WO2010116681A1 - Procédé de récupération de polyhydroxyalcanoate - Google Patents

Procédé de récupération de polyhydroxyalcanoate Download PDF

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WO2010116681A1
WO2010116681A1 PCT/JP2010/002304 JP2010002304W WO2010116681A1 WO 2010116681 A1 WO2010116681 A1 WO 2010116681A1 JP 2010002304 W JP2010002304 W JP 2010002304W WO 2010116681 A1 WO2010116681 A1 WO 2010116681A1
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polyhydroxyalkanoate
pha
enzyme
surfactant
suspension
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PCT/JP2010/002304
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English (en)
Japanese (ja)
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柳田義文
山口貴生
滝田昌輝
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a method for recovering polyhydroxyalkanoate produced by a microorganism.
  • PHA Polyhydroxyalkanoate
  • PHA Polyhydroxyalkanoate
  • a thermoplastic polyester that is produced and stored as an energy storage substance in the cells of many microbial species.
  • PHA produced by microorganisms using natural organic acids and oils and fats as a carbon source is completely biodegraded by microorganisms in soil and water and taken into the natural carbon cycle process. Therefore, it can be said that it is an environmentally conscious plastic material that has almost no adverse effects on the ecosystem.
  • synthetic plastics have become serious social problems from the viewpoint of environmental pollution, waste disposal, and petroleum resources, and PHA has been attracting attention as an environmentally friendly green plastic, and its practical use is eagerly desired.
  • biocompatible plastics such as implant materials and drug carriers that do not require collection, and is expected to be put to practical use.
  • PHA produced by microorganisms is normally accumulated in microbial cells as granules, in order to use PHA as plastic, a process of separating and removing PHA from the microbial cells is required.
  • Known methods for separating and purifying PHA from microbial cells include a method for extracting PHA from microbial cells using an organic solvent in which PHA is soluble, and cell components other than PHA by chemical treatment or physical treatment. It is divided into a method of separating PHA by solubilizing or removing by crushing.
  • Non-patent Document 1 discloses that microbial cell components other than PHA can be treated by treating the microbial cell suspension with sodium hypochlorite. A method for solubilizing and separating PHA is described. In this method, since significant decomposition of PHA is caused simultaneously with solubilization of microbial cell constituents other than PHA, processing into a product is limited. Further, since chlorine that cannot be ignored remains in the PHA, it is not suitable for practical use because it is not preferable as a polymer product.
  • Patent Document 6 discloses a method for recovering PHA using a heat treatment at 100 ° C. or more and a digestion treatment using a proteolytic enzyme or a surfactant.
  • the suspension becomes very viscous due to the liberated nucleic acid. Therefore, the suspension is heated in advance at 100 ° C. or higher to decompose the nucleic acid.
  • heating at 100 ° C. or higher significantly reduces the molecular weight of PHA, making it impossible to apply to products.
  • this method is very complicated and requires many steps, the purity of the obtained PHA is about 88% and about 97% at the maximum.
  • the nucleic acid released from the bacterial cells is treated with hydrogen peroxide at 80 ° C. for 3 hours to decompose and isolate 99% purity PHA (see Patent Document 7) ) And a method of separating PHA after heating the cell suspension to 50 ° C. or higher under strong acidity of less than pH 2 (see Patent Document 8). Under these heat treatment conditions, the molecular weight of PHA is remarkably lowered, so that even if the purity is improved, it cannot be applied to products.
  • Non-Patent Document 2 describes that after adding alkali to a poly-3-hydroxybutyrate (PHB) -containing cell suspension, the pH is returned to neutral and then high-pressure crushing is performed. Although there is no description of the polymer purity or recovery rate in this document, it is expected that the bacterial cell constituents remain in the PHB fraction because of high-pressure crushing under neutral conditions, and the purity is not high.
  • PHB poly-3-hydroxybutyrate
  • Patent Document 9 discloses a method in which an alkali is added to bacterial cells, heated to 80 ° C. and stirred for 1 hour, and then the polymer is recovered by centrifugation.
  • Patent Document 10 discloses a method of high-pressure crushing at 70 ° C. .
  • patent document 11 the method of performing high pressure crushing at 70 degreeC or more after adding an alkali is disclosed. In these methods, since the treatment is performed at a high temperature, the molecular weight of PHA tends to be remarkably lowered depending on the conditions, and the polymer purity is as low as about 66 to 85%.
  • Patent Document 12 and Non-Patent Document 3 disclose a method of separating a polymer by crushing a microbial cell and then treating it with a combination of an enzyme and a surfactant. Therefore, it cannot be applied to an actual industrial process.
  • the object of the present invention is to solve the above-mentioned problems in the prior art, and from a PHA-containing microbial cell with a high purity from a PHA-containing microbial cell by a simple and inexpensive method suitable for industrial production and with a small number of steps without causing a significant decrease in molecular weight. It is to provide a PHA recovery method capable of separating PHA.
  • the present inventors have performed an enzyme treatment on an aqueous suspension of microbial cells containing PHA, and then, under basic conditions, in the presence of a surfactant.
  • a surfactant By performing physical crushing treatment at a relatively low temperature, the crushing effect is dramatically improved, and the recovered PHA is found to have a reduced molecular weight and high purity, thereby completing the present invention. It came to.
  • the first aspect of the present invention is a method for recovering polyhydroxyalkanoate produced by a microorganism from the inside of a microbial cell, comprising the step (a): an aqueous suspension of a polyhydroxyalkanoate-containing microbial cell, an enzyme, And a step of performing an enzyme treatment by adding an alkali and / or a surfactant to obtain an enzyme treatment solution, step (b): physical treatment in the presence of a surfactant under basic conditions with respect to the enzyme treatment solution.
  • step (c) Performing a disruptive treatment to disrupt the cells and solubilize or emulsify cellular substances other than the polyhydroxyalkanoate in the cells to obtain a polyhydroxyalkanoate suspension, step (c): And a step of separating the polyhydroxyalkanoate from the polyhydroxyalkanoate suspension.
  • the enzyme in step (a) is preferably a proteolytic enzyme and / or a cell wall degrading enzyme.
  • the physical crushing treatment is preferably performed with a high-pressure homogenizer.
  • the alkali used in step (a) and / or (b) is preferably at least one selected from the group consisting of sodium hydroxide, sodium carbonate, potassium hydroxide and lithium hydroxide.
  • step (c) the pH of the suspension when the polyhydroxyalkanoate is separated from the suspension is preferably adjusted to 8.0 to 13.0.
  • the surfactant used in the step (a) and / or (b) is at least one selected from the group consisting of an anionic surfactant, a cationic surfactant, an amphoteric surfactant and a nonionic surfactant.
  • the polyhydroxyalkanoate is 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxypropionate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxypentenoate Copolymerizing at least two monomers selected from the group consisting of 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate and 3-hydroxydocanoate A formed copolymer is preferable.
  • a second aspect of the present invention is a polyhydroxyalkanoate obtained by the polyhydroxyalkanoate recovery method, wherein a sheet having a thickness of 0.5 mm obtained from the polyhydroxyalkanoate by pressing at 160 ° C. is 15 Relates to polyhydroxyalkanoates having a yellowness index (YI value) of less than or equal to.
  • YI value yellowness index
  • a PHA recovery method capable of separating high-purity PHA from PHA-containing microbial cells by a simple and inexpensive method suitable for industrial production and with a small number of steps without causing a significant decrease in molecular weight.
  • the method for recovering a polyhydroxyalkanoate according to the present invention is a method for recovering a polyhydroxyalkanoate produced by a microorganism from the inside of a microbial cell, comprising the step (a): converting the polyhydroxyalkanoate-containing microbial cell into an aqueous suspension.
  • step (b) presence of a surfactant under basic conditions with respect to the enzyme-treated solution
  • step (c) Separating the polyhydroxyalkanoate from the polyhydroxyalkanoate suspension.
  • the enzyme treatment is performed in the presence of an alkali and / or a surfactant before the physical disruption treatment is performed on the PHA-containing microbial cells, and the subsequent physical disruption treatment is performed under a basic condition.
  • the agent By carrying out in the presence of the agent, cell disruption is achieved, and cells other than PHA are solubilized or emulsified to obtain an aqueous PHA suspension. PHA can be easily separated from the aqueous PHA suspension.
  • PHA is a general term for hydroxyalkanoate polymers.
  • the hydroxyalkanoate is not particularly limited, and examples thereof include 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxypropionate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5 -Hydroxyvalerate, 3-hydroxypentenoate, 3-hydroxyhexanoate (3HH), 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate, 3-hydroxydocanoate, etc. It is done.
  • the PHA in the present invention may be a homopolymer composed of only one of these hydroxyalkanoates, or may be a copolymer formed by copolymerizing two or more hydroxyalkanoates.
  • PHBV which is a homopolymer of 3HB
  • PHBV which is a binary copolymer of 3HB and 3HV
  • PHBH of 3HB and 3HH
  • Japanese Patent No. 2777757 3HB, 3HV and 3HH
  • PHBHV see Japanese Patent No. 277757
  • the copolymer tends to decrease in molecular weight due to hydrolysis due to heating or the like, the present invention having the advantage that the molecular weight hardly decreases as will be described later is significant in recovering the copolymer. .
  • a copolymer having 3HH as a monomer unit is preferable, and PHBH is more preferable from the viewpoint of degradability as a biodegradable polymer and soft properties.
  • the composition ratio of each monomer unit constituting PHBH is not particularly limited, but from the viewpoint of showing good processability, the content of 3HH units with respect to the total units is preferably 1 to 99 mol%, more preferably. 3 to 30 mol%.
  • the composition ratio of each monomer unit constituting PHBHV is not particularly limited.
  • the content of 3HB units is 1 to 95 mol% with respect to the total units, and the content of 3HV units is 1 to 96 mol%, 3HH units.
  • the content of is preferably 1 to 30 mol%.
  • the weight average molecular weight of PHA using polystyrene as a molecular weight standard is required to be 10,000 or more by gel chromatography. It is preferably 50,000 or more, more preferably 100,000 or more, further preferably 200,000 or more, particularly preferably 200,000 to 2,000,000, very preferably 200,000 to 1,500,000, most preferably 200,000 to 1,000,000. When the molecular weight exceeds 2 million, fluidity may be lowered when melted and processed, and handling may be poor.
  • the microorganism used in the present invention is not particularly limited as long as it is a microorganism that generates PHA in cells.
  • a microorganism isolated from nature, a microorganism deposited at a depositary of a strain (for example, IFO, ATCC, etc.), or a mutant or transformant that can be prepared from them can be used.
  • the genus Capriavidus, the genus Alcaligenes, the genus Ralstonia, the genus Pseudomonas, the genus Bacillus, the genus Azotobacter, the genus Nocardia Examples include bacteria.
  • strains such as A. lipolytica, A. latus, Aeromonas caviae, A.
  • hydrophila, C. necator, etc. is preferred.
  • a transformant obtained by introducing a target PHA synthase gene and / or a mutant thereof into the microorganism can also be used.
  • the PHA synthase gene used for the preparation of such a transformant is not particularly limited, but a PHA synthase gene derived from Aeromonas caviae is preferred. By culturing these microorganisms under appropriate conditions, microbial cells in which PHA is accumulated in the cells can be obtained.
  • the culture method is not particularly limited, and for example, the method described in JP-A No. 05-93049 is used.
  • the present invention aims to recover PHA from microbial cells, it is preferable that the PHA content in the cultured microbial cells is high. In industrial level application, it is preferable that 50% by weight or more of PHA is contained in the dried cells, and the PHA content is preferably 60% by weight or more in consideration of the subsequent separation operation, the purity of the separated polymer, etc. More preferably, it is 70 weight% or more.
  • the PHA produced by the microorganism is recovered by the steps including the following steps (a), (b) and (c).
  • Step (a) in the present invention is a step of performing enzyme treatment by adding an enzyme and an alkali and / or a surfactant to an aqueous suspension of PHA-containing microbial cells. By this step, an enzyme treatment solution is obtained.
  • the PHA-containing microbial cells used in the step (a) may be a culture broth itself containing PHA-containing microbial cells after completion of the culture.
  • the cells are similarly collected.
  • an aqueous suspension of PHA-containing microbial cells can be prepared by adding water or the like.
  • the temperature for heating the culture broth is preferably 50 ° C. to 80 ° C.
  • step (a) in the present invention before the physical disruption treatment in the step (b), an enzyme and an alkali and / or a surfactant are added to the aqueous suspension of PHA-containing microbial cells to carry out the enzyme treatment. It is important to do.
  • the enzyme treatment of the PHA-containing microbial cells before the physical disruption treatment, the cell wall can be decomposed in advance and a higher disruption effect can be obtained by the next physical disruption treatment.
  • the enzyme used in the step (a) is not particularly limited as long as it can be used for industrial products, but for the purpose of degrading the cell wall to obtain a higher crushing effect, a proteolytic enzyme (protease) Cell wall degrading enzymes are preferred. In terms of supply stability and cost, industrially, a proteolytic enzyme is more preferable. Only one type of enzyme may be used, or two or more types may be used in combination. One of proteolytic enzymes and cell wall degrading enzymes may be used, or both enzymes may be used in combination.
  • proteolytic enzymes include alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase and the like.
  • cell wall degrading enzymes include lysozyme, amylase, cellulase, maltase, saccharase, ⁇ and ⁇ -glycosinase.
  • an enzyme composition containing an enzyme stabilizer, a surfactant, or a recontamination preventive agent in addition to the enzyme may be used, and the enzyme composition is not limited to the use of only the enzyme.
  • commercially available enzyme detergents for laundry can be used.
  • lysozyme is preferable from the viewpoint of lysis effect.
  • proteolytic enzymes include, for example, “Protease A”, “Protease P”, “Protease N” (above, manufactured by Amano Enzyme), “Alcalase”, “Esperase”, “Zabinase”, “Everase” (above) , Manufactured by Novozymes) and the like can be used industrially, and can also be preferably used from the viewpoint of decomposition activity.
  • Specific cell wall degrading enzymes include, for example, “lysozyme” (manufactured by Huayuan Economic Trade, Shandongzhou), “biozyme A”, “cellulase A“ Amano ”3”, “cellulase T“ Amano ”4”, “ ⁇ -Glucosidase “Amano” (above, Amano Enzyme), "Tarmamyl”, “Cellsoft” (above, Novozymes) etc. can be used industrially.
  • the treatment with these enzymes is preferably performed in the presence of a surfactant in that a higher purification effect can be obtained.
  • the enzyme treatment is preferably performed at the optimum temperature and pH of the enzyme used.
  • temperature: 50-60 ° C. and pH: 8-9 are preferable
  • Esperase manufactured by Novozymes
  • temperature: 55-65 ° C., pH: 8-10 are preferable.
  • lysozyme is used, the temperature is preferably 40-50 ° C. and the pH is 6-7.
  • the enzyme treatment is preferably continued until the required degree of treatment is achieved, and this time is usually 0.5 to 8 hours.
  • the amount of enzyme added depends on the type and activity of the enzyme.
  • the enzyme treatment is preferably performed while stirring the aqueous suspension.
  • step (a) the pH of the aqueous suspension of PHA-containing microbial cells is adjusted to the optimum pH of the enzyme, and / or the cell walls of the PHA-containing microorganisms are destroyed to bring the PHA in the cells out of the cells.
  • an alkali to the aqueous suspension and perform the enzyme treatment in the presence of the alkali.
  • Alkali means a substance whose aqueous solution is basic when dissolved in water. Both organic and inorganic compounds are included.
  • the alkali that is an inorganic compound is not particularly limited, but examples thereof include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, and sodium hydrogen carbonate.
  • Alkali metal hydrogen carbonates such as potassium hydrogen carbonate, organic metal alkali metal salts such as sodium acetate and potassium acetate, alkali metal borates such as borax, trisodium phosphate, disodium hydrogen phosphate, phosphorus
  • alkali metal phosphates such as tripotassium acid and dipotassium hydrogen phosphate
  • alkaline earth metal hydroxides such as barium hydroxide, and aqueous ammonia.
  • step (a) sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide are preferable in terms of industrial production and price.
  • the amount of alkali added in step (a) can be appropriately determined in consideration of the optimum pH of the enzyme used. If the pH of the aqueous suspension is already within the optimum pH range of the enzyme used before adding the alkali, it is not necessary to add the alkali in step (a). In addition, in the step (a), the pH of the aqueous suspension varies as the enzyme treatment proceeds, and therefore it is preferable to carry out the enzyme treatment while controlling the pH to be within the optimum pH range.
  • the temperature of the aqueous suspension when adding alkali to the aqueous suspension is desirably around the optimum temperature of the enzyme used. Specifically, a range of 20 to 60 ° C. is preferable. When alkali is added at a temperature higher than 60 ° C., the molecular weight of PHA may be significantly reduced. If an alkali is added at a temperature lower than 20 ° C., the viscosity of the aqueous suspension may be improved and stirring may be difficult.
  • the step (a) in order to destroy the cell wall of the PHA-containing microorganism and facilitate the outflow of PHA in the cell, and / or to wash and remove proteins, fatty acids, fats and oils contained in the cell, PHA In order to improve the purity, it is preferable to add a surfactant to the aqueous suspension and perform the enzyme treatment in the presence of the surfactant.
  • the alkali and the surfactant may be used together or may not be used together.
  • the surfactant used in the present invention include an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant.
  • anionic surfactants and / or nonionic surfactants are preferred.
  • anionic surfactant for the purpose of washing and removing proteins and the like, it is preferable to use an anionic surfactant, and for the purpose of washing and removing fatty acids and fats and oils, it is preferable to use a nonionic surfactant.
  • the weight ratio of the anionic surfactant / nonionic surfactant is preferably from 1/100 to 100/10, more preferably from 5/100 to 100/20, and from 5/100 to 100/100. Further preferred is 5/100 to 50/100.
  • anionic surfactant examples include alkyl sulfates, alkylbenzene sulfonates, alkyl or alkenyl sulfate sulfates, alkyl or alkenyl ether sulfates, ⁇ -olefin sulfonates, ⁇ -sulfo fatty acid salts or esters thereof. , Alkyl or alkenyl ether carboxylates, amino acid type surfactants, N-acyl amino acid type surfactants, and the like.
  • alkyl sulfates having an alkyl group having 12 to 14 carbon atoms linear alkylbenzene sulfonates having an alkyl group having 12 to 16 carbon atoms, alkyl sulfate esters or alkyl ethers having an alkyl group having 10 to 18 carbon atoms.
  • Sulfuric acid ester salts are preferred.
  • the counter ion contained in the anionic surfactant is preferably an alkali metal such as sodium or potassium, an alkaline earth metal such as magnesium, or an alkanolamine such as monoethanolamine, diethanolamine or triethanolamine.
  • nonionic surfactant examples include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyalkylene alkyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether, and the like. .
  • a highly hydrophilic compound and a compound that has a low ability to form a liquid crystal when mixed with water or a compound that does not generate such a liquid crystal are preferable.
  • polyoxyethylene alkyl ether and polyoxyalkylene alkyl ether are preferable from the viewpoint of relatively good biodegradability.
  • Examples of the cationic surfactant include alkyl trimethyl ammonium salts and dialkyl dimethyl ammonium salts.
  • amphoteric surfactant examples include carbobetaine type and sulfobetaine type surfactants.
  • anionic surfactants such as sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate and sodium oleate, polyoxyethylene alkyl ether as nonionic surfactant And polyoxyalkylene alkyl ethers are preferred from the viewpoints of price, amount used and effect of addition. Two or more of these surfactants may be used in combination.
  • the amount of the surfactant added in the step (a) is not particularly limited, but is preferably 0.001 to 10 parts by weight with respect to 100 parts by weight of the PHA produced by the microorganism, and further 5 parts by weight from the viewpoint of cost. The following is preferred.
  • Step (b) the enzyme-treated solution obtained in the step (a) is subjected to a physical crushing treatment in the presence of a surfactant under basic conditions, and the cells contained in the enzyme-treated solution. And solubilizing or emulsifying cellular substances other than PHA in the cells. This step yields an aqueous PHA suspension.
  • the physical disruption treatment in step (b) is performed by adjusting the enzyme treatment solution to basic conditions in order to dissolve cellular materials other than PHA in the cells in water. For this reason, it is preferable to add an alkali with respect to the enzyme treatment liquid obtained at the process (a).
  • the alkali added in the step (b) the compounds exemplified for the alkali that can be used in the step (a) can be used. Of these, sodium hydroxide, sodium carbonate, potassium hydroxide, and lithium hydroxide are preferred because they are inexpensive and suitable for industrial production. It is preferable to use these alkalis and adjust the enzyme-treated solution obtained in step (a) under basic conditions of pH 8.0 to 13.0.
  • the basic condition is more preferably pH 8.5 to 12.5, and still more preferably pH 9.0 to The basic condition is 12.5, and the basic condition is particularly preferably pH 10.0 to 12.5.
  • the amount of alkali added in step (b) can be appropriately determined in consideration of the pH range. When the pH of the enzyme treatment solution is already within the above pH range before adding the alkali, the alkali addition in the step (b) can be omitted.
  • the temperature of the enzyme treatment solution when adding alkali to the enzyme treatment solution is preferably adjusted to a range of 20 to 40 ° C.
  • the molecular weight of PHA may be significantly reduced.
  • insoluble matter derived from cells may not be solubilized.
  • the physical crushing process in the step (b) is performed in order to destroy the cell wall of the PHA-containing microorganism to facilitate the outflow of the PHA in the cell and / or to wash the protein, fatty acid, fat and oil contained in the cell.
  • the enzyme treatment solution contains a surfactant. If a surfactant has already been added in step (a), the enzyme treatment solution already contains a surfactant, so there is no need to add a new surfactant in step (b). It may be added.
  • the surfactant to be added in the step (b) the compounds exemplified for the surfactant that can be used in the step (a) can be used. Among these, anionic surfactants and / or nonionic surfactants are preferable from the viewpoint of detergency. A preferable addition amount is the same as the range described in the step (a).
  • a physical crushing treatment is performed on the enzyme treatment solution which shows a basic pH and contains a surfactant.
  • a high pressure homogenizer for example, a high pressure homogenizer, an ultrasonic crusher, an emulsification disperser, a bead mill etc. are mentioned.
  • the use of a high-pressure homogenizer is preferable from the viewpoint of crushing efficiency, and a type in which the liquid to be treated is introduced from a pressure-resistant container having a micro-opening and pushed out from the opening by applying high pressure is more preferable.
  • a high-pressure homogenizer model “PA2K type” manufactured by Nirosoavi Co., Ltd. may be mentioned.
  • PA2K type manufactured by Nirosoavi Co., Ltd.
  • a high-pressure homogenizer When a high-pressure homogenizer is used, a large shearing force acts on the microbial cells, so that the microbial cells are efficiently destroyed and the separation of PHA is improved. Since such devices are subjected to high pressure at the opening and instantaneously become high temperature, if necessary, the processing liquid is cooled by a general low-temperature constant temperature circulation tank to prevent the temperature from rising and at 20 to 40 ° C. It is preferable to perform a physical crushing process. In the range of 20 to 40 ° C., the physical crushing treatment can be performed with almost no decrease in the molecular weight of PHA.
  • the crushing pressure during the physical crushing treatment is preferably 30 to 60 MPa. If the pressure is lower than 30 MPa, the crushing efficiency is lowered, and the microbial cells may not be destroyed even if the crushing operation is repeated many times. When the pressure is higher than 60 MPa, the crushing efficiency is increased, but the high-pressure homogenizer becomes an ultra-high pressure specification, and the manufacturing cost increases because the equipment price is expensive.
  • the physical crushing process in step (b) may be performed a plurality of times in order to increase crushing efficiency.
  • Step (c) in the present invention is a step of separating PHA from the PHA aqueous suspension obtained in step (b). Thereby, PHA produced by the microorganism can be recovered.
  • a conventionally known method such as centrifugation or membrane separation can be used.
  • centrifugation is preferable because it can be processed industrially and can be used continuously.
  • a centrifugal sedimentator having a rotating container without holes is preferable, and types include a separation plate type, a cylindrical type, and a decanter type.
  • the PHA particles have a small specific gravity difference with water
  • a separation plate type intermittent discharge type, nozzle discharge type
  • the nozzle discharge type is particularly preferable.
  • the decanter type is generally unsuitable when the acceleration is low and the specific gravity difference between the solid and liquid is small, but the decanter type can also be used by changing the particle diameter of the PHA.
  • the decanter type includes a model having a separation plate and a large separation and sedimentation area. Such a model may be usable without changing the particle diameter.
  • the cellular material other than the PHA contained in the recovered PHA can be removed by suspending the PHA with water and washing with water.
  • the pH at the time of washing with water is preferably 8.0 to 12.5.
  • the pH is lower than 8.0, solubilization of cellular substances as impurities may not proceed, and when the pH is higher than 12.5, the molecular weight reduction of PHA may increase.
  • the temperature of water used for washing at this time is preferably 25 to 40 ° C. When the temperature is lower than 25 ° C, the solubilization of the cellular material does not proceed.
  • the purity of PHA can be improved by performing the above water washing treatment a plurality of times.
  • PHA can be recovered from PHA-containing microbial cells without performing heat treatment at a high temperature.
  • the recovery method of the present invention can be carried out under the condition that the temperature to which PHA is exposed throughout the entire process is 80 ° C. or less, thereby avoiding a significant decrease in molecular weight of PHA.
  • high-purity PHA can be recovered while avoiding a significant decrease in molecular weight of PHA.
  • the PHA recovered by the recovery method of the present invention can be formed into various shapes after drying. For example, it can be melt-pressed at 160 ° C. and formed into a sheet shape. Since the PHA obtained according to the present invention is highly pure, the yellowness index (YI value) of the sheet shows a very low value. Thereby, it turns out that the said PHA is a high quality thing with little coloring.
  • a specific YI value is 15.0 or less when the sheet has a thickness of 0.5 mm.
  • the YI value required for PHA varies depending on the intended use, but the YI value is preferably 15 or less, more preferably 10 or less, and even more preferably 5 or less. When it exceeds 15, coloring will be strong when processed into a transparent sheet or bottle, and the use without coloring will be limited.
  • the protein content remaining in the PHA was evaluated by the amount of residual nitrogen.
  • the crushed liquid sample obtained in the examples was washed with a sufficient amount of water, and then PHBH was collected by centrifugation.
  • the obtained PHBH was dried under reduced pressure and used for measurement of the residual nitrogen amount.
  • the amount of residual nitrogen was calculated by a coloration method. First, 5 M NaOH was added to each sample, and a hydrolysis reaction was performed at 95 ° C. This hydrolyzed solution was neutralized with an equal amount of 60% aqueous acetic acid solution, and an acetic acid buffer solution and a ninhydrin solution were added to perform a color reaction at 100 ° C. The absorbance of this color reaction solution was measured with a ratio beam spectrophotometer “U-1800 type” manufactured by Hitachi, Ltd. The amount of residual nitrogen was calculated by comparing this absorbance with a calibration curve prepared using a leucine sample.
  • ⁇ Measurement of YI value> The dry PHA (3.0 g) obtained in the examples was sandwiched between 15 cm square metal plates, and metal plates with a thickness of 0.5 mm were inserted into the four corners of the metal plate. Company H-15 type). After heating at 160 ° C. for 7 minutes, the sheet was pressed while heating at about 5 MPa for 2 minutes to obtain a sheet having a thickness of 0.5 mm. After pressing, the PHA is cured by allowing it to stand at room temperature, and then, using a color difference meter “SE-2000” (manufactured by Nippon Denshoku), a press sheet is placed using a 30 mm measuring plate, and a white standard plate is placed thereon. The YI value was measured by covering.
  • SE-2000 color difference meter
  • Example 1 Ralstonia eutropha KNK-005 described in [0049] of WO08 / 010296 is cultured by the method described in [0050] to [0053], and 3-hydroxybutyrate and 3-hydroxyhexanoate are cultured. A cell culture broth containing polyhydroxyalkanoate (PHBH) composed of an ate copolymer was obtained. The weight average molecular weight of PHBH at the end of the culture was 1,840,000. Ralstonia and Eutropha are now classified as Capriavidas Necka.
  • PHBH polyhydroxyalkanoate
  • Example 2 1000 ml of the cell culture solution obtained in Example 1 was sterilized at 70 to 75 ° C. for 1 hour, cooled to 50 ° C., sodium hydroxide was added to pH 8.5, and then 2.5 L of Alcalase (Novozymes) Was added so as to be 1.0% by weight of the amount of PHBH contained in the bacterial cell culture solution and stirred for 4 hours while controlling the pH at 8.5. Then, it cooled to 25 degreeC and 20.0g of sodium dodecyl sulfate was added. Sodium hydroxide was added so that the pH was 10.0, and high pressure was controlled while controlling the temperature at 25 to 35 ° C.
  • Alcalase Novozymes
  • Example 1 1000 ml of the cell culture solution obtained in Example 1 was sterilized at 70 to 75 ° C. for 1 hour, cooled to 25 ° C., 4.0 g of sodium dodecyl sulfate was added, and sodium hydroxide was added to adjust the pH to 13.2. Added and stirred for 1 hour. Thereafter, high-pressure crushing was carried out three times with a high-pressure crusher (high-pressure homogenizer model “PA2K type” manufactured by Nirosoavi) at a pressure of 45 to 55 MPa while controlling the temperature at 25 to 35 ° C. The operation of recovering PHBH by centrifuging this crushed liquid and washing with water was repeated three times.
  • a high-pressure crusher high-pressure homogenizer model “PA2K type” manufactured by Nirosoavi
  • Example 2 1000 g of the bacterial cell culture solution obtained in Example 1 was sterilized at 70 to 75 ° C. for 1 hour, cooled to 50 ° C., 4.0 g of sodium dodecyl sulfate was added, and sodium hydroxide was added so that the pH became 8.5. After the addition, 2.5 L of Alcalase (Novozymes) was added so as to be 1.0% by weight of the amount of PHBH contained in the cell culture medium, and the mixture was stirred for 4 hours while controlling at pH 8.5. Then, after cooling to 25 ° C.
  • Alcalase Novozymes
  • Table 1 shows the results of measuring the molecular weight, protein content, and YI value of the press sheet of PHBH obtained in Examples 1 and 2 and Comparative Examples 1 and 2.

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Abstract

Un PHA (polyhydroxyalcanoate) de pureté élevée est séparé à partir de cellules microbiennes contenant du PHA par une méthode commerciale simple et économique qui comporte un nombre réduit de procédés, n'entraînant pas de diminution significative du poids moléculaire. Le procédé de récupération de polyhydroxyalcanoate spécifiquement décrit dans la présente invention comprend : (a) une étape de traitement enzymatique consistant à ajouter une enzyme et un alcali et/ou un tensioactif à une suspension aqueuse de cellules microbiennes contenant un polyhydroxyalcanoate, pour obtenir ainsi un liquide de traitement enzymatique ; (b) une étape de soumission du liquide de traitement enzymatique à un procédé de broyage physique dans des conditions basiques en présence d'un tensioactif, de façon que les cellules soient broyées et que les substances cellulaires autres que le polyhydroxyalcanoate dans les cellules soient solubilisées ou émulsifiées, pour obtenir ainsi une suspension de polyhydroxyalcanoate ; et (c) une étape de séparation du polyhydroxyalcanoate à partir de la suspension de polyhydroxyalcanoate.
PCT/JP2010/002304 2009-03-30 2010-03-30 Procédé de récupération de polyhydroxyalcanoate WO2010116681A1 (fr)

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CN102133511A (zh) * 2011-01-14 2011-07-27 清华大学 两亲性蛋白—聚羟基脂肪酸酯颗粒结合蛋白PhaP的新用途
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
TWI573306B (zh) * 2010-12-28 2017-03-01 半導體能源研究所股份有限公司 發光單元，發光裝置，及燈光裝置
WO2017221755A1 (fr) 2016-06-23 2017-12-28 株式会社カネカ Procédé de fabrication d'acide polyhydroxyalcanoïque
EP3608415A4 (fr) * 2017-04-05 2021-01-13 Kaneka Corporation Particules de polyhydroxyalcanoate et dispersion aqueuse de celles-ci
WO2021251162A1 (fr) * 2020-06-09 2021-12-16 株式会社カネカ Procédé de production d'une feuille de polyhydroxyalcanoate et son utilisation
WO2022113530A1 (fr) * 2020-11-24 2022-06-02 株式会社カネカ Procédé de production de poly(3-hydroxyalcanoate)
CN115807044A (zh) * 2022-11-09 2023-03-17 华南理工大学 一种高效提取并纯化高纯度聚羟基脂肪酸酯的方法
CN116144046A (zh) * 2022-06-06 2023-05-23 北京蓝晶微生物科技有限公司 一种聚羟基脂肪酸酯凝集体的制备方法
WO2024003698A1 (fr) * 2022-06-27 2024-01-04 Versalis S.P.A. Procédé de récupération et de purification de polyhydroxyalcanoates à partir d'un bouillon de fermentation

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JP6709781B2 (ja) * 2015-04-24 2020-06-17 株式会社カネカ 微生物産生樹脂の製造方法及び微生物産生樹脂
JP6864585B2 (ja) * 2017-08-30 2021-04-28 株式会社カネカ ポリヒドロキシアルカノエートの製造方法
CN111019108B (zh) * 2020-01-07 2021-03-05 清华大学 一种提取并纯化聚羟基脂肪酸酯的方法
WO2021186872A1 (fr) * 2020-03-18 2021-09-23 株式会社カネカ Procédé de production d'une résine d'acide polyhydroxybutyrique
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110980A (en) * 1989-04-06 1992-05-05 Ecole Polytechnique Separation of poly-β-hydroxyalkanoic acid from microbial biomass
JP2001046094A (ja) * 1999-08-10 2001-02-20 Kanegafuchi Chem Ind Co Ltd ポリ−３−ヒドロキシアルカン酸の分離精製方法
WO2004029266A1 (fr) * 2002-09-30 2004-04-08 Kaneka Corporation Methode de purification d'un copolymere d'acide 3-hydroxyalcanoique
WO2005085461A1 (fr) * 2004-03-04 2005-09-15 Kaneka Corporation Methode de degradation de l'acide nucleique et son utilisation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110980A (en) * 1989-04-06 1992-05-05 Ecole Polytechnique Separation of poly-β-hydroxyalkanoic acid from microbial biomass
JP2001046094A (ja) * 1999-08-10 2001-02-20 Kanegafuchi Chem Ind Co Ltd ポリ−３−ヒドロキシアルカン酸の分離精製方法
WO2004029266A1 (fr) * 2002-09-30 2004-04-08 Kaneka Corporation Methode de purification d'un copolymere d'acide 3-hydroxyalcanoique
WO2005085461A1 (fr) * 2004-03-04 2005-09-15 Kaneka Corporation Methode de degradation de l'acide nucleique et son utilisation

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TWI573306B (zh) * 2010-12-28 2017-03-01 半導體能源研究所股份有限公司 發光單元，發光裝置，及燈光裝置
US9905632B2 (en) 2010-12-28 2018-02-27 Semiconductor Energy Laboratory Co., Ltd. Light-emitting unit, light-emitting device, and lighting device
CN102133511A (zh) * 2011-01-14 2011-07-27 清华大学 两亲性蛋白—聚羟基脂肪酸酯颗粒结合蛋白PhaP的新用途
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
US11440823B2 (en) 2016-06-23 2022-09-13 Kaneka Corporation Method for producing polyhydroxyalkanoic acid
WO2017221755A1 (fr) 2016-06-23 2017-12-28 株式会社カネカ Procédé de fabrication d'acide polyhydroxyalcanoïque
EP3608415A4 (fr) * 2017-04-05 2021-01-13 Kaneka Corporation Particules de polyhydroxyalcanoate et dispersion aqueuse de celles-ci
WO2021251162A1 (fr) * 2020-06-09 2021-12-16 株式会社カネカ Procédé de production d'une feuille de polyhydroxyalcanoate et son utilisation
WO2022113530A1 (fr) * 2020-11-24 2022-06-02 株式会社カネカ Procédé de production de poly(3-hydroxyalcanoate)
CN116144046A (zh) * 2022-06-06 2023-05-23 北京蓝晶微生物科技有限公司 一种聚羟基脂肪酸酯凝集体的制备方法
WO2023236718A1 (fr) * 2022-06-06 2023-12-14 北京蓝晶微生物科技有限公司 Procédé de préparation d'agrégat de polyhydroxyalcanoate
CN116144046B (zh) * 2022-06-06 2024-01-23 北京蓝晶微生物科技有限公司 一种聚羟基脂肪酸酯凝集体的制备方法
WO2024003698A1 (fr) * 2022-06-27 2024-01-04 Versalis S.P.A. Procédé de récupération et de purification de polyhydroxyalcanoates à partir d'un bouillon de fermentation
CN115807044A (zh) * 2022-11-09 2023-03-17 华南理工大学 一种高效提取并纯化高纯度聚羟基脂肪酸酯的方法
CN115807044B (zh) * 2022-11-09 2023-10-13 华南理工大学 一种高效提取并纯化高纯度聚羟基脂肪酸酯的方法

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