 mPa s, at a shear rate of 10 1 / s at 40° C. The shear viscosity of the PHA aqueous suspension is measured by the method described in the Examples.

[0047] The solids concentration (i.e., PHA concentration) of the PHA aqueous suspension to be subjected to the filtration step is not particularly limited, but from the viewpoint of improving the fluidity of the PHA aqueous suspension, it is preferably 5 to 30% by weight, and more preferably 10 to 20% by weight.

[0048] (Method of manufacturing PHA aqueous suspension) ·PHA aqueous suspension preparation process The PHA aqueous suspension to be subjected to the filtration step is preferably a PHA aqueous suspension derived from a culture broth of a PHA-producing microorganism. Such a PHA aqueous suspension derived from a culture broth of a PHA-producing microorganism can be prepared, for example, by the following method: (1) culturing a microorganism capable of producing PHA; (2) inactivating the culture broth of the microorganism by heating to obtain an inactivated culture broth; (3) treating the inactivated culture broth with hydrogen peroxide to reduce the viscosity of the culture broth; (4) treating the hydrogen peroxide-treated inactivated culture broth with alkali; (5) adding a lytic enzyme (a cell wall-degrading enzyme) to the alkali-treated inactivated culture broth to lyse the microorganism cells and disperse the intracellular substances, including PHA, in the culture broth; (6) adding a protease to the culture broth to decompose substances derived from the microorganism cells other than PHA (particularly proteins); and (7) further adjusting the pH of the culture broth and adding a surfactant to decompose substances derived from the microorganism cells other than PHA (particularly cell membranes).

[0049] The present production method may include, prior to the filtration step, a step of culturing a PHA-producing microorganism and preparing a PHA aqueous suspension from the culture solution, which includes one or more of the above-mentioned operations (a PHA aqueous suspension preparation step). Furthermore, when the present production method includes two or more of the above-mentioned operations, the order in which the operations are performed is not limited to the above-mentioned order.

[0050] Centrifugation process The aqueous PHA suspension obtained in the above-mentioned PHA aqueous suspension preparation step is an unpurified aqueous PHA suspension. Unpurified aqueous PHA suspensions typically contain a large amount of impurities, typically 60,000 ppm or more. Therefore, in order to perform more efficient filtration, the present production method preferably centrifugally controls the amount of impurities in the aqueous polyhydroxyalkanoate suspension prior to the filtration step, thereby adjusting the amount of impurities to a level suitable for the filtration step (preferably 6,000 to 30,000 ppm). That is, the present production method preferably includes a centrifugation step in which the aqueous PHA suspension is centrifuged prior to the filtration step.

[0051] As used herein, the term "unpurified PHA aqueous suspension" refers to a PHA aqueous suspension produced by using a culture medium of a PHA-producing microorganism or an aqueous solution containing a PHA-producing microorganism as a raw material, disrupting the PHA-containing microorganism cells in the raw material and / or solubilizing biological components other than PHA, and dispersing the PHA in the microorganism cells in water, preferably by disrupting the PHA-containing microorganism cells and solubilizing biological components other than PHA, and which has not undergone any purification treatment, specifically, centrifugation or filtration. The method for disrupting the PHA-containing microorganism cells and / or solubilizing biological components other than PHA is not particularly limited, and known methods can be used, such as methods using enzymes such as lysozyme and / or alcalase and other protease enzymes.

[0052] In addition, when the purification history of a certain aqueous PHA suspension is unknown, whether the aqueous suspension is unpurified or not can be determined, for example, based on the amount of impurities, particularly the amount of protein, in the aqueous suspension. Specifically, in this specification, if the protein content in a certain aqueous PHA suspension is 60,000 ppm or more, the aqueous suspension is considered to be an unpurified PHA aqueous suspension. In addition, a PHA aqueous suspension that has undergone some processing but still has a protein content of 60,000 ppm or more is also considered to be essentially an "unpurified PHA aqueous suspension." Needless to say, even if the protein content of a PHA aqueous suspension is less than 60,000 ppm, the aqueous suspension is also considered to be an unpurified PHA aqueous suspension if the aqueous suspension is unpurified. The protein content of a PHA aqueous suspension is measured by the method described in the Examples.

[0053] The centrifugation operation performed in the centrifugation step is not particularly limited as long as it can remove impurities (particularly proteins) from the crude PHA aqueous suspension, and can be performed by any centrifugation method known in the technical field of the present invention.

[0054] Such known centrifugation methods include, for example, centrifugation using a centrifugal settler or a centrifugal dehydrator.

[0055] Examples of centrifugal settlers that can be used in the centrifugation step include separation plate type (e.g., disk type, self-cleaning type, nozzle type, screw decanter type, skimming type, etc.) and cylindrical type centrifugal settlers. Furthermore, both palindrome type and continuous type centrifugal settlers can be used. Similarly, either palindrome type or continuous type centrifugal dehydrators can be used.

[0056] The number of centrifugation steps in the centrifugation step is not particularly limited, but from the viewpoint of reducing the amount of wastewater generated by the present production method and reducing the environmental load, it is preferably two or less times, and may be one time. In this specification, one centrifugation step refers to a series of operations in which a target liquid (e.g., an unpurified PHA aqueous suspension) is centrifuged under any conditions, followed by removal of a portion of the supernatant (e.g., an amount equivalent to 40% by volume or more of the liquid subjected to centrifugation) or all of the supernatant. When two or more centrifugations are performed, the liquid to be centrifuged may be a concentrated liquid (concentrated PHA aqueous suspension) obtained by removing the supernatant from the above-mentioned centrifugation step, or may be a liquid obtained by adding an aqueous solvent to the concentrated liquid and adjusting the concentration to any desired level.

[0057] The centrifugation conditions (rotation speed, rotation time) in the centrifugation step are not particularly limited as long as an aqueous PHA suspension with a desired protein content can be obtained with the desired number of centrifugations, but for example, the rotation speed can be 3000 to 6000 rpm and the rotation time can be 1 to 30 minutes per centrifugation.

[0058] ·pH adjustment process In one embodiment of the present production method, if the pH of the aqueous suspension to be subjected to the filtration step is not within the desired pH range (e.g., a range of 3.0 to 11.0), the pH of the PHA aqueous suspension may be adjusted to the desired pH range (e.g., a range of 3.0 to 11.0) before the filtration step. That is, the present production method may include a pH adjustment step of adjusting the pH of the PHA aqueous suspension to be subjected to the filtration step. ·Mixing process In one embodiment of the present production method, the method may include a mixing step of mixing an organic solvent with the PHA aqueous suspension. In the mixing step, the organic solvent is mixed so that the content of the organic solvent in the PHA aqueous suspension after mixing is 0.01 to 1 wt % relative to 100 wt % of the total amount of the PHA aqueous suspension after mixing (i.e., the total amount of the PHA aqueous suspension before mixing and the organic solvent to be mixed).

[0059] In the mixing step, the method for mixing the organic solvent into the PHA aqueous suspension is not particularly limited as long as it allows the organic solvent to be mixed uniformly into the PHA aqueous suspension, and known devices or methods can be used.

[0060] The timing of performing the mixing step is not particularly limited as long as it is performed before the filtration step, and for example, it may be performed before or after the centrifugation step, but it is preferable to perform it after the centrifugation step because the effect of improving the impurity removal efficiency due to the presence of the organic solvent can be achieved with a smaller amount of organic solvent used. In other words, the mixing step is preferably performed after the centrifugation step and before the filtration step.

[0061] (Drainage volume) Typically, in the production of PHA, multiple purification (impurity removal) procedures are performed to obtain PHA from which impurities have been sufficiently removed, and wastewater is generated for each purification procedure. Compared to conventional PHA production methods, the present production method can remove impurities (proteins) more efficiently, i.e., it is possible to provide PHA from which impurities have been sufficiently removed with fewer purification procedures (filtration procedures in the present production method). As a result, the present production method can also reduce the amount of wastewater generated in the production process compared to conventional methods.

[0062] The amount of wastewater generated in this production method is not particularly limited, but from the viewpoint of reducing the amount of wastewater generated throughout the entire production process and thereby reducing the environmental impact, it is preferably 12.00 (kg / kg-resin) or less, more preferably 11.00 (kg / kg-resin) or less, and even more preferably 10.50 (kg / kg-resin) or less. Note that the unit of wastewater amount, "kg / kg-resin," means the amount of wastewater (kg) generated to obtain 1 kg of resin (PHA in this production method).

[0063] (filter cake) In this production method, the PHA aqueous suspension is filtered in the filtration step to obtain a filter cake as a residue. The filter cake is then subjected to a purification step including the filtration step to become a PHA aggregate from which impurities have been sufficiently removed.

[0064] The amount of impurities in the filter cake obtained by this manufacturing method can be evaluated based on the protein content of the filter cake.Specifically, the lower the protein content of the filter cake obtained through the filtration process, the more impurities are removed in the filtration process.Specifically, the protein content of the filter cake obtained through the filtration process is preferably 5,500 ppm or less, more preferably 5,000 ppm or less, more preferably 4,000 ppm or less, even more preferably 3,000 ppm or less, and even more preferably 4,000 ppm or less.

[0065] The filter cake obtained by the filtration step can be dried by a known method to obtain PHA particles or PHA powder with a sufficiently reduced content of impurities. Such PHA particles or PHA powder can be used as a molded article by a known molding method, such as injection molding, extrusion molding, blow molding, or compression molding. They can also be used as a foamed molded article by foaming using a known method and then molding. These molded articles and foamed molded articles made from PHA particles or PHA powder can be used for various purposes, such as paper, film, sheet, tube, plate, rod, container (e.g., bottle container), bag, parts, etc.

[0066] 2. Methods for reducing protein In another aspect of the present invention, there is provided a method for reducing protein in a PHA aqueous suspension, the method comprising: a mixing step of mixing an organic solvent with a protein-containing PHA aqueous suspension; and a protein reduction step of reducing protein in the PHA aqueous suspension, wherein the organic solvent comprises at least one selected from the group consisting of alcohol solvents, ketone solvents, and ether solvents, and the mixing step involves mixing the organic solvent so that the content of the organic solvent in the PHA aqueous suspension after mixing is 0.01 to 1 wt % relative to the total weight of the PHA aqueous suspension after mixing (100 wt %). Hereinafter, the "method for reducing protein in a PHA aqueous suspension according to one embodiment of the present invention" may be referred to as the "present reduction method."

[0067] In this reduction method, the proteins to be reduced from the PHA aqueous suspension are basically intended to be derived from bacterial cells, but are not particularly limited as long as they are proteins.

[0068] (Mixing process) This reduction method includes a mixing step of mixing an organic solvent with a protein-containing PHA aqueous suspension. The definitions and specific aspects of the PHA aqueous suspension and organic solvent used in the mixing step of this reduction method are as described in the above section [1. PHA production method].

[0069] In the mixing step, the organic solvent is mixed so that the content of the organic solvent in the PHA aqueous suspension after mixing is 0.01 to 1 wt % relative to 100 wt % of the total amount of the PHA aqueous suspension after mixing (i.e., the total amount of the PHA aqueous suspension before mixing and the mixed organic solvent).

[0070] In the mixing step, the method for mixing the organic solvent into the PHA aqueous suspension is not particularly limited as long as it allows the organic solvent to be mixed uniformly into the PHA aqueous suspension, and known devices or methods can be used.

[0071] (Protein reduction process) The present reduction method includes a protein reduction step of reducing proteins in the PHA aqueous suspension mixed with an organic solvent in the mixing step.

[0072] In the protein reduction step, the method for reducing the protein in the PHA aqueous suspension is not particularly limited, but a method of filtering the PHA aqueous suspension can be preferably applied. When a method of filtering the PHA aqueous suspension is applied as a method for reducing the protein in the PHA aqueous suspension, the description of the filtering step in the above section [1. PHA production method] applies appropriately to specific aspects of the method of filtering the PHA aqueous suspension.

[0073] In the protein reduction step, the operation of reducing the protein in the PHA aqueous suspension may be carried out only once or may be carried out multiple times. For example, when a method of filtering the PHA aqueous suspension is applied as a method of reducing the protein, the filtration operation may be carried out only once or may be carried out two or more times in the reduction step. Furthermore, one or more filtration operations may be combined with other operations.

[0074] In the protein reduction step, the amount of protein reduced from the PHA aqueous suspension is not particularly limited, but it is preferable that the protein content in the reduced PHA aqueous suspension (or filter cake) is reduced by 2,000 ppm or more compared to the PHA aqueous suspension before reduction. The greater the amount of protein reduced in the protein reduction step, the more preferable it is, more preferably 3,000 ppm or more, even more preferably 4,000 ppm or more, and even more preferably 5,000 ppm or more. Note that when the protein reduction step includes multiple reduction operations, the amount of protein reduced in the protein reduction step is the total amount of protein reduced by such multiple reduction operations.

[0075] According to this reduction method, the protein content in a protein-containing PHA aqueous suspension can be efficiently reduced, and therefore, this reduction method can be suitably used as one step in the production of PHA. [Example]

[0076] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0077] [Measurement method] Measurements in the examples and comparative examples were carried out by the following methods.

[0078] (pH of PHA aqueous suspension) The pH of the PHA aqueous suspension was measured using a pH meter (9652-10D, manufactured by HORIBA).

[0079] (Shear viscosity of PHA aqueous suspension) The shear viscosity of the PHA aqueous suspension was measured by the following method. Specifically, the shear viscosity was measured using a coaxial double cylinder with an Anton Paar MCR302. The PHA aqueous suspension was poured into a 20 mL cylinder, and the liquid temperature was adjusted to 40°C or 50°C. After reaching the target shear rate (10 1 / s), the viscosity was measured when the change in torque with time became less than 1%.

[0080] (Protein content of PHA aqueous suspension) The protein content of the PHA aqueous suspension was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, an amount of PHA aqueous suspension equivalent to 2 mg of PHA in solid content was placed in a 15 mL Falcon tube, 2 mL of the kit's reagent was added, and the suspension was shaken at 60°C for 30 minutes. 30 minutes after the end of shaking, the suspension was cooled to 25°C, and the absorbance at a wavelength of 562 nm was measured. The protein content of the PHA aqueous suspension was calculated based on the measured absorbance.

[0081] (Measurement of filter media air permeability) The air permeability of the filter material (filter cloth) used in the filtration process was measured using a TEXTEST INSTRUMENTS FX3345 Flex Air.

[0082] (Filtrate permeation rate during filtration process) Filtration was carried out under the conditions described in the filtration step of each Example and Comparative Example, and the amount of filtrate was measured for 1 minute from the start of the filtration. The filtrate permeation rate (LMH) was calculated based on the following formula: Filtrate permeation rate (LMH) = Filtrate volume (L) / Filtration area (m 2 ) / filtration time (h).

[0083] (Protein content of filter cake) The protein content of the filter cake was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, the filter cake was dried to obtain PHA dried particles, and 2 mg of the dried particles was placed in a 15 mL Falcon tube, 2 mL of the kit's reagent was added, and the tube was shaken at 60 ° C for 30 minutes. 30 minutes after the end of shaking, the tube was cooled to 25 ° C, and the absorbance at a wavelength of 562 nm was measured. The protein content of the filter cake was calculated based on the measured absorbance.

[0084] Example 1 (Preparation of PHA aqueous suspension) Preparation of bacterial culture medium Ralstonia eutropha, as described in International Publication No. WO 2019 / 142717, was cultured using the method described in paragraphs

[0041] to

[0048] 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 (3HB unit / 3HH unit composition ratio) was 99 / 1 to 92 / 8 (mol / mol).

[0085] Inactivation treatment The bacterial cell culture solution obtained above was sterilized by heating and stirring at an internal temperature of 60 to 70°C for 7 hours to obtain an inactivated culture solution. The weight-average molecular weight of PHA in the obtained inactivated culture solution was 1.8 million. The solids concentration of the inactivated culture solution was 30% by weight.

[0086] Hydrogen peroxide treatment Hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the inactivated culture medium obtained above to a concentration of 0.66%, and the inactivated culture medium was treated with hydrogen peroxide. After treatment, the shear viscosity of the inactivated culture medium at a temperature of 50°C and a shear rate of 10 1 / s was 5.01 mPa s.

[0087] Alkali treatment To the inactivated culture solution with reduced viscosity obtained above, a 30% aqueous solution of sodium hydroxide was added to adjust the pH to 11.0. While maintaining the solution at 60°C, the pH was maintained at 11.0 for 1.5 hours by continuing to add the 30% aqueous solution of sodium hydroxide, thereby obtaining an aqueous PHA suspension.

[0088] Neutralization and enzyme treatment The pH of the resulting PHA aqueous suspension was adjusted to 7.0±0.2 by adding 95% sulfuric acid. The solids concentration of this PHA aqueous suspension was measured and found to be 30% by weight. After adding sulfuric acid, lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), an enzyme that degrades sugar chains (peptidoglycans) in cell walls, was added to a liquid 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 protease, was added to a liquid concentration of 300 ppm, and then 30% sodium hydroxide was added at 50°C to adjust the pH to 8.5, and the mixture was maintained for 2 hours.

[0089] Surfactant addition treatment Sodium dodecyl sulfate (SDS, manufactured by Kao) was added to the resulting enzyme-treated PHA aqueous suspension to a concentration of 0.6-1.0 wt%. The pH was then adjusted to 11.0±0.2 using aqueous sodium hydroxide. After holding the suspension at 40°C for 1 hour, the PHA aqueous suspension was diluted with aqueous sodium hydroxide to obtain a PHA aqueous suspension with a solids concentration of 15 wt% and a pH of 11 (unpurified PHA aqueous suspension). The shear viscosity of the resulting PHA aqueous suspension at a liquid temperature of 40°C and a shear rate of 10 1 / s was 8.58 mPa·s.

[0090] Centrifugation process The resulting PHA aqueous suspension was centrifuged (4500 rpm, 10 minutes), and then an amount of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed, yielding a 2x concentrated PHA aqueous suspension. To this PHA aqueous suspension, an amount of sodium hydroxide solution equal to the amount of the removed supernatant was added, and the suspension was centrifuged again (4500 rpm, 10 minutes), and an amount of supernatant equivalent to 50% by volume of the aqueous suspension centrifuged was removed. To the PHA aqueous suspension obtained after these two centrifugation operations, an additional amount of sodium hydroxide solution was added, yielding a PHA aqueous suspension with a pH of 11.0 and a solids concentration of 30 wt%. The protein content of the resulting PHA aqueous suspension is shown in Table 1.

[0091] (filtration process) The PHA aqueous suspension after the centrifugation was mixed with isopropanol, an organic solvent, to obtain a PHA aqueous suspension with an isopropanol concentration of 0.1% by weight (mixing step). 18.2 g of this PHA aqueous suspension was weighed out, and its pH was adjusted to 11.0 with an aqueous sodium hydroxide solution. The air permeability was 0.16 cc / cm. 2 Dead-end filtration was performed using a filter cloth of 1 / sec to obtain a filter cake. Specifically, the filter cloth was placed in a filter having an inner diameter of 40 mm, and attached to a suction bottle (2 L, manufactured by SHIBATA). After that, the PHA aqueous suspension was introduced into the filter while applying suction to -90 kPa with a vacuum pump, thereby performing suction filtration (first filtration).

[0092] The filter cake obtained in the first filtration was dispersed in an aqueous sodium hydroxide solution to obtain an aqueous suspension with a solids concentration of 30 wt% and a pH of 11.0. This aqueous suspension was then filtered again under the same conditions as the first filtration to obtain a filter cake that had undergone two filtration operations in total (second filtration). Table 1 shows the protein content of the filter cake obtained in each filtration operation and the amount of wastewater generated (amount per kg of resin (PHA)) until the filter cake was obtained.

[0093] [Table 1]

[0094] (Comparative Example 1) Except for not mixing an organic solvent with the PHA aqueous suspension after centrifugation in the filtration step, a filter cake was obtained after two filtration operations in total in the same manner as in Example 1. The protein content of the filter cake obtained in each filtration operation and the amount of wastewater generated until the filter cake was obtained (amount per kg of resin (PHA)) are shown in Table 2.

[0095] [Table 2]

[0096] 〔summary〕 A comparison of Example 1 and Comparative Example 2, in which PHA was produced by the same procedure except for the addition of an organic solvent, clearly shows that Example 1, in which an organic solvent was added to the PHA aqueous suspension, had a lower protein content, i.e., more efficient protein removal, in both the first and second filtrations. These results demonstrate that protein reduction (filtration) can be more efficiently achieved by adding an organic solvent to the PHA aqueous suspension. [Industrial Applicability]

[0097] The present production method can more efficiently reduce the protein content of a PHA aqueous suspension and is therefore suitable for use in the production of PHA.The PHA produced by the present production method can be suitably used, for example, as a molded product in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

Claims

1. a filtration step of subjecting the aqueous suspension of polyhydroxyalkanoate to dead-end filtration, The polyhydroxyalkanoate aqueous suspension contains at least one organic solvent selected from the group consisting of alcohol-based solvents, ketone-based solvents, and ether-based solvents, A method for producing a polyhydroxyalkanoate, wherein the content of the organic solvent in the aqueous suspension of polyhydroxyalkanoate is 0.01 to 1% by weight, based on 100% by weight of the total amount of the aqueous suspension of polyhydroxyalkanoate.

2. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the polyhydroxyalkanoate aqueous suspension contains, as the organic solvent, at least one selected from the group consisting of ethanol, isopropyl alcohol, methyl ethyl ketone, and acetone.

3. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the protein content of the aqueous suspension of polyhydroxyalkanoate is 6,000 to 30,000 ppm.

4. 2. The method for producing a polyhydroxyalkanoate according to claim 1, further comprising a centrifugation step of centrifuging the crude aqueous suspension of polyhydroxyalkanoate prior to the filtration step.

5. 5. The method for producing a polyhydroxyalkanoate according to claim 4, further comprising a mixing step of mixing the organic solvent with the aqueous suspension of polyhydroxyalkanoate obtained in the centrifugation step after the centrifugation step and before the filtration step.

6. 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein the aqueous suspension of polyhydroxyalkanoate has a shear viscosity at 40° C. and 10 1 / s of 4 to 15 mPa·s.

7. In the filtration step, the air permeability is 0.25 cc / cm ２ 2. The method for producing a polyhydroxyalkanoate according to claim 1, wherein dead-end filtration is carried out using a filter medium having a flow rate of 1 / sec or less.

8. a mixing step of mixing an organic solvent with an aqueous suspension of polyhydroxyalkanoate containing a protein; a protein reduction step of reducing proteins in the polyhydroxyalkanoate aqueous suspension, the organic solvent includes at least one selected from the group consisting of alcohol-based solvents, ketone-based solvents, and ether-based solvents; In the mixing step, the organic solvent is mixed so that the content of the organic solvent in the aqueous polyhydroxyalkanoate suspension after mixing is 0.01 to 1 wt % relative to the total amount (100 wt %) of the aqueous polyhydroxyalkanoate suspension after mixing.

Images