JP2009207380A - Method for purifying polyhydroxyalkanoic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a purification method for efficiently obtaining a high-purity polyhydroxyalkanoic acid by cleaning and extracting a biomass of a polyhydroxyalkanoic acid-containing microorganism by using an organic solvent miscible with water. <P>SOLUTION: The method for purifying the polyhydroxyalkanoic acid (PHA) includes a step for carrying out pretreatment of the biomass such as the removal of impurities by cleaning the biomass containing the polyhydroxyalkanoic acid by a mixed solvent of the organic solvent miscible with the water and the water, and a succeeding step of extracting the polyhydroxyalkanoic acid for carrying out the extraction and separation of the PHA by using the same solvent. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for purifying PHA produced using fungi having polyhydroxyalkanoic acid (PHA) production ability.

  Currently, plastic waste is processed by incineration, landfill, etc., but these treatment methods have problems such as global warming and ground relaxation of landfill. For this reason, recycling systems are being developed along with increasing social awareness of plastic recycling. However, recyclable uses are limited, and as a matter of fact, many plastic disposal methods cannot be handled by incineration, landfilling, and recycling alone, and are often left in nature. Therefore, biodegradable plastics, which are taken into the natural material circulation after disposal and do not harm the decomposition products, are attracting attention, and their practical application is eagerly desired.

  Among these biodegradable plastics, polyhydroxyalkanoic acid (hereinafter referred to as PHA) is a biodegradable thermoplastic polyester that is produced and stored as an energy storage substance in the cells of many microbial species (Non-patent Document 1). ), Because it is expected to have almost no adverse effects on the ecosystem because it is incorporated into the natural carbon cycle process. Also in the medical field, it is considered that it can be used as an implant material and a drug carrier that do not require collection (Non-patent Documents 2 and 3).

  For convenience, PHA produced by microorganisms is short chain PHA (scl-PHA) mainly composed of hydroxyalkanoic acid having 6 or less carbon atoms and medium and long chain PHA (mcl) mainly composed of hydroxyalkanoic acid having 6 or more carbon atoms. -PHA). Furthermore, microbial production PHA is known to vary greatly in composition, structure, and molecular weight depending on the type of microorganism used in production, the composition of the culture medium, culture conditions, and so on. For the purpose of improving the physical properties of PHA so far Various studies have been made. To date, over 90 types of structures have been reported as units to be introduced into PHA (Non-Patent Document 4).

For example, Capiliavidus necator H16 strain ( Cupriavidusnecator H16, ATCC17699) and its mutant strains can be obtained by changing the carbon source during culturing to produce 3-hydroxybutanoic acid (3-HB) and 3-hydroxypentanoic acid (3-HV ) In various compositions (Patent Document 1, Patent Document 2, Patent Document 3, and Non-Patent Document 5). Alcaligeneseutrophus H16 strain uses 4-hydroxybutanoic acid (4-HB) or γ-butyronolactone as a carbon source, and Comamonas acidovorans uses gluconic acid and 1,4-butanediol. It is reported that when cultured, a copolymer containing 3-HB and 4-HB is produced (Non-patent Document 6). Aeromonas caviae (Aeromonas caviae) generates a random copolymer comprising a fat or fatty acid from 3-HB and C 6 of the 3-hydroxy hexanoic acid (3-HH) as a carbon source. Furthermore, Pseudomonas putida (Pseudomonas putida) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) bacteria belonging to the genus Pseudomonas, such as metabolize various compounds such as sugars or organic acids, alkanes, long chain 3-hydroxyalkanoic acid in the C6~C12 PHA (mcl-PHA) having a monomer unit as a monomer unit is synthesized (Non-patent Document 1). Further, PHA composed of a wide range of 3-hydroxyalkanoic acids from C4 to C16 is also known (Patent Document 5).

  PHA produced by microorganisms forms granules and accumulates in the cells, and in order to use them as plastics, it is necessary to separate and remove them from the cells of the microorganisms. As a known method for separating and purifying PHA from microbial cells, broadly speaking, a method of extracting PHA in an organic solvent in which PHA is soluble, and a method of solubilizing cell components other than PHA, etc. There is a method of obtaining PHA by removing.

  As solvents conventionally used for solvent extraction of PHA, chloroform (Patent Document 4), methylene chloride (Patent Document 4), pyridine (Patent Document 6) and the like are known. However, since these solvents cannot efficiently extract PHA from wet cells, a step of previously drying the cells obtained from the culture solution before extraction is required. In addition, a solvent such as methanol must be added as a poor solvent to the extract obtained after extraction to precipitate PHA. In this case, a large amount of poor solvent is required, which requires a very large production facility. It is economically disadvantageous. Further, since it is a two-solvent system, it is disadvantageous from the viewpoint of solvent recovery and reuse.

  Patent Document 7 describes a method for extracting PHA from wet cells, but all of the solvents used here are special and are industrially insufficient in terms of economy and the like.

  On the other hand, in the purification method in which components other than PHA are solubilized and removed with an enzyme or a surfactant, the effects of the enzyme, the surfactant, etc. vary greatly depending on the strain, and many steps are required to obtain high-purity PHA. It may be necessary.

In particular, development of an industrial purification method for mcl-PHA has not progressed compared to scl-PHA. As an example, a method using acetone or isopropanol has been reported. In the acetone extraction method, washing with methanol or NaOH is necessary as a pretreatment, and methanol, which is a poor solvent, is required for precipitation of mcl-PHA (Non-patent Document 7). Therefore, recycling of the solvent requires separation of acetone and methanol, which is industrially disadvantageous. Extraction with isopropanol does not require the addition of methanol for the precipitation of mcl-PHA, but there is a problem that the recovery rate is extremely low (Non-patent Document 8). In any method, a step of freeze-drying the bacterial cells is necessary.
Japanese National Patent Publication No. 6-15604 JP 7-14352 Gazette Japanese National Patent Publication No. 8-19227 JP-A-57-65193 Japanese Patent Laid-Open No. 5-30980 US Patent No. 3036959 JP-A-2-69187 Microbiological Reviews, 450-472, 1990 Tissue engineering, 6, 183-8, 2000 Journal of controlled release, 59, 207-217, 1999 FEMS Microbiology Letters, 128, 219-228, 1995 Applied Microbiology and Biotechnology, 28, 330-334, 1988 Applied and Environmental Microbiology, 64, 3437-3443, 1998 Journal of Microbiological Methods, 67, 212-219, 2006 Journal of Microbiological Methods, 69, 206-213, 2007

  It is an object of the present invention to provide a method for obtaining PHA by pretreating wet cells with an inexpensive and readily available solvent and / or extracting PHA with the same solvent in a purification process of PHA from biological cells. It is to provide.

  In order to solve the above-mentioned problems, the present inventors have intensively studied a method capable of industrially producing PHA, and as a result, surprisingly, an organic solvent capable of mixing PHA-containing biological cells with water and water. It was found that PHA can be efficiently purified by pretreatment with the mixed solution. Furthermore, it has been found that PHA can be satisfactorily extracted by performing extraction with the same solvent, and the present invention has been completed.

That is, the present invention has been completed based on such knowledge and includes the following inventions. [1]
A method for producing PHA by separating and purifying it from a biological cell containing polyhydroxyalkanoic acid (PHA), comprising a step of pretreating the biological cell with an organic solvent that can be mixed with water. A characteristic PHA manufacturing method.
[2]
The method for producing PHA according to [1], wherein the pretreatment step is a step of washing biological cells containing PHA with a mixed solution of the organic solvent and water.
[3]
The method for producing PHA according to [1] or [2], comprising a step of aggregating biological cells before the pretreatment step.
[4]
The PHA production method according to any one of [1] to [3], wherein the mixed solvent of the organic solvent and water contains the organic solvent in an amount of 1 wt% to 99 wt%.
[5]
The organic solvent is contained in the mixed solution of the organic solvent and water in the range of 30% by weight to 99% by weight.
[6]
The organic solvent is contained in the range of 40 wt% to 80 wt% in the mixed solution of the organic solvent and water, The PHA production method according to [5].
[7]
The PHA production method according to any one of [1] to [6], wherein the organic solvent used is selected from the group of solvents consisting of alcohols, ketones, ethers and nitriles.
[8]
The organic solvent to be used is selected from ethanol, isopropanol, and acetone, The PHA production method according to [7].
[9]
Any one of [1] to [8], including a step of extracting PHA using an organic solvent mixed with water and using the same organic solvent as the step of pretreating the biological cells. The PHA manufacturing method as described.
[10]
The PHA production method according to [9], wherein the extraction temperature of PHA with the organic solvent is 10 ° C to 130 ° C.
[11]
The PHA production method according to any one of [1] to [10], wherein the PHA is a polyester composed of a component selected from 3-, 4-, or 5-hydroxyalkanoic acids having 4 to 16 carbon atoms.
[12]
The PHA production method according to [11], wherein the PHA is a polyester comprising a constituent selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.
[13]
The PHA production according to any one of [1] to [10], wherein the PHA is a copolyester composed of a component selected from 3-, 4-, or 5-hydroxyalkanoic acids having 4 to 16 carbon atoms. Method.
[14]
The PHA production method according to [13], wherein the PHA is a copolyester composed of a constituent selected from at least three types of 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.
[15]
The PHA production method according to [13], wherein the PHA is a copolyester containing at least three types of 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.
[16]
The method for producing PHA according to [13], wherein the PHA is a copolyester containing at least 5 types of 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.
[17]
[1] to [10], wherein the PHA is a copolyester composed of a component selected from 4-, 16-, and 5-hydroxyalkanoic acids having 4 to 16 carbon atoms and containing at least one unsaturated bond. The PHA manufacturing method according to any one of the above.
[18]
The method for producing PHA according to [17], wherein the PHA is a copolyester comprising a constituent selected from 4-hydroxy alkanoic acids having 4 to 16 carbon atoms and containing at least one unsaturated bond.
[19]
The method for producing PHA according to [1], wherein the biological cell containing PHA is bacteria or yeast.
[20]
PHA organism cells containing Bacillus (Bacillus) genus, Ralstonia (Ralstonia) genus Cupriavidus (Cupriavidus) genus Wauterushia (Wautersia) genus Aeromonas (Aeromonas) genus Escherichia (Esherichia) genus Pseudomonas (Pseudomonas) genus, Candy da (Candida) genus Alcaligenes (Alcaligenes) genus Comamonas (Comamonas) PHA production method of [19], wherein the a microorganism belonging to any genus.
[21]
The method for producing PHA according to [20], wherein the biological cell containing PHA is a microorganism belonging to the genus Pseudomonas .
[22]
The method for producing PHA according to any one of [1] to [21], further comprising a step of changing the molecular weight of PHA.
[23]
Furthermore, the PHA production method according to any one of [1] to [22], further including a step of moving the container of PHA having a concentration of 80% or higher at a temperature of 10 ° C. or higher.
[24]
A PHA obtained by the PHA production method according to any one of [1] to [23].

  According to the present invention, before extraction of PHA from wet biological cells containing PHA, the biological cells are pretreated with a mixed solvent of water and an organic solvent that can be mixed with water. The amount of impurities contained in the PHA can be removed, and a high-purity PHA can be obtained. Moreover, since the extraction after the biological cell pretreatment can be performed using the same solvent as the pretreatment, the solvent can be easily recovered and reused. The present invention greatly contributes to improving the efficiency of industrial production of PHA by microorganisms and the like and reducing the cost by solvent recycling.

  The polyhydroxyalkanoic acid (PHA) in the present invention is not particularly limited as long as it accumulates in biological cells. As a constituent component of polyester, 3-, 4-, or 5-hydroxyalkanoic acid having 4 to 16 carbon atoms can be used as a monomer unit. PHA containing 3-hydroxyalkanoic acid as a constituent is preferable.

  The 3-hydroxyalkanoic acid having 4 to 16 carbon atoms is 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxytetradecanoic acid, 3- Examples include hydroxyhexadecanoic acid. These monomer units can contain one or more unsaturated bonds. As what contains an unsaturated bond, 3-hydroxy-5-cis-dodecenoate, 3-hydroxy-7-cis-tetradecenoate, etc. are mentioned, for example. Moreover, the monomer unit which has substituents, such as a phenyl group, a phenoxy group, a benzoyl group, a thienyl group, may be included. For example, those having a phenyl group include 5-phenylvaleric acid, those having a benzoyl group include 5-benzoylvaleric acid, and those having a thienyl group include 5-thienylvaleric acid. These may be a homopolyester composed of a single component or a copolyester composed of a plurality of monomer units. PHA belonging to mcl-PHA is preferable from the viewpoint of solubility in an organic solvent, and more preferable is a PHA containing three or more, more preferably five or more constituent monomers, in terms of low crystallinity.

The biological cells to PHA was accumulated to be used in the present invention, for example Kapiriabidasu-Neketa (Cupriavidusnecator) Kapiriabidasu genus such as Alcaligenes Ratasu (A. Latas) genus Alcaligenes such as (Alcaligenes), Pseudomonas genus (Pseudomonas), Bacillus (Bacillus), Azotobacter sp (Azotobacter), the genus Nocardia (Nocardia), Aeromonas (Aeromonas), Ralstonia (Ralstonia) genus microorganisms include accumulating PHA in the cells, such as Wauterushia (Wautersia) genus (Microbiological Reviews , 450-472, 1990).

Among them, Pseudomonas putida (Pseudomonas putida), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas Rejinoboransu (Pseudomonas resinovorans), Pseudomonas oleovorans (Pseudomonas oleovorans) is preferably, Pseudomonas fluorescens ( Pseudomonas fluorescens), Pseudomonas Rejinoboransu (Pseudomonas resinovorans) and Pseudomonas oleovorans (Pseudomonas oleovorans) safety, more preferred in terms of productivity.

In addition to these microorganisms, biological cells that have been modified to artificially produce PHA by introducing PHA synthase or the like using genetic engineering techniques can also be used. In this case, for example, Gram-negative bacteria such as Esherichia genus, Gram-positive bacteria such as Bacillus genus, yeast such as Candida genus, and biological cells such as plants can be used (Advances in biochemical engineering biotechnology, 71, 209-40, 2001, WO2005085415, etc.). It is preferable to use a microorganism in that a large amount of PHA can be accumulated.

  The production method of PHA in these biological cells is not particularly limited, but a production method using a culture tank is realistic.

  The method for culturing PHA-producing bacteria in the culture tank is not particularly limited as long as PHA can be efficiently accumulated in the microbial cells. For example, Appl Environ Microbiol. 1992, Biomacromoecules, Vol. 2, 211-216, 2001, Appl Environ Microbiol., 62, 536-543, 2003, and the like. Hereinafter, PHA-containing biological cells are referred to as cells.

  In the present invention, the cells may be pretreated by directly adding an organic solvent that can be mixed with water to the culture solution after completion of the culture. However, the pretreatment is performed after the water content of the culture solution is once reduced. May be economical. As a method for reducing the amount of water in the culture solution, methods well known to those skilled in the art such as a centrifugal separation method, a decantation method, and a filtration method can be used. At this time, a flocculant of the microbial cells such as a polymer flocculant and an inorganic flocculant can also be used for the purpose of improving the sedimentation and filterability of the microbial cells.

  Examples of the polymer flocculant include polyacrylamide, sodium polyacrylate, polymethacrylate, polyacrylate flocculants, etc., and inorganic flocculants include sulfate band, polyaluminum chloride, chloride. Examples thereof include ferric iron and polyferric sulfate. These may be used alone or in combination. In addition, the cells can be aggregated by adjusting the pH. When producing high-purity PHA, it is preferable to add water again to the bacterial cell culture solution containing PHA with reduced water content, and stir and wash. If economically reasonable, the water may be further reduced by heating or a reduced pressure method.

  The present invention is characterized by pretreating a culture solution containing water, or a culture solution concentrate or washed cells using an organic solvent mixed with water. Here, the culture solution containing water means that 100% by weight or more and 10,000% by weight or less of water is included with respect to the dry cell mass, and more preferably 100% by weight or more and 5000% by weight in terms of pretreatment efficiency. % Of water is included. The organic solvent that can be used for the pretreatment of the present invention is not particularly limited as long as it is mixed with water and PHA can be dissolved. However, alcohols, ketones, ethers, Nitrile solvents are preferred. Examples of alcohols include methanol, ethanol, propanol, and isopropanol. Examples of ketones include acetone and ethyl methyl ketone. Examples of ethers include 1,4-dioxane. Examples of nitriles include acetonitrile. Particularly preferred are methanol, ethanol, propanol, isopropanol, and acetone. When these organic solvents are used for pretreatment, they are used as a mixed solvent with water. At that time, the concentration of the organic solvent is 1% by weight or more, preferably 30% by weight or more, more preferably 40% by weight or more as a lower limit with respect to the weight of the treated bacterial cell liquid as the final concentration. The upper limit may be 99% by weight or less, preferably 80% by weight or less. This is to ensure a sufficient cleaning effect and collect more PHA. At this time, taking into consideration the water content of the treated cell fluid, an organic solvent not containing water can be added to the cell solution to obtain a desired concentration, or an organic solvent containing water can be added to obtain a desired concentration. You can also. Further, the optimized concentration can be determined in consideration of the solvent to be used and the composition / molecular weight of the target PHA. In the case where a plurality of pretreatments are performed, the concentration of the organic solvent used can be changed within the concentration range of the present invention each time.

  Although there is no restriction | limiting in particular in the quantity of the organic solvent to be used, and the frequency | count of pre-processing, It is preferable to use in the quantity of 0.5 times or more and 10 times or less with respect to a process microbial cell liquid volume. The number of pretreatments is not particularly limited, but is preferably 1 to 5 times. The pretreatment can be performed, for example, by mixing and stirring the above-mentioned organic solvent in the treated bacterial cell liquid and separating the bacterial cells containing PHA by a method such as a centrifugal separation method, a decantation method, or a filtration method. There is no particular limitation on the stirring time and the stirring strength, and it is sufficient if it can be sufficiently uniformized. Repair may be performed without stirring. It is preferable to remove the water-containing organic solvent so that the final concentration is 1000% by weight or less with respect to the dry cells.

  PHA is extracted from the cells subjected to the pretreatment as described above using an extraction solvent. As the extraction solvent, any solvent may be used as long as it is an organic solvent capable of dissolving the target PHA. For example, chloroform, methylene chloride, normal hexane, ethyl acetate, acetone, isopropanol and the like can be mentioned. More preferably, the same solvent as the organic solvent used in the pretreatment can be used as the extraction solvent. By using the same solvent, the cost on equipment can be reduced, which is also a second feature of the present invention.

  The amount of extraction solvent to be used is not particularly limited, but if it is too much, it will cost to recover PHA, and if it is too little, it will not be sufficiently extracted or the liquid after extraction will become too viscous. Processing may be difficult. Preferably, the lower limit of the PHA concentration is 1% or more, preferably 3% or more, and the upper limit is 50% or less, more preferably 30% or less. The extraction temperature may be in the range of −20 ° C. to 200 ° C., and extraction using a pressurizer is possible above the boiling point of the organic solvent used. Preferably it is 10 degreeC-130 degreeC, More preferably, it is below the boiling point under the atmospheric pressure of the organic solvent to be used, and is performed in the range of 50 degreeC-90 degreeC. A smaller number of extractions is desirable, but there is no particular limitation.

  Next, the PHA dissolved in the above-mentioned organic solvent is used to remove the cell residue. In the PHA purification method of the present invention, methods such as a centrifugal separation method, a decantation method, and a filtration method can be used. The filtration method is preferable because it can be processed in a large amount. When performing separation by filtration, it is desirable to preheat the filter to the same temperature as the extraction temperature. A method well known to those skilled in the art can be used as a method for isolating PHA by removing the solvent from an extraction solution in which PHA separated from the cell residue is dissolved. Although PHA can be precipitated and recovered by adding a so-called poor solvent, it is more preferably concentrated by, for example, evaporation of the solvent by reduced pressure or heating, precipitation of PHA by cooling or addition of water as a poor solvent, etc. (Separate). These methods can be used in combination. PHA is dried by a method well known to those skilled in the art, for example, airflow drying, vacuum drying, or the like.

  Depending on the monomer composition of PHA, the solubility in organic solvents may be low and may not dissolve even at high temperatures and pressures. In such a case, solubility in an organic solvent can be modified by decomposing PHA limitedly and reducing the molecular weight.

  The decomposition of PHA can be performed using heat or an acid, preferably sulfuric acid, hydrochloric acid, or the like, but a method using an alkali is more preferable. The alkali is not particularly limited as long as it can be used for the decomposition of PHA. For example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, and calcium hydroxide; alkali metal carbonates such as sodium carbonate, potassium carbonate, and calcium carbonate; sodium hydrogen carbonate, potassium hydrogen carbonate, etc. Alkali metal hydrogen carbonates; alkali metal salts of organic acids such as sodium acetate and potassium acetate; alkali metal borates such as borax; trisodium phosphate, disodium hydrogen phosphate, tripotassium phosphate, phosphorus Examples thereof include, but are not limited to, phosphates of alkali metals such as dipotassium hydrogen hydride; hydroxides of alkaline earth metals such as barium hydroxide; ammonia water. These may be used alone or in combination of two or more. Of these, alkali metal hydroxides and alkali metal carbonates are preferable from the viewpoint of industrial production and cost, and more preferably sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, sodium carbonate, etc. It is.

  In the present invention, an alkali hydrolysis step can be added before or after the step of performing the pretreatment using an organic solvent mixed with water. The decomposition of PHA can be performed in parallel with the extraction of PHA with an organic solvent, or can be performed after the extraction.

  When alkaline hydrolysis is performed before the pretreatment step using an organic solvent mixed with water, a predetermined alkaline solution is added to the culture solution or the previously washed culture solution with water, and the mixture is heated or kept at room temperature. The decomposition reaction is carried out by stirring.

  The decomposition reaction is preferably a method in which alkali is continuously or intermittently added to the cell suspension while controlling the pH to a desired value. After reaching the desired molecular weight, the reaction is stopped by bringing the pH to neutral or lower, and then pretreatment is performed using an organic solvent mixed with water.

  Similarly, when alkaline hydrolysis is performed after the pretreatment step using an organic solvent mixed with water, a predetermined alkaline solution is added to the pretreated cells and stirred at room temperature or at room temperature. Decomposition reaction. The decomposition reaction can also be performed by controlling the pH. After reaching the desired molecular weight, the reaction is stopped by setting the pH to neutral or lower, and then the bacterial cell components and PHA are precipitated and collected using a centrifugal method, a filtration method, or the like. Thereafter, extraction can be performed as described above using an organic solvent.

  When the PHA is decomposed in parallel with the extraction of the PHA with an organic solvent, the solution containing the bacterial cell components from which PHA is extracted or being extracted is decomposed with alkali in the same manner as described above, and filtered. Separated from bacterial components. At this time, the reaction may be stopped by neutralizing the pH either before or after separation from the bacterial cell components. For the PHA separated from the bacterial cell components, the organic solvent may be removed under reduced pressure as it is. However, the organic solvent can be removed after stationary separation or precipitation by adding water. Furthermore, a salt can also be removed by adding a water washing process.

  When PHA is decomposed after extraction with an organic solvent of PHA, the solution in which the PHA separated from the bacterial cell components is dissolved is decomposed to the desired molecular weight with alkali in the same manner as described above, and the reaction is performed with neutral pH. Stop. The organic solvent may be removed under reduced pressure as it is, but the organic solvent can be removed after stationary separation or addition of water and precipitation concentration. Furthermore, salt can also be removed by adding water washing.

  A PHA having desired organic solvent solubility can also be prepared by modifying a PHA production system such as a microorganism. For example, PHA having a reduced molecular weight can be produced by excessively expressing PHA synthase.

  The PHA purified as described above is finally collected in another container. The upper limit of the discharge temperature at this time is not particularly limited, but the lower limit is preferably 10 ° C. or higher, preferably 30 ° C. or higher, more preferably 50 ° C. or higher because the operability is remarkably improved.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Measurement method of average molecular weight of PHA)
About 10 mg of the recovered PHA was dissolved in 5 ml of chloroform, and insoluble matters were removed by filtration. This solution was analyzed using chloroform as a mobile phase using a Shimadzu GPC system equipped with Shodex K805L (300 × 8 mm, 2 linked) (Showa Denko). A commercially available standard polystyrene was used as the molecular weight standard sample.

(Quantitative method of PHA)
The gravimetric method or the HPLC method was used. For the HPLC method, an Shimadzu HPLC system equipped with Shodex GF-310HQ (300 × 7.6 mm, manufactured by Showa Denko KK) was used, and ethyl acetate was analyzed as a mobile phase. An RI detector was used for detection. As the standard sample, the cells cultured by the method described in Example 1 and purified as follows were used. The cells were washed with water, freeze-dried, extracted with chloroform, the residue was filtered, concentrated with an evaporator, and methanol was added for precipitation. The dissolution with chloroform and precipitation with methanol were repeated three more times. After removing the methanol supernatant, it was dried in a vacuum dryer at 40 ° C. for 3 days to obtain a standard product.

Example 1
(culture)
Pseudomonas renonovans (ATCC14235) strain was cultured as follows.

The composition of the seed medium is 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v% Na ₂ PO ₄ · 12H ₂ O, 0.15 w / v% KH ₂ PO ₄ (pH 6.8).

The composition of the preculture medium is 1.1 w / v% Na ₂ HPO ₄ · 12H ₂ O, 0.19 w / v% KH ₂ PO ₄ , 1.29 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w _{/ v% MgSO 4 · 7H 2} O, 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl _{/ v% FeCl 3 · 6H 2} O, 1w / v% CaCl 2 · 2H 2 O, 0.02 w / v% CoCl ₂ .6H ₂ O, 0.016 w / v% CuSO ₄ .5H ₂ O, 0.012 w / v% NiCl ₂ .6H ₂ O. As the carbon source, palm oil was used at a concentration of 2.5 w / v%.

The composition of the polyester production medium is 0.385 w / v% Na ₂ HPO ₄ · 12H ₂ O, 0.067 w / v% KH ₂ PO ₄ , 0 · 291 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w / _{v% MgSO 4 · 7H 2 O} , 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl _{/ v% FeCl 3 · 6H 2} O, 1w / v% CaCl 2 · 2H 2 O, 0 0.02 w / v% CoCl ₂ · 6H ₂ O, 0.016 w / v% CuSO ₄ · 5H ₂ O, 0.012 w / v% NiCl ₂ · 6H ₂ O)), 0.05 w / v% BIOSPUREX 200K (antifoaming agent: manufactured by Cognis Japan) was used.

  A glycerol stock (50 μl) of Pseudomonas renonovans (ATCC 14235) strain was inoculated into a seed medium (10 ml) and cultured for 24 hours. A Sakaguchi flask containing 300 ml of preculture medium was inoculated with 3 v / v%. The operating conditions were a culture temperature of 30 ° C. and a shaking toe speed of 100 rpm for 24 hours.

  In the polyester production culture, a 10 L jar fermenter (MDL-1000 model manufactured by Maruhishi Bioengine) containing 6 L of production medium was inoculated with 2.5 v / v% of the preculture seed. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 400 rpm, an aeration rate of 6 L / min, and a pH controlled between 6.7 and 6.8. A 7% aqueous ammonia solution was used for pH control. Palm oil as a carbon source was added at a rate of 10 g / H with a peristaltic pump throughout the culture period. The culture was stopped 50 hours after the start of the culture, and the culture solution was collected. A bacterial cell containing about 50% by weight of PHA was obtained.

(Purification)
After adding 2.5 g of sulfuric acid band to 500 mL of the cultured bacterial cell solution containing 17.5 g of PHA and stirring for a while, the pH was adjusted to 7 to aggregate the bacterial cells. Thereafter, the cells were precipitated by centrifugation (3000 g, 10 min), and the supernatant was removed to obtain 200 g of wet cells. This wet cell was washed twice by redispersing and centrifuging with 400 mL of water to obtain 200 g of a washed cell solution. 400 mL of an aqueous solution containing 80% by weight of isopropanol was added thereto, followed by redispersion and centrifugation. This operation was repeated twice to obtain 180 g of wet cells. Extraction was performed by adding 400 mL of 100% isopropanol to the wet cells and stirring at 80 ° C. for 30 minutes. Thereafter, filtration with a filter paper was performed, and 400 mL of 100% isopropanol was further added to the residue, followed by stirring at 80 ° C. for 30 minutes to perform re-extraction. After each solvent was distilled off, the amount of the extract obtained and the PHA content were measured. The results are shown in Table 1.

  PHA having a purity of 94% or more (molecular weight: 130000) was obtained at a recovery rate of 82%.

(Example 2)
In Example 1, the same operation was performed except that the washing solvent was changed to 80% acetone aqueous solution and the extraction solvent was changed to 100% acetone. The results are shown in Table 2.

  PHA having a purity of 88% or more (molecular weight: 130000) was obtained with a recovery rate of 82%.

(Example 3)
6 g of a sulfuric acid band was added to 300 mL of a cultured bacterial cell solution containing 6.3 g of PHA in the bacterial cells and stirred for a while, and then the pH was adjusted to 7 to aggregate the bacterial cells. Thereafter, the cells were precipitated by centrifugation (3000 g, 10 min), and the supernatant was removed to obtain 100 g of wet cells. The wet cells were redispersed and centrifuged with 100 mL of water, and this operation was further repeated. Next, the wet cells were resuspended in 90% ethanol and centrifuged. This operation was repeated twice. The obtained wet cells were resuspended in 300 mL of water, 2N KOH was added to maintain the pH at 11, and a hydrolysis reaction was performed at 80 ° C. After 2 hours, hydrochloric acid was added to adjust the pH of the reaction solution to 7, and the hydrolysis reaction was stopped. Thereafter, the reaction mixture was centrifuged (3000 g, 10 min) to collect a precipitate fraction. 300 mL of 100% ethanol was added to the precipitate fraction, and extraction was performed at 78 ° C. Thereafter, filtration with filter paper was performed, and the residue was further extracted with 100% ethanol. After each solvent was distilled off, the amount of the extract obtained and the PHA content were measured. The results are shown in Table 3.

  PHA (molecular weight: 20000) having a purity of 89% or more was obtained with a recovery rate of 39%.

Example 4
In Example 3, the same operation was performed except that the solvent was changed from ethanol to acetone. The results are shown in Table 4.

  PHA having a purity of 82% or more (molecular weight: 20000) was obtained at a recovery rate of 50%.

(Comparative Example 1)
After adding 0.5 g of a sulfuric acid band to 100 mL of a cultured bacterial cell solution containing 2.7 g of PHA and stirring for a while, the pH was adjusted to 7 to aggregate the bacterial cells. Thereafter, the cells were precipitated by centrifugation (3000 g, 10 min), and the supernatant was removed to obtain 50 g of wet cells. The wet cells were washed twice by redispersing and centrifuging with 200 mL of water. 250 mL of ethyl acetate was added thereto, and extraction was performed at room temperature for 2 hours. Thereafter, filtration with filter paper was performed, and the residue was further extracted with 250 mL of ethyl acetate for 2 hours at room temperature. After combining each solvent, the solvent was distilled off and the amount of PHA contained in the obtained extract was measured. When the recovery rate and purity of the obtained PHA were calculated, the recovery rate was 32.6% and the purity was 19.5%.

(Comparative Example 2)
After adding 0.5 g of a sulfuric acid band to 100 mL of a cultured bacterial cell solution containing 2.7 g of PHA and stirring for a while, the pH was adjusted to 7 to aggregate the bacterial cells. Thereafter, the cells were precipitated by centrifugation (3000 g, 10 min), and the supernatant was removed to obtain 50 g of wet cells. The wet cells were washed twice by redispersing and centrifuging with 200 mL of water. Then, 10 g of dry cells were obtained by freeze-drying the wet cells. 100 mL of isopropanol was added to the dried cells and extracted at 80 ° C. for 1 hour. The residue was filtered through filter paper, and the residue was further extracted with 100 mL of isopropanol at 80 ° C. for 1 hour. After combining each solvent, the solvent was distilled off and the amount of PHA contained in the obtained extract was measured. When the recovery rate and purity of the obtained PHA were calculated, the recovery rate was 74% and the purity was 45%.

(Comparative Example 3)
In Comparative Example 2, the same operation was performed except that the solvent was changed from isopropanol to acetone and the extraction temperature was room temperature. When the recovery rate and purity of the resulting PHA were calculated, the recovery rate was 81% and the purity was 48%.

Claims (24)

  A method for producing PHA by separating and purifying it from a biological cell containing polyhydroxyalkanoic acid (PHA), comprising a step of pretreating the biological cell with an organic solvent that can be mixed with water. A characteristic PHA manufacturing method.   The method for producing a PHA according to claim 1, wherein the pretreatment step is a step of washing biological cells containing PHA with a mixed solution of the organic solvent and water.   The method for producing PHA according to claim 1, comprising a step of aggregating biological cells before the pretreatment step.   The PHA production method according to any one of claims 1 to 3, wherein the organic solvent is contained in the mixed solution of the organic solvent and water in the range of 1 wt% to 99 wt%.   5. The PHA production method according to claim 4, wherein an organic solvent is contained in a range of 30 wt% to 99 wt% in the mixed solution of the organic solvent and water.   6. The method for producing PHA according to claim 5, wherein the organic solvent and water are contained in a range of 40 wt% to 80 wt% in the mixed solution of the organic solvent and water.   The method for producing PHA according to any one of claims 1 to 6, wherein the organic solvent used is selected from the group of solvents consisting of alcohols, ketones, ethers and nitriles.   The method for producing PHA according to claim 7, wherein the organic solvent used is selected from ethanol, isopropanol, and acetone.   The method according to any one of claims 1 to 8, further comprising a step of extracting PHA using an organic solvent mixed with water and using the same organic solvent as the step of pretreating the biological cells. PHA manufacturing method.   The method for producing PHA according to claim 9, wherein the extraction temperature of the PHA with the organic solvent is 10 to 130 ° C.   The method for producing PHA according to any one of claims 1 to 10, wherein the PHA is a polyester comprising a constituent component selected from 3-, 4-, or 5-hydroxyalkanoic acid having 4 to 16 carbon atoms.   The method for producing PHA according to claim 11, wherein the PHA is a polyester comprising a constituent selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. The method for producing PHA according to any one of claims 1 to 10, wherein the PHA is a copolyester composed of a constituent selected from 3-, 4-, or 5-hydroxyalkanoic acids having 4 to 16 carbon atoms.
  The method for producing PHA according to claim 13, wherein the PHA is a copolyester comprising a constituent selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.   The method for producing PHA according to claim 13, wherein the PHA is a copolyester containing at least three types of 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.   The method for producing PHA according to claim 13, wherein the PHA is a copolyester containing at least 5 kinds of 4-hydroxyalkanoic acids having 4 to 16 carbon atoms.   The said PHA is a copolyester which consists of a structural component selected from C4-C16 3-, 4-, or 5-hydroxyalkanoic acid containing an at least 1 unsaturated bond. A method for producing PHA as described above.   The method for producing PHA according to claim 17, wherein the PHA is a copolyester composed of a component selected from 4-hydroxy alkanoic acids having 4 to 16 carbon atoms and containing at least one unsaturated bond.   The method for producing PHA according to claim 1, wherein the biological cells containing PHA are bacteria or yeast. PHA organism cells containing Bacillus (Bacillus) genus, Ralstonia (Ralstonia) genus Cupriavidus (Cupriavidus) genus Wauterushia (Wautersia) genus Aeromonas (Aeromonas) genus Escherichia (Esherichia) genus Pseudomonas (Pseudomonas) genus, Candy da (Candida) genus Alcaligenes (Alcaligenes) genus Comamonas (Comamonas) PHA production method according to claim 19 characterized in that it is a microorganism belonging to any genus. 21. The method for producing PHA according to claim 20, wherein the biological cell containing PHA is a microorganism belonging to the genus Pseudomonas .   Furthermore, the process of changing the molecular weight of PHA is included, The PHA manufacturing method in any one of Claims 1-21 characterized by the above-mentioned.   Furthermore, the PHA manufacturing method in any one of Claims 1-22 including the process of moving a container for PHA with a density | concentration of 80% or more at the temperature of 10 degreeC or more. PHA acquired by the PHA manufacturing method in any one of Claims 1-23.