JP2002256064A - New polyhydroxyalknoate containing 3-hydroxy-5- phenoxyvaleric unit and 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 hardly been contained conventionally in 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] and 3-hydroxy-5-phenoxyvaleric unit shown by chemical formula [2], of 1% or above, respectively, by culturing microbes in media containing compounds shown by formulae [4] and [5] and separating them from cells after culturing to be refined.

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, the “D-
"D-3-" converted from "3-hydroxyacyl-ACP"
"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 product 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) PHA having a phenoxy group or a partially substituted phenoxy group 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.

[0028]

[Table 1] Japanese Patent No. 2989175 discloses 3-hydroxy-5-
(Monofluorophenoxy) pentanoate (3H5 (MF
A homopolymer consisting of (P) P) units or 3-hydroxy-5- (difluorophenoxy) pentanoate (3H5 (DFP) P) units, at least 3H5 (MFP) P
Copolymers containing a unit or 3H5 (DFP) P unit; Pseudomonas putida for synthesizing these polymers; and a method for producing the above polymer 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.

In Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996), Pseudomonas oleovoran (Pseudomonas oleovoran)
s) ATCC 29347 strain and Pseudomonas putida
omonas putida) using KT2442 strain, octanoic acid and p-
Production of PHA containing cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as a substrate and containing 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit 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.

[0033]

Among them, PHA having a cyanophenoxy group is very important because the cyano group can exhibit various functions or can be used for further conversion reactions. It is considered to be a functional group. However, in a PHA containing a 3-hydroxy-p-cyanophenoxyvaleric acid unit, the introduction ratio is only 1% or less, and the physical properties such as a low melting point and a low glass transition point and a very soft property are insufficient. Only what was obtained.

An object of the present invention is to provide a novel PHA containing a 3-hydroxy-p-cyanophenoxyvaleric acid unit more than before and having physical properties as a plastic, and a method for producing the same.

[0035]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, have reached the following invention.

That is, the first aspect of the present invention is to provide a 3-hydroxy-5- (4-cyanophenoxy) valeric acid unit represented by the chemical formula [1] and a 3-hydroxy-5 represented by the chemical formula [2].
-A novel polyhydroxyalkanoate containing at least 1 unit% of each phenoxyvaleric acid unit.

[0037]

Embedded image

[0038]

Embedded image In the polyhydroxyalkanoate of the present invention, the units other than the unit represented by the chemical formula [1] and the unit represented by the chemical formula [2] are linear 3-hydroxyalkanoic acid units represented by the chemical formula [3].

[0039]

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.

A second aspect of the present invention is represented by a chemical formula [4]:
Culture of a microorganism capable of synthesizing a polyhydroxyalkanoate containing a unit derived from each compound in a medium containing 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid represented by the chemical formula [5] A method for producing a polyhydroxyalkanoate, comprising culturing a microorganism having a step of carrying out the method.

[0041]

Embedded image

[0042]

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

That is, the present inventors have developed a strain having the above-mentioned synthetic system as a fatty acid having a functional group other than linear aliphatic alkyl added to a terminal thereof using polypeptone, glucose or the like as a substrate. When cultured with (cyanophenoxy) valeric acid and 5-phenoxyvaleric acid, 3-hydroxy-
5-cyanophenoxyvaleric acid unit and 3-hydroxy
At least one -5-phenoxyvaleric acid unit
% Of "unusual PHA" which is simultaneously contained, has led to the present invention.

Also, in the present invention, the P
Preferably, the method further comprises a step of isolating HA.

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

Hereinafter, microorganisms used in the present invention, culturing steps, and the like will be described.

(Microorganism) Microorganisms used in the method of the present invention include:
Any microorganism capable of producing a polyester simultaneously containing 1% or more of the units represented by the chemical formulas [1] and [2] by culturing in a medium containing the compounds represented by the chemical formulas [4] and [5] There may be. 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 a microorganism belonging to the genus Pseudomonas. More specifically, the microorganism is Pseudomonas cichorii YN2;
MBP-7375), Pseudomonas chicory eye H45 strain (P
seudomonas cichorii H45, FERM BP-7374), Pseudomonas jessenii P161 strain (Pseudomonas jes
senii P161, FERM BP-7376). These three types of microorganisms have been deposited with the Ministry of Economy, Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology, and are disclosed in Japanese Patent Application No. 11-371863.
The microorganisms described in No.

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 <Bacteriological properties of strain P161> (1) Morphological properties Cell shape and size: spherical, φ0.6 μm rod, 0.6 μm ×
1.5-2.0 μm Cell polymorphism: Yes (elongated) Motility: Yes Sporulation: No Gram stain: Negative Colony shape: Round, whole edge smooth, low convex, surface layer smooth, pale yellow (2) Physiology 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 on 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) For normal cultivation of microorganisms used in the method for producing PHA according to the present invention, for example, for the preparation of a preserved strain, growth for ensuring the number of bacteria and active state required for PHA production, etc. A medium containing components necessary for the growth of the microorganism to be used is appropriately selected and 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 and 3-hydroxy-5-phenoxyvaleric acid as a monomer unit using the PHA-producing microorganism as described above, it is necessary to produce PHA. 5- (4-cyanophenoxy) valeric acid represented by the above chemical formula [4] corresponding to the monomer unit, 5-phenoxyvaleric acid represented by the above chemical formula [5], and a microorganism And an inorganic medium containing at least a growth carbon source. 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid are each 0.01% to 1% (w / v), more preferably 0.02%, per medium.
It is desirable that it be contained at a rate of 0.2% to 0.2%.

Although the water solubility of these compounds is not necessarily good, there is no problem even if they are 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, the concentration of the solvent is 3% with respect to the medium solution.
It is necessary to:

As the growth carbon source, nutrients such as yeast extract, polypeptone, and meat extract can be used. In addition, one or two steps from saccharides, organic acids generated as intermediates in the TCA cycle, and the 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.

Further, 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 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid from 0.01% each.
The cells are cultured in an inorganic medium or the like containing about 1.0%, and the cells can be recovered at the time from the late logarithmic growth phase to the stationary phase to extract a desired PHA. 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 have been 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
The cells are cultured from the late log phase to the stationary phase in an inorganic medium containing about 0.01% to 1.0% of 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid, and the cells are centrifuged. After collection, 5- (4-cyanophenoxy) valeric acid and 5-phenoxy valeric acid were added in 0.01 parts each.
There is a method of further culturing in an inorganic medium containing about 1% to 1.0% of a nitrogen source with 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.

The inorganic medium used in the above culture method includes 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.

[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, the usual method of extracting chloroform 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, a method of recovering only PHA by removing intracellular components other than PHA by treatment with a surfactant such as SDS, treatment with an enzyme such as lysozyme, or treatment with a chemical such as EDTA. Can also be taken.

The culture 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 above-mentioned methods. Absent.

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

[0065]

[Example 1] Polypeptone 0.5% and 5- (4-
A colony of the YN2 strain on an agar plate was inoculated into 200 mL of M9 medium containing 0.05% each of (cyanophenoxy) valeric acid and 5-phenoxyvaleric acid, and cultured in a 500 mL shake flask at 30 ° C. for 24 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 170
mg, and the weight of the obtained polymer was 26 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, two peaks were observed, the former having Mn =
2100000, Mw = 2600000, and the latter is Mn = 71000,
Mw = 150000. UV absorption (254 nm) was only in the latter, indicating that the target polymer was 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; nuclide measured: ¹ H; solvent used: C
DCl ₃ ; reference: capillary-encapsulated TMS / CDCl ₃ ;
Measurement temperature: room temperature). FIG. 1 shows the ¹ H-NMR chart. Calculated from the integrated value of each assigned peak, the ratio of the units shown in the following equation is A: B: C: D = 8:
25: 5: 62.

[0068]

Embedded image Example 2 0.5% of glucose and 0.05% of 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid respectively
200 ml of the M9 medium contained was inoculated with a colony of the YN2 strain on an agar plate, and placed in a 500 ml shake flask.
The culture was performed at 72 ° C. for 72 hours. After the culture, cells were harvested by centrifugation, and glucose was added to M9 medium containing no NH ₄ Cl component.
The cells were transferred to a medium prepared by adding 0.05% each of 0.5% and 5- (4-cyanophenoxy) valeric acid and 5-phenoxyvaleric acid, and cultured at 30 ° C. for 50 hours. After the culture, the cells were harvested by centrifugation, washed with methanol, and lyophilized. After weighing the dried cells, chloroform was added, and
Extracted the polymer 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 dried cells is 2
00 mg and the weight of the obtained polymer was 61 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, Mn was 76,000 and Mw was 180,000.

The structure of the obtained polymer was determined by ¹ H-NM
R (FT-NMR: Bruker DPX400; ¹
H resonance frequency: 400 MHz; nuclide measured: ¹ H; solvent used: CD
Cl ₃ ; reference: TMS / CDCl ₃ enclosed in a capillary; measurement temperature: room temperature). FIG. 2 shows the ¹ H-NMR chart. Calculated from the integral of each assigned peak,
The ratio of the unit shown in the following formula is A: B: C: D = 30: 3:
It was 10:54.

[0071]

Embedded image [Example 3] A polymer was obtained in the same manner as in Example 2 except that the strain was changed to H45 strain. The weight of the dried cells was 180 mg, and the weight of the obtained polymer was 49 mg. ¹ H-NMR was measured in the same manner as in Examples 1 and 2. As a result, the ratio of units represented by the following formula was A: B: C: D = 14: 9: 9: 68.

[0072]

Embedded image [Example 4] A polymer was obtained in the same manner as in Example 2 except that the strain was changed to P161 strain. The weight of the dried cells is 200mg,
The weight of the obtained polymer was 53 mg. ¹ H-NMR was measured in the same manner as in Examples 1 and 2. As a result, the ratio of units represented by the following formula was A: B: C: D = 20: 5: 9: 66.

[0073]

Embedded image

[0074]

According to the method of the present invention, 3-hydroxy-5
-Phenoxyvaleric acid unit and 3-hydroxy-5- (4-
Production of novel polyhydroxyalkanoates containing (cyanophenoxy) valeric acid units has become possible.

[Brief description of the drawings]

FIG. 1 shows the ¹ H-NMR chart of polyhydroxyalkanoate and the assignment thereof in Example 1.

FIG. 2 shows a ¹ H-NMR chart of polyhydroxyalkanoate and its assignment 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 Inside (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo F-term (reference) 4B064 AC21 AD30 AE01 4J029 AA02 AB01 AB04 AC02 EB01 ED05A EF01

Claims (11)

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