JP2002256066A - New polyhydroxyalknoate containing 3-hydroxy-5-(4- cyanophenoxy)valeric unit, and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymer capable of raising a content of 3-hydroxy-5-(4- cyanophenoxy)valeric unit which has hitherto been hardy incorporated with PHA and its production method, and to provide a production method of this polymer. SOLUTION: To obtain PHA having 3-hydroxy-5-(4-cyanophenoxy)valeric unit shown by chemical formula [1], of 1% or above by culturing microbes in media containing a compound shown by formula [4] and separating it from cells after culturing for refining.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel polyhydroxyalkanoate (hereinafter sometimes abbreviated as PHA). Further, the present invention relates to a very efficient method for producing PHA using a microorganism having an ability to produce PHA and accumulate in the cells.

[0002]

2. Description of the Related Art It has been reported that many microorganisms produce poly-3-hydroxybutyric acid (hereinafter, sometimes abbreviated as PHB) or other PHA and accumulate in the cells ("Non-biotics"). Biodegradable Plastics Handbook ”, edited by Biodegradable Plastics Study Group, NTT Co., Ltd., P
178-197). These polymers can be used for production of various products by melt processing or the like, similarly to conventional plastics. Furthermore, because it is biodegradable, it has the advantage of being completely degraded by microorganisms in nature,
It does not remain in the natural environment and cause pollution unlike many conventional synthetic polymer compounds. It is also excellent in biocompatibility and is expected to be used as a medical soft member.

[0003] Such PHA produced by microorganisms depends on the type of microorganism used for the production, the composition of the medium, the culture conditions, and the like.
It is known that various compositions and structures can be obtained, and studies on such composition and structure control have been made mainly from the viewpoint of improving the physical properties of PHA.

[1] First, 3-hydroxybutyric acid (hereinafter referred to as 3H
Examples of the biosynthesis of PHA obtained by polymerizing a monomer unit having a relatively simple structure such as B) are as follows.

(A) 3HB and 3-hydroxyvaleric acid (hereinafter referred to as 3
HV) containing, for example, Alcaligenes eutrophus H16 strain (Alc
aligenes eutropus H16, ATCC No. 17699) and its mutants can be obtained by changing the carbon source at the time of cultivation to obtain 3-hydroxybutyric acid (hereinafter sometimes abbreviated as 3HB) and 3-hydroxyvaleric acid (hereinafter, referred to as 3HB). It has been reported to produce copolymers (which may be abbreviated as 3HV) in various composition ratios (Japanese Patent Application Laid-Open No. Hei 6-15604, Japanese Patent Application Laid-Open No.
No. 14,352, Japanese Patent Application Laid-Open No. Hei 8-19227).

[0006] JP-A-5-74492 discloses a genus Methylobacterium (Methylobacterium sp.), A genus Paracoccus (Paracoccus sp.), And a genus Alcaligene.
s sp.) and a method of producing a copolymer of 3HB and 3HV by contacting a microorganism of the genus Pseudomonas sp. with a primary alcohol having 3 to 7 carbon atoms.

(B) those containing 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as 3HHx) JP-A-5-93049 and JP-A-7-2650
No. 65 discloses Aeromonas caviae.
caviae) is cultured using oleic acid or olive oil as a carbon source to produce 3HB and 3-hydroxyhexanoic acid.
(Hereinafter sometimes abbreviated as 3HHx) is disclosed.

(C) 3HB and 4-hydroxybutyric acid (hereinafter referred to as 4H
Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IFO 13852 strain (Comamonas acidovorans
IFO 13852) has gluconic acid and 1,4-
It is disclosed that a polyester having 3HB and 4-hydroxybutyric acid as monomer units is produced by culturing using butanediol.

(D) Containing 3-hydroxyalkanoate having 6 to 12 carbon atoms In Patent Publication No. 2642937, Pseudomonas oleovorans ATCC 29347 (Pseudomonas oleovo)
rans ATCC 29347) discloses that by providing an acyclic aliphatic hydrocarbon as a carbon source, a PHA having a monomer unit of 3-hydroxyalkanoate having 6 to 12 carbon atoms is produced.

(E) Biosynthesis using a single fatty acid as a carbon source.
The product is almost the same as (d). Appl. Environ. Microbiol, 58 (2), 746 (1992) shows that Pseudomonas resnobolans
sinovorans), using octanoic acid as the sole carbon source, 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid (1: 15: 7 ratio).
5: 9) as a monomer unit, and 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid (quantity ratio) using hexanoic acid as a single carbon source. 8: 62: 23: 7)
It has been reported to produce polyesters having as a unit.

All of these (a) to (e) are as follows: (1) β-oxidation of hydrocarbons or the like by microorganisms (2) Synthesized by fatty acid synthesis from sugar, PHA comprising a monomer unit having a group, that is, "usual PH"
A ".

(1) Normally, PHA biosynthesis is performed using PHA synthase (PHA synthase) using "D-3-hydroxyacyl-CoA", which is generated as an intermediate of various metabolic pathways in cells, as a substrate.
synthase).

Here, “CoA” means “coenzyme A (coenzy
me A) ". Then, as described in the prior art of (d) and (e) above, when a fatty acid such as octanoic acid or nonanoic acid is used as a carbon source, biosynthesis of PHA occurs during the “β oxidation cycle”. D-3-hydroxyacyl-CoA "
Is believed to be performed as a starting material.

In the following, P via the “β oxidation cycle”
1 shows a reaction until HA is biosynthesized.

[0015]

Embedded image

(2) On the other hand, when PHA is biosynthesized using a saccharide such as glucose as a substrate, "D-hydroxyacyl-ACP" generated in the "fatty acid synthesis pathway" is converted into "D-hydroxyacyl-ACP". "3-Hydroxyacyl-CoA" is said to be performed as a starting material.

Here, "ACP" means "acyl carrier protein". In the above (e), 3HA having a longer chain length than the raw material fatty acid
It is considered that the monomer unit passes through this fatty acid synthesis pathway.

[2] However, such a microorganism-produced PHA
In consideration of a wider range of applications of, for example, application as a functional polymer, a PHA "unusual PHA" in which a substituent other than an alkyl group is introduced into a side chain is expected to be extremely useful. Examples of the substituent include those containing an aromatic ring (phenyl group, phenoxy group, benzoyl group, etc.),
Examples include unsaturated hydrocarbons, ester groups, allyl groups, cyano groups, halogenated hydrocarbons, and epoxides.

(F) Among them, there are the following reports on unsaturated hydrocarbons which are an extension of the above [1].

Int. J. Biol. Macromol., 16 (3), 119
(1994) include Pseudomonas sp.
sp. 61-3 strain), using sodium gluconate as a single carbon source, 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid. It has been reported to produce polyesters having units of 3-hydroxyalkenoic acid such as hydroxyalkanoic acid and 3-hydroxy-5-cis-decenoic acid, and 3-hydroxy-5-cis-dodeceneic acid. In this document, the synthesis using the fatty acid synthesis pathway of the above (2) is described in detail.

[3] Among the "unusual PHAs",
Research on PHA having an aromatic ring has been actively conducted.

(G) those containing a phenyl group or a partially substituted derivative thereof: Makromol. Chem., 191, 1957-1965 (1990) and Mac
Romolecules, 24, 5256-5260 (1991), reported that Pseudomonas oleovorans produces PHA containing 5-hydroxy-5-phenylvaleric acid as a unit using 5-phenylvaleric acid as a substrate. ing.

More specifically, Pseudomonas oleovorans uses 5-phenylvaleric acid (hereinafter sometimes abbreviated as PVA) and nonanoic acid as substrates (molar ratio 2: 1, total concentration
10mmol / L), 3HV, 3-hydroxyheptanoic acid, 3-
Hydroxynonanoic acid, 3-hydroxyundecanoic acid, 3-
Hydroxy-5-phenylvaleric acid (hereinafter sometimes abbreviated as 3HPV) as a monomer unit, 0.6: 16.0: 4
PHA containing 1.1: 1.7: 40.6 in a quantity ratio of 160 mg per liter of culture solution (dry weight ratio to bacterial cells: 31.6%) was produced, and PVA and octanoic acid were used as substrates (molar ratio 1: 1,
PHA containing 3HHx, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3HPV as monomer units in a ratio of 7.3: 64.5: 3.9: 24.3 (total concentration 10 mmol / L)
200 mg per liter of culture solution (dry weight ratio to cells)
39.2%). The PHA in this report is considered to have been synthesized mainly via the β-oxidation pathway because nonanoic acid and octanoic acid are used.

Macromolecules, 29, 1762-1766 (1996)
Pseudomonas oleovorans (Pseudomonas oleovorans) with 5- (4'-tolyl) valeric acid as substrate
It has been reported to produce PHA containing -hydroxy-5- (4'-tolyl) valeric acid as a unit.

Macromolecules, 32, 2889-2895 (1999)
Pseudomonas oleovorans (5- (2 ', 4'-dinitrophenyl) valeric acid as substrate)
leovorans) has been reported to produce PHA containing 3-hydroxy-5- (2 ', 4'-dinitrophenyl) valeric acid and 3-hydroxy-5- (4'-nitrophenyl) valeric acid as units. I have.

(H) Containing Phenoxy Group or Partially Substituted PHA PHA having a phenoxy group in the side chain is more thermally stable than PHA having a normal side chain of an aliphatic hydrocarbon. Because of its excellent properties, attempts have been made to impart further functionality by introducing a substituent into the phenoxy group.

Macromol. Chem. Phys., 195, 1665-
In 1672 (1994), Pseudomonas oleovorans (Pseudomonas o.
leovorans) with 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-5-phenoxyvaleric acid
It has been reported to produce PHA copolymers of hydroxy-9-phenoxynonanoic acid.

Also, Macromolecules, 29,3432-3435
(1996), using Pseudomonas oleovorans, PHA containing units of 6-phenoxyhexanoic acid to 3-hydroxy-4-phenoxybutyric acid and 3-hydroxy-6-phenoxyhexanoic acid as a unit from 8-phenoxyoctanoic acid. 3-hydroxy-4-phenoxybutyric acid and 3-
PH containing hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid as units
A is converted from 11-phenoxyundecanoic acid to 3-hydroxy
It has been reported to produce PHA containing -5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid as units. Excerpts of the polymer yields in this report are as follows.

[0029]

[Table 1]

Japanese Patent No. 2989175 discloses 3-hydroxy-5- (monofluorophenoxy) pentanoate
(3H5 (MFP) P) unit or 3-hydroxy-5
-(Difluorophenoxy) pentanoate (3H5 (DF
P) Homopolymer consisting of P) units, at least 3H
Copolymers containing 5 (MFP) P units or 3H5 (DFP) P units; Pseudomonas putida for synthesizing these polymers; and a method for producing the above polymers using Pseudomonas sp.

These productions are performed in the following "two-stage culture".
It is done in. Culture time: 1st stage, 24 hours; 2nd stage, 96 hours The substrate in each stage and the resulting polymer are shown below. (1) Obtained polymer: 3H5 (MFP) P homopolymer First substrate: citric acid, yeast extract Second substrate: monofluorophenoxyundecanoic acid (2) Obtained polymer: 3H5 (DFP) P homopolymer Second substrate: citric acid, yeast extract Second substrate: difluorophenoxyundecanoic acid (3) Obtained polymer: 3H5 (MFP) P copolymer First substrate: octanoic acid or nonanoic acid, yeast extract Second substrate : Monofluorophenoxyundecanoic acid (4) Polymer obtained: 3H5 (DFP) P copolymer First substrate: Octanoic acid or nonanoic acid, yeast extract Second substrate: Difluorophenoxyundecanoic acid Phenoxy group in which the side chain end is substituted with 1 or 2 fluorine atoms It is possible to synthesize a polymer having a high melting point and to give stereoregularity and water repellency while maintaining good workability.

In addition to such a fluorine-substituted product, a cyano- or nitro-substituted product has also been studied.

Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996) include:
Pseudomonas oleovor
ans) ATCC 29347 strain and Pseudomonas putida (Pse
udomonas putida) using KT 2442
PHAs containing p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as a substrate and 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit
Production has been reported.

These reports are different from general PHA in which the side chain is an alkyl group, all of which have an aromatic ring in the side chain of PHA, and are useful in obtaining a polymer having physical properties derived therefrom. is there.

[0035]

When attention is paid to phenoxyalkanoic acid substituted with a cyano group, the content of PHA containing 3-hydroxy-6-cyanophenoxyhexanoic acid as a unit is 24.2%. Optical harmonic second generation (Second Harmonic Genera) which is a characteristic of nonlinear optical materials
(SHG) is confirmed.

On the other hand, a P-containing unit containing 3-hydroxy-5-cyanophenoxyvaleric acid having one less carbon atom as a unit
Regarding HA, there is no report that the content of the unit is 1% or more.

An object of the present invention is to provide a PHA containing at least 1% of a 3-hydroxy-5-cyanophenoxyvaleric acid unit,
It is to provide the production method.

[0038]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have reached the following invention. That is, the first aspect of the present invention is a polyhydroxyalkanoate itself containing 1 unit% or more of the unit represented by the chemical formula [1].

[0039]

Embedded image

The polyhydroxyalkanoate of the present invention comprises units other than the unit represented by the chemical formula [1]:
It is a polyhydroxyalkanoate which is a unit represented by the chemical formula [2].

[0041]

Embedded image

Further, the polyhydroxyalkanoate of the present invention may contain a unit represented by the chemical formula [3] as a unit other than the units represented by the chemical formulas [1] and [2].

[0043]

Embedded image

The polyhydroxyalkanoate of the present invention is a polyhydroxyalkanoate having a number average molecular weight in the range of 10,000 to 300,000.

The second aspect of the present invention comprises a step of culturing a microorganism capable of synthesizing a polyhydroxyalkanoate containing a unit derived from the compound in a medium containing the compound represented by the chemical formula [4]. A method for producing polyhydroxyalkanoate, characterized in that:

[0046]

Embedded image

Preferably, after the step of culturing the microorganism, a step of recovering polyhydroxyalkanoate from cells of the cultured microorganism is included.

[0048]

DETAILED DESCRIPTION OF THE INVENTION That is, the present inventors have determined that a strain having the above-mentioned synthetic system is a fatty acid having a functional group other than linear aliphatic alkyl added to its terminal using polypeptone or glucose as a substrate. When cultured with-(4-cyanophenoxy) valeric acid, "unus containing at least 1% or more of 3-hydroxy-5-cyanophenoxyvaleric acid units is contained.
ual PHA "was obtained, and the present invention has been accomplished.

Further, according to the present invention, the P produced by a microorganism
Preferably, the method further comprises a step of isolating HA.

The PHA of the present invention is generally composed of only the R-isomer and is an isotactic polymer.

Hereinafter, the microorganism used in the present invention, the culturing step, and the like will be described.

(Microorganism) The microorganism used in the method of the present invention includes:
Any microorganism can be used as long as it can produce a polyester containing 1% or more of the unit represented by the chemical formula [1] by culturing in a medium containing the compound represented by the chemical formula [4]. In addition, a plurality of microorganisms can be mixed and used as needed within a range where the object of the present invention can be achieved. One example is Pseudomonas.
nas). For more information,
The microorganism is Pseudomonas chicory eye YN2 strain (Pseudo
monas cichorii YN2; FERM BP-7375), Pseudomonas cichorii strain H45 (Pseudomonas cichorii)
H45, FERM BP-7374), Pseudomonas jessenii P161, FER
MBP-7376). These three types of microorganisms have been deposited with the Ministry of Economy, Trade and Industry at the Institute of Biotechnology and Industrial Technology, and are the microorganisms described in Japanese Patent Application No. 11-371863.

The details of the YN2 strain, H45 strain and P161 strain are shown below. <Bacteriological properties of YN2 strain> (1) Morphological properties Cell shape and size: bacilli, 0.8 μm × 1.5-2.0 μm Cell polymorphism: no Motility: yes Sporulation: no Gram stain : Negative Colony shape: Circular, whole edge smooth, low convex, surface smooth, glossy, translucent (2) Physiological properties Catalase: Positive oxidase: Positive O / F test: Reduction of oxidized (non-fermentative) nitrate: Negative Indole production: Positive Glucose acidification: Negative Arginine dihydrolase: Negative urease: Negative Esculin hydrolysis: Negative Gelatin hydrolysis: Negative β-galactosidase: Negative Fluorescent dye production on King's B agar: Positive 4% NaCl Growth: Positive (weak growth) Accumulation of poly-β-hydroxybutyric acid: Negative (*) Hydrolysis of Tween80: Positive * Judged by staining nutrient agar colonies with Sudan Black. (3) Substrate utilization glucose: positive L-arabinose: positive D-mannose: negative D-mannitol: negative N-acetyl-D-glucosamine: negative maltose: negative potassium gluconate: positive n-capric acid: positive adipic acid : Negative dl-malic acid: Positive Sodium citrate: Positive Phenyl acetate: Positive <Bacteriological properties of H45 strain> (1) Morphological properties Cell shape and size: Bacillus, 0.8 μm × 1.0-1.2 μm cells Polymorphism of: No Motility: Yes Sporulation: No Gram stain: Negative Colony shape: Round, whole edge smooth, low convex, surface layer smooth, gloss, cream color (2) Physiological properties Catalase: Positive oxidase: Positive O / F test: Oxidized nitrate reduction: Negative Indole formation: Negative Glucose acidification: Negative Arginine dihydrolase: Negative urease: Negative Esculin hydrolysis: Negative gelatin Hydrolysis: Negative β-galactosidase: Negative Fluorescent dye production on King's B agar: Positive Growth with 4% NaCl: Negative Accumulation of poly-β-hydroxybutyric acid: Negative (3) Substrate utilization glucose: Positive L- Arabinose: Negative D-Mannose: Positive D-mannitol: Positive N-acetyl-D-glucosamine: Positive Maltose: Negative potassium gluconate: Positive n-Capric acid: Positive Adipic acid: Negative dl-malic acid: Positive sodium citrate: Positive Phenyl acetate: Positive (2) Physiological properties Catalase: Positive oxidase: Positive O / F test: Reduction of oxidized nitrate: Positive Indole formation: Negative Glucose acidification: Negative Arginine dihydrolase: Positive urease: Negative Esculin hydrolysis: Negative gelatin hydrolysis : Negative β-galactosidase: Negative Fluorescent dye production in King's B agar: Positive (3) Substrate utilization glucose: Positive L-arabinose: Positive D-mannose: Positive D-mannitol: Positive N-acetyl-D-glucosamine : Positive maltose: negative potassium gluconate: positive n-capric acid: positive adipic acid: negative dl-malic acid: positive sodium citrate: positive phenyl acetate: positive (culture step) Normal culture of, for example, the preparation of stock strains, ensuring the number and activity of bacteria required for PHA production The like growth for, appropriately selected and used medium containing ingredients necessary for the growth of the microorganisms used. For example, any medium such as a general natural medium (meat broth, yeast extract, etc.) or a synthetic medium to which a nutrient is added can be used, as long as it does not adversely affect the growth and survival of the microorganism. Culture conditions such as temperature, aeration and stirring are appropriately selected according to the microorganism used.

In order to produce PHA containing 3-hydroxy-5-cyanophenoxyvaleric acid as a monomer unit using the PHA-producing microorganism as described above, it is necessary to prepare PHA.
As a raw material for HA production, an inorganic medium or the like containing at least 5- (4-cyanophenoxy) valeric acid corresponding to the monomer unit represented by the above chemical formula [4] and a carbon source for growing microorganisms is used. Can be used. 5- (4-cyanophenoxy) valeric acid is 0.01% to 1% per medium
(w / v), more preferably 0.02% to 0.2%.

Although the water solubility is not always good, there is no problem even if it is in a suspended state by using the microorganism of the present invention. In some cases, it may be contained in the medium in a form dissolved or suspended in a solvent such as 1-hexadecene or n-hexadecane. in this case,
It is necessary that the concentration of the solvent be 3% or less based on the medium solution.

As the carbon source for growth, nutrients such as yeast extract, polypeptone, and meat extract can be used. In addition, one or two steps from sugars, organic acids generated as intermediates in the TCA cycle, and TCA cycle. Can be selected as appropriate from the usefulness as a substrate for the strain to be used, from organic acids or salts thereof, amino acids or salts thereof generated through the biochemical reaction of the above.

Among these, saccharides include glyceraldehyde, erythrose, arabinose, xylose,
Aldoses such as glucose, galactose, mannose and fructose, glycerol, erythritol,
One or more compounds selected from alditols such as xylitol, aldonic acids such as gluconic acid, uronic acids such as glucuronic acid and galacturonic acid, and disaccharides such as maltose, sucrose and lactose can be suitably used.

As the organic acid or a salt thereof, one or more compounds selected from pyruvic acid, malic acid, lactic acid, citric acid, succinic acid, oxaloacetic acid, isocitric acid, ketoglutaric acid, fumaric acid and salts thereof are used. It can be suitably used.

As the amino acid or a salt thereof, one or more compounds selected from glutamic acid, aspartic acid and a salt thereof can be suitably used. Among these, it is preferable to use polypeptone or a saccharide, and among the saccharides, it is more preferable that at least one selected from the group consisting of glucose, fructose, and mannose. These substrates are usually 0.1% to 5% per medium.
% (W / v), more preferably 0.2 to 2%.

As an example, D-glucose is 0.1%
From about 5.0%, and about 0.01% to 1.0% of 5- (4-cyanophenoxy) valeric acid in an inorganic medium or the like. PHA can be extracted. The same amount of yeast extract may be given instead of D-glucose.

As a method for producing and accumulating PHA in microorganisms, once the cells are sufficiently grown, the cells are transferred to a medium having a limited nitrogen source such as ammonium chloride, and a compound serving as a substrate for the target unit is added. Further culturing in this state may improve the productivity. Specifically, adoption of a multi-stage system in which the above-described steps are connected in a plurality of stages is exemplified. For example, D-glucose is about 0.1% to 5.0%, and
5- (4-cyanophenoxy) valeric acid from 0.01% to 1.0%
After culturing from the late logarithmic growth phase to the stationary phase in an inorganic medium containing a certain amount of cells, the cells were collected by centrifugation, etc.
There is a method of further culturing on an inorganic medium containing about 0.01% to 1.0% of (cyanophenoxy) valeric acid and having a limited or almost no nitrogen source.

The cultivation temperature may be any temperature at which the above strain can be satisfactorily grown, for example, 15 to 40 ° C., preferably 20 to 35 ° C., and more preferably about 20 to 30 ° C.

The culture can be carried out by any culture method, such as liquid culture and solid culture, in which the microorganism grows and PHA is produced. Further, types such as batch culture, fed-batch culture, semi-continuous culture, and continuous culture are not limited. As a form of the liquid batch culture, there are a method of supplying oxygen by shaking with a shaking flask and a method of supplying oxygen by stirring and aeration using a jar fermenter.

As the inorganic medium used in the above-mentioned culture method, a phosphorus source (for example, phosphate), a nitrogen source (for example,
Any one containing components necessary for the growth of the microorganism, such as ammonium salts and nitrates, may be used, and examples thereof include an MSB medium and an M9 medium.

The inorganic salt medium (M9) used in one method of the present invention
The composition of the medium is shown below.

[M9 medium] 6.2 g of Na ₂ HPO ₄ 3.0 g of KH ₂ PO ₄ 0.5 g of NaCl 1.0 g of NH ₄ Cl (pH 7.0 in 1 liter of medium) Further, good growth and production of PHA For the above, the above-mentioned trace component solution 0.3% (v /
v) It is necessary to add about.

[0067] [minor component solution] nitrilotriacetic _{acid: 1.5; MgSO 4: 3.0;} MnSO 4: 0.5
_{; NaCl: 1.0; FeSO 4:} 0.1; CaCl 2: 0.1; CoC
_{l 2: 0.1; ZnSO 4:} 0.1; CuSO 4: 0.1; AlK (S
O ₄ ) ₂ : 0.1; H ₃ BO ₃ : 0.1; Na ₂ MoO ₄ : 0.1; NiCl
₂ : 0.1 (pH of 1 in 1 liter of medium, pH 7.0) For obtaining PHA from the culture solution according to the present invention, a commonly used method can be applied. When PHA is secreted into the culture solution, an extraction and purification method from the culture solution is used, and when PHA is accumulated in the cells, an extraction and purification method from the cells is used. For example, for the recovery of PHA from cultured microbial cells of microorganisms, extraction with an organic solvent such as chloroform, which is usually performed, is the simplest, but dioxane, tetrahydrofuran, acetonitrile, and acetone may be used in addition to chloroform. . In an environment where an organic solvent is difficult to use, treatment with a surfactant such as SDS, treatment with an enzyme such as lysozyme,
By removing bacterial components other than PHA by treatment with an agent such as EDTA, P
A method of recovering only HA can be adopted.

The cultivation of the microorganism of the present invention, the production and accumulation of PHA by the microorganism of the present invention, and the recovery of PHA from the bacterial cells in the present invention are not limited to the methods described above. Absent.

Examples will be described below. In the following, “%” is based on weight, unless otherwise specified.

[0070]

[Example 1] Polypeptone 0.5% and 5- (4-
(Cyanophenoxy) 200 mL of M9 medium containing 0.1% valeric acid
Then, a colony of the YN2 strain on an agar plate was inoculated into
The cells were cultured in a mL shake flask at 30 ° C for 96 hours. After the culture, the cells were harvested by centrifugation, washed with methanol, and lyophilized. After weighing the dried cells, chloroform was added and the polymer was extracted at 60 ° C for 24 hours. After chloroform from which the polymer was extracted was filtered and concentrated by an evaporator, a portion precipitated and solidified with cold methanol was collected and dried under reduced pressure to obtain a target polymer. The weight of the dried cells was 120 mg, and the weight of the obtained polymer was 7 mg.

The molecular weight of the obtained polymer was measured by gel permeation chromatography (GPC).
(Tosoh HLC-8220 GPC, column: Tosoh TSK-
GEL Super HM-H, solvent: chloroform, converted to polystyrene). As a result, the obtained polymer had Mn = 45000,
Mw = 80,000.

The structure of the obtained polymer was determined by ¹ H-NM.
R (FT-NMR: Bruker DPX400;
¹ H resonance frequency: 400 MHz; measured nuclide: ¹ H; solvent used: C
DCl ₃ ; reference: capillary-encapsulated TMS / CDCl ₃ ;
Measurement temperature: room temperature). FIG. 1 shows the spectrum chart and the assignment of each peak.

As a result of ¹ H-NMR assignment, 3-hydroxy-
It was shown that at least 2% or more of the 5- (4-cyanophenoxy) valeric acid unit was introduced.

Example 2 Glucose 0.5% and 5- (4-
Cyanophenoxy) M9 medium containing valeric acid 0.05% 200m
L was inoculated with a colony of the YN2 strain on an agar plate,
The cells were cultured in a 0 mL shake flask at 30 ° C. for 45 hours. After the culture, the cells were harvested by centrifugation, and the cells were transferred to a medium prepared by adding 0.5% of glucose and 0.05% of 5- (4-cyanophenoxy) valeric acid to M9 medium containing no NH ₄ Cl component. And incubated at 30 ° C. for 48 hours. After the culture, the cells were harvested by centrifugation, washed with methanol, and lyophilized. After weighing the dried cells, chloroform was added and the mixture was added at 60 ° C.
The polymer was extracted for 4 hours. After the chloroform from which the polymer was extracted was filtered and concentrated by an evaporator,
The portion precipitated and solidified with cold methanol was collected and dried under reduced pressure to obtain a target polymer. Weight of dried cells is 200
mg, and the weight of the obtained polymer was 46 mg.

The molecular weight of the obtained polymer was measured by gel permeation chromatography (GPC).
(Tosoh HLC-8220GPC, column: Tosoh TSK-G
EL Super HM-H, solvent: chloroform, converted to polystyrene). As a result, the obtained polymer had two molecular weight peaks, and Mn = 2400000 and Mw = 3400000, respectively.
And Mn = 40,000 and Mw = 70,000. Since UV (254 nm) absorption was observed only in the latter peak, the target polymer is considered to be the latter.

The structure of the obtained polymer was determined by ¹ H-NM
R (FT-NMR: Bruker DPX400;
¹ H resonance frequency: 400 MHz; measured nuclide: ¹ H; solvent used: C
DCl ₃ ; reference: capillary-encapsulated TMS / CDCl ₃ ;
Measurement temperature: room temperature). FIG. 2 shows the spectrum chart and the assignment of each peak.

As a result of ¹ H-NMR assignment, 3-hydroxy-
It was shown that at least 8% or more of the 5- (4-cyanophenoxy) valeric acid unit was introduced.

Example 3 A polymer was obtained in the same manner as in Example 1 except that the strain was changed to H45 strain. The weight of the dried cells is
120 mg and the weight of the obtained polymer was 4 mg. As a result of measuring ¹ H-NMR in the same manner as in Examples 1 and 2, 3
It was shown that at least 3% or more of the -hydroxy-5- (4-cyanophenoxy) valeric acid unit had been introduced.

Example 4 A polymer was obtained in the same manner as in Example 1 except that the strain was changed to P161 strain. The weight of the dried cells was 110 mg, and the weight of the obtained polymer was 7 mg.
As a result of measuring ¹ H-NMR in the same manner as in Examples 1 and 2,
It was shown that at least 3% or more of the 3-hydroxy-5- (4-cyanophenoxy) valeric acid unit had been introduced.

[0080]

According to the method of the present invention, 3-hydroxy-5
A polyhydroxyalkanoate containing 1% or more of-(4-cyanophenoxy) valeric acid unit and a method for producing the same are provided.

[Brief description of the drawings]

FIG. 1 is a ¹ H-NMR spectrum of a polyhydroxyalkanoate obtained in Example 1.

FIG. 2 shows the ¹ H-NMR spectrum of the polyhydroxyalkanoate obtained in Example 2.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tsukasa Homma 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Etsuko Sugawa 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo F-term in Canon Inc. (Reference) 4B064 AD83 BA15 BH04 CA02 CD12 DA20 4J029 AA02 AB01 AB04 AC02 AD01 AE06 EA05 ED00 EF01

Claims (13)

[Claims] 1. A polyhydroxyalkanoate containing 1 unit% or more of the unit represented by the chemical formula [1]. Embedded image 2. The polyhydroxyalkanoate according to claim 1, wherein the unit other than the unit represented by the chemical formula [1] is a unit represented by the chemical formula [2]. Embedded image 3. The polyhydroxyalkanoate according to claim 1, wherein the unit other than the units represented by the chemical formulas [1] and [2] is a unit represented by the chemical formula [3]. Embedded image 4. A number average molecular weight of 10,000 to 3,000.
The polyhydroxyalkanoate according to any one of claims 1 to 3, which is in the range of 00. 5. A method for culturing a microorganism capable of synthesizing a polyhydroxyalkanoate containing a unit derived from the compound in a medium containing the compound represented by the chemical formula [4]. A method for producing an alkanoate. Embedded image 6. The method according to claim 5, further comprising a step of recovering polyhydroxyalkanoate from cells of the cultured microorganism after the step of culturing the microorganism. 7. The microorganism according to claim 5, wherein the microorganism is capable of producing a polyhydroxyalkanoate containing 1 unit% or more of the unit represented by the chemical formula [1] from the compound represented by the chemical formula [4]. The production method described in 1. Embedded image 8. The method according to claim 7, wherein the polyhydroxyalkanoate contains a unit represented by the chemical formula [2]. Embedded image 9. The method according to claim 7, wherein the polyhydroxyalkanoate contains a unit represented by the chemical formula [3]. Embedded image 10. The method according to claim 10, wherein the microorganism is Pseudomonas.
The method according to any one of claims 5 to 9, which is a microorganism belonging to the genus nas). 11. The method according to claim 11, wherein said microorganism is Pseudomonas cichorii YN2; FERM.
BP-7375). 12. The method according to claim 11, wherein the microorganism is Pseudomonas cichorii H45, FERM.
BP-7374). 13. The method according to claim 11, wherein said microorganism is Pseudomonas jessenii P161, F161.
The method according to claim 10, which is ERM BP-7376).