Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
  • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
  • C08G63/688—Polyesters containing atoms other than carbon, hydrogen and oxygen containing sulfur
  • C08G63/6882—Polyesters containing atoms other than carbon, hydrogen and oxygen containing sulfur derived from hydroxy carboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
  • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

• the present invention relates to a polyhydroxy alkanoate (hereinafter also abbreviated as PHA) copolymer including a novel unit having a double bond and a producing method therefor utilizing microorganisms, also polyhydroxy alkanoate copolymer including a novel unit having a carboxyl group or a salt thereof, derived from the aforementioned copolymer, and a producing method therefor.
• PHA polyhydroxy alkanoate
• the present invention relates to a polyhydroxy alkanoate copolymer including a novel unit having an ester group and a producing method therefor utilizing microorganisms, also polyhydroxy alkanoate copolymer including a novel unit having ' a carboxyl group or a salt thereof, derived from the aforementioned copolymer, and a producing method therefor.
• PHA poly-3-hydroxybutyric acid
• PHA poly-3-hydroxyalkanoate
• PHA having a substituent other than alkyl group in the side chain namely "unusual PHA"
• examples of hopeful substituents for this purpose include a group containing an aromatic ring (phenyl group, phenoxy group etc.), an unsaturated hydrocarbon group, an ester group, an allyl group, a cyano group, a halogenated hydrocarbon group and an epoxide present on the side chain.
• PHA having an aromatic ring is actively investigated as follows : (a) PHA containing a phenyl group or a partially substituted group thereof:
• Japanese Patent No. 2989175 discloses inventions relating to a homopolymer constituted of a 3-hydroxy-5- (monofluorophenoxy) pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5- (difluorophenoxy) pentanoate (3H5(DFP)P) unit, a copolymer containing either a 3H5(MFP)P unit or a 3H5(DFP)P unit or both, a novel strain of Pseudomonas putida capable of producing these polymers, and a method for producing the aforementioned polymers utilizing bacteria of genus Pseudomonas .
• PHA polymer having a phenoxy group substituted with 1 or 2 fluorine atoms at the end of the side chain can be biosynthesized from a long-chain fatty acid having a fluorine substituent and that thus produced PHA has a high melting point and is capable of providing stereoregularity and water repellency while maintaining satisfactory working properties.
• fluorine-substituted PHA having a fluorine substitution on the aromatic ring in the unit there are also investigated PHA having a cyano group or a nitro group on the aromatic ring in the unit.
• Microbiol., 41, 32-43(1995) and Polymer International, 39, 205-213(1996) report production of PHA, containing 3-hydroxy-6- (p-cyanophenoxy) hexanoic acid or 3-hydroxy-6- (p-nitrophenoxy) hexanoic acid as the monomer unit, by Pseudomonas oleovorans ATCC 29347 strain and Pseudomonas putida KT2442 strain, from octanoic acid and 6- (p-cyanophenoxy) hexanoic acid or 6- (p-nitrophenoxy) hexanoic acid as a substrate.
• Polymer, 41, 1703-1709(2000) reports producing PHA, having 3-hydroxyalkenic acid with an unsaturated bond (vinyl group) at an end of a side chain as a monomer unit, by Pseudomonas oleovorans with 10- undecenoic acid as a substrate, followed by an oxidation reaction with potassium permanganate to synthesize 3-hydroxyalkanoic acid having a diol at the end of the side chain, which PHA is reported to show ⁇ such a change in solubility in solvents, as becoming soluble in polar solvents such as methanol, an acetone-water (80/20, v/v) or dimethylsulfoxide and insoluble in non-polar solvents such as chloroform, tetrahydrofuran or acetone.
• solvents such as becoming soluble in polar solvents such as methanol, an acetone-water (80/20, v/v) or dimethylsulfoxide and insoluble in non-polar solvents such as chlor
• Macromolecules, 31, 1480-1486(1996) reports production of a polyester, including a unit having vinyl group in a side chain by Pseudomonas oleovorans and epoxylating the vinyl group to obtain a polyester having an epoxy group in the side chain.
• Polymer, 35, 2090-2097(1994) reports a crosslinking reaction within the polyester molecule utilizing the vinyl group in the side chain of polyester, thereby improving physical properties of polyester.
• Macromolecular chemistry, 4, 289-293(2001) reports producing PHA, including 3-hydroxy-10- undecenoic acid as a monomer unit, from 10-undecenoic acid as a subsrate, and then executing an oxidation reaction with potassium permanganate to obtain PHA including 3-hydroxy-10-carboxydecanoic acid as a monomer unit, and reports an improvement in a decomposition thereof.
• Such PHA having a highly reactive active group such as a bromo group or a vinyl group can be subjected to introduction of various functional groups or chemical modification, and such a group can be a crosslinking point for a polymer, so that it is very useful means for realizing multiple functions in PHA.
• Also technologies related to the present invention include a technology of oxidizing a carbon- carbon double bond with an oxidant to obtain a carboxylic acid (Japanese Patent Application Laid- Open No. S59-190945, J. Chem. Soc, Perkin. Trans. 1, 806(1973), Org. Synth., 4, 698(1963), J. Org. Chem., 46, 19(1981), and J. Am. Chem. Soc, 81, 4273(1959).
• Macromol. Chem. Phys., 195, 1405-1421(1994) reports production of a polyhydroxy alkanoate including a unit having an ester group in a side chain, employing Pseudomonas oleovorans as a production microorganism and an alkanoate ester. Also University of Massachusetts Ph. D. Dissertation Order Number 9132875 (1991) reports production of a polyhydroxy alkanoate including a unit having a benzylester structure, also employing Pseudomonas oleovorans as a production microorganism.
• the copolymers in the foregoing reports are comprised of a monomer unit having a carboxyl group or an ester group at the end of a side chain and a monomer unit having a linear alkyl group (usual PHA) having a low glass transition temperature.
• a monomer unit having a carboxyl group or an ester group at the end of a side chain and a monomer unit having a linear alkyl group (usual PHA) having a low glass transition temperature.
• PHA linear alkyl group
• PHA having a vinyl group as an active group is a PHA copolymer with a monomer unit having a linear alkyl group (usual PHA), its low glass transition temperature and low melting point are undesirable properties in the working and the use of the polymer.
• the present inventors have found a method of synthesizing a PHA formed by copolymerization of a unit having a vinyl group, an ester group or a carboxyl group of a high reactivity, and a unit having either one of a phenyl structure, a thienyl structure and a cyclohexyl structure which can contribute to an improvement of physical properties of the polymer, and have thus made the present invention.
• the present invention is outlined in the following.
• n represents an integer selected within a range indicated in the chemical formula; and in case plural units are present, n is the same or different for each unit; [Chemical Formula (2) ]
• m represents an integer selected within a range indicated in the chemical formula
• R represents a residue having any of a phenyl structure or a thienyl structure; and in case plural units are present, and R are the same or different for each unit;
• Rx being a substituent on a cyclohexyl group represents a hydrogen atom, a CN group, a N0 2 group, a halogen atom, a CH 3 group, a C 2 Hs group, a C 3 H 7 group, a CF 3 group, a C 2 Fs group, or a C 3 F 7 group;
• k represents an integer selected within a range indicated in the chemical formula; and in case plural units are present, Ri and k may be the same or different for each unit.
• n 1-8 (19) in which n represents an integer selected within a range indicated in the chemical formula; Ris represents an H atom, a Na atom or a K atom: and in case plural units are present, n and R ⁇ 8 may be the same or different for each unit; and [Chemical Formula (32)]
• n represents an integer selected within a range indicated in the chemical formula
• R 2 represents any of residues indicated in the chemical formula
• n and R 27 may be the same or different for each unit.
• H 2 C HC (CH 2 ) P CH 2 CH 2 C II OH
• R 23 includes a residue having a phenyl structure or a thienyl structure
• R 24 represents a substituent on a cyclohexyl group and represents an H atom, a CN group, a N0 2 group, a halogen atom, a CH 3 group, a C 2 H 5 group, a
• a method for producing a polyhydroxy alkanoate copolymer including at least a 3-hydroxy- ⁇ - carboxyalkanoic acid unit represented by the chemical formula (19) in a molecule, and simultaneously at least a 3-hydroxy- ⁇ -alkanoic acid unit represented by the chemical, formula (2)' or a 3-hydroxy- ⁇ - cyclohexylalkanoic acid unit represented by the chemical formula (3) in the molecule comprising the steps of : preparing a polyhydroxy alkanoate copolymer including at least a 3-hydroxy- ⁇ -alkenoic acid unit represented by the chemical formula (1) in a molecule, and simultaneously at least a 3-hydroxy- ⁇ -alkanoic acid unit represented by the chemical formula (2) or a 3-hydroxy- ⁇ -cyclohexylalkanoic acid unit represented by the chemical formula (3) in the molecule as
• a method for producing a polyhydroxy alkanoate copolymer characterized in employing a polyhydroxy alkanoate copolymer including at least a 3-hydroxy- ⁇ - alkoxycarbonylalkanoic acid unit represented by a chemical formula (32) in a molecule, and simultaneously at least a 3-hydroxy- ⁇ -alkanoic acid unit represented by the chemical formula (2) or a 3- hydroxy- ⁇ -cyclohexylalkanoic acid unit represented by the chemical formula (3) in the molecule as a starting material, and executing a hydrolysis in the presence of an acid or an alkali or executing a hydrogenolysis including a catalytic reduction, thereby generating a polyhydroxy alkanoate copolymer including at least a 3-hydroxy- ⁇ -carboxyalkanoic acid unit represented by the chemical formula (19) in a molecule, and simultaneously at least a 3-hydroxy- ⁇ - alkanoic acid unit represented by the chemical formula (2) or a 3-hydroxy- ⁇ -cyclohexylalkano
• Fig. 1 is a 1 H-NMR spectrum of a polyester obtained in Example 1.
• Fig. 2 is a 1 H-NMR spectrum of a polyester obtained in Example 2.
• Fig. 3 is a 1 H-NMR spectrum of a polyhydroxy alkanoate copolymer obtained in Example 11, and including 3-hydroxy-5- (phenylsulfanyl) valeric acid represented by a chemical formula (58) , a 3-hydroxy- 10-undecenoic acid represented by a chemical formula (5) , 3-hydroxy-8-noneic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenic acid represented by a chemical formula (7).
• Fig. 3 is a 1 H-NMR spectrum of a polyhydroxy alkanoate copolymer obtained in Example 11, and including 3-hydroxy-5- (phenylsulfanyl) valeric acid represented by a chemical formula (58) , a 3-hydroxy- 10-undecenoic acid represented by a chemical formula (5) , 3-hydroxy-8-noneic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenic acid represented by a chemical formula (7).
• Example 4 is a 1 H-NMR spectrum of a polyhydroxy alkanoate copolymer obtained in Example 11, and including 3-hydroxy-5- (phenylsulfonyl) valeric acid represented by a chemical formula (59) , a 3-hydroxy- 9-carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- -5-carboxyvaleric acid represented by a chemical formula (56) .
• Fig. 5 is a GC-MS TIC spectrum of a methylation decomposite of a polyester obtained in Example 34.
• Fig. 6 is a mass spectrum of a peak derived from a unit shown by a chemical formula (80) of a methylation decomposite of the polyester, obtained in Example 34.
• Fig. 7 is a mass spectrum of a peak derived from a unit shown by a chemical formula (81) of a methylation decomposite of the polyester obtained in Example 34.
• Fig. 8 is a mass spectrum of a peak derived from a unit shown by a chemical formula (82) of a methylation decomposite of the polyester obtained in Example 34.
• Ri represents a substituent on a cyclohexyl group and represents a hydrogen atom, a CN group, a N0 2 group, a halogen atom, a CH 3 group, a C 2 H 5 group, a C 3 H 7 group, a CF 3 group, a C 2 F 5 group, or a C 3 F 7 group;
• k represents an integer selected within a range indicated in the chemical formula; and in case plural units are present, Ri and k may be the same or different for each unit.
• R in the chemical formula (2) represents a residue having a phenyl structure or a thienyl structure selected from the group consisting of chemical formulas (8), (9), (10), (11), (12), (13), (14), (15), (16), (17) and (18): the chemical formula (8) :
• R represents a group of non-substituted or substituted phenoxy groups in which R represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a CH 3 group, a C 2 H 5 group, a C 3 H 7 group, a SCH 3 group, a CF 3 group, a C 2 Fs group, or a C 3 F 7 group; and in case plural units are present, R 4 may be the same or different for each unit; the chemical formula (10) :
• R 5 represents a group of non-substituted or substituted benzoyl groups in which R 5 represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a CH 3 group, a C 2 H 5 group, a C 3 H group, a CF 3 group, a C 2 F 5 group, or a C 3 F 7 group; and in case plural units are present, R 5 may be the same or different for each unit; the chemical formula (11)
• R 6 represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a C00R group, a S0 8 group
• R 7 represents either one of H, Na, K, CH 3 and C2H5
• R 8 represents either one of OH, ONa, OK, a halogen atom, OCH 3 and OC 2 H 5
• R ⁇ may be the same or different for each unit; the chemical formula (12) :
• R 9 represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a COORio group, a S0 2 Rn group (Rio represents either one of H, Na, .
• Rn represents either one of OH, ONa, OK, a halogen atom, OCH 3 and OC 2 H 5 ) , a CH 3 group, a C 2 H5 group, a C 3 H 7 group, a (CH 3 ) 2 -CH group or a (CH 3 ) 3 -C group; and in case plural units are present, R 9 may be the same or different for each unit; the chemical formula (13) :
• R ⁇ 2 represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a COOR X3 group, a S0 2 R ⁇ group (R ⁇ 3 represents either one of H, Na, K, CH 3 and C 2 H5; and R ⁇ represents either one of OH, ONa, OK, a halogen atom, 0CH 3 and OC 2 H 5 ) , a CH 3 group, a C 2 H 5 group, a C 3 H 7 group, a (CH 3 ) 2 -CH group or a (CH 3 ) 3 -C group; and in case plural units are present, R 12 may be the same or different for each unit; the chemical formula (17) :
• R 15 represents a substituent on an aromatic ring and represents an H atom, a halogen atom, a CN group, a N0 2 group, a COOR16 group, a S0 2 R ⁇ group (R ⁇ 6 represents either one ⁇ 'of H, Na, K, CH 3 and C 2 H 5 ; and R ⁇ 7 represents either one of OH, ONa, OK, a halogen atom, OCH 3 and 0C 2 H 5 ) , a CH 3 group, a C 2 H 5 group, a C 3 H 7 group, a (CH 3 ) 2 -CH group or a (CH 3 ) 3 -C group; and in case plural units are present, R i5 may be the same or different for each unit; and the chemical formula (18):
• the producing methods therefor are mainly classified to: a method of oxidizing a double bond portion in a polyhydroxy alkanoate copolymer (hereinafter also called a precursor vinyl PHA) including a 3-hydroxy- ⁇ -alkenoic acid unit having a carbon-carbon double bond at an end of a side chain as represented in a chemical formula (1)
• n represents an integer selected within a range indicated in the chemical formula; and in case plural units are present, such units may be mutually different
• a unit represented by a chemical formula (2) or a chemical formula (3) and a method of hydrolyzing an alkoxycarbonyl portion in a polyhydroxy alkanoate copolymer (hereinafter also called an alkoxycarbonyl PHA) including a 3-hydroxy- ⁇ -alkoxyalkanoic acid unit having an ester group at an end of a side chain as represented in a chemical formula (48):
• n 1-8 ( 8) in which n represents an integer selected within a range indicated in the chemical formula; R 47 represents any of residues indicated in the chemical formula; and in case plural units are present, n and R 4 ⁇ may be the same or different for each unit, and a unit represented by a chemical formula (2) or a chemical formula (3) .
• the precursor vinyl PHA and the precursor alkoxycarbonyl PHA may be collectively called a precursor PHA.
• a producing method for such precursor PHA is not particularly restricted, but there can be employed a microbial production using microorganisms, a method using a genetically modified plant, or a chemical polymerization. Preferably a method by microbial production is employed.
• the precursor vinyl PHA and the precursor ester (alkoxycarbonyl) PHA were synthesized for the first time by the present inventors, and the present invention therefore includes also the precursor vinyl PHA and the precursor ester PHA themselves, and a production process thereof by microorganisms. Also such precursor vinyl PHA and precursor ester PHA can be effectively utilized not only for the carboxyl PHA which is an object of the present invention but also for introducing other functional groups .
• the precursor vinyl PHA can be producing by culturing a microorganism in a culture medium including an ⁇ -alkenoic acid represented by a chemical formula (24) and a compound represented by a chemical formula (25) or an ⁇ -cyclohexylalkanoic acid ⁇ represented by a chemical formula (26) .
• the precursor alkoxycarbonyl PHA can be producing by culturing a microorganism in a culture medium including a carboxylic acid monoester compound represented by a chemical formula (49) and a compound represented by the chemical formula (25) or an ⁇ -cyclohexylalkanoic acid represented by the chemical formula (26) .
• R 24 is a substituent on the cyclohexyl group and represents a hydrogen atom, a CN group, a N0 2 group, a halogen atom, a CH 3 group, a C 2 H 5 group, a C 3 H 7 group, a CF 3 group, a C 2 F 5 group, or a C 3 F 7 group; and r is an integer selected within a range indicated in the chemical formula.
• each precursor PHA can be more advantageously prepared by culturing a microorganism in a culture medium containing respective raw material compounds, namely, for the precursor vinyl PHA, a combination of at least one ⁇ - alkenoic acid represented by the chemical formula (24) and at least one compound represented by the chemical formula (25) or at least one ⁇ - cyclohexylalkanoic acid represented by the chemical formula (26) ; and for the precursor alkoxycarbonyl PHA, a combination of at least one carboxylic acid monoester compound represented by the chemical formula (49) and at least one compound represented by the chemical formula (25) or at least one ⁇ - cyclohexylalkanoic acid represented by the chemical formula (26) , and further- containing at least one of ⁇ peptide, yeast extract, organic acid or salt thereof, amino acid or a salt thereof, sugar, and linear alkanoic acid with 4 to 12 carbon atoms or salt thereof.
• the peptide being polypeptone; one or more organic acids selected from a group of piruvic acid, oxaloacetic acid, citric acid, isocitric acid, ketoglutaric acid, succinic acid, fumaric acid, malic acid, lactic acid and salts thereof; one or more amino acids selected from a group of glutamie acid, aspartic acid and salts thereof; and one or more sugars selected from a group of glyceraldehyde, erythrose, arabinose, xylose, glucose, galactose, mannose, fructose, glycerol, erythritol, xylitol, gluconic acid, glucuronic acid, galacturonic acid, maltose, sucrose and lactose.
• organic acids selected from a group of piruvic acid, oxaloacetic acid, citric acid, isocitric acid, ketoglutaric acid, succinic acid, fumaric acid,
• the raw material compound for each precursor PHA namely, for the precursor vinyl PHA, a combination of at least an ⁇ -alkenoic acid represented by the chemical formula (24) and at least a compound represented by the chemical formula (25) or at least an ⁇ -cyclohexylalkanoic acid represented by the chemical formula (26) ; or for the precursor alkoxycarbonyl PHA, a combination of at least a carboxylic acid monoester compound represented by the chemical formula (49) and at least a compound represented by the chemical formula (25) or at least an ⁇ -cyclohexylalkanoic acid represented by the chemical formula (26) , is preferably contained in the culture medium in a proportion of 0.01 to 1 % (w/v), further preferably 0.02 to 0.2 % .
• the aforementioned nutrients as a carbon source and a nitrogen source for proliferation, and as an energy source for polyhydroxy alkanoate production are preferably added to the culture medium in a proportion of 0.1 to 5 % (v/v) per medium, more preferably 0.2 to 2 % .
• the culture temperature can be any temperature at which the microorganism can satisfactorily proliferate, and is usually within a range of 15 to 37°C, preferably 20 to 30°C.
• the culture may be carried out by any culture method so long as the microorganisms can proliferate and produce PHA, such as a liquid culture or a solid culture. Also it may be batch culture, fed batch culture, semi-continuous culture or continuous culture. For example, for liquid batch culture, the oxygen supply method may be shaking using a shaking flask or agitation aeration in a jar fermenter.
• the method for producing precursor vinyl PHA of the present invention may comprise the steps of: culturing a production microorganism under the aforementioned conditions, and recovering produced PHA from the cells, the PHA copolymer produced by the microorganism at least containing a 3-hydroxy- ⁇ - alkenoic acid unit represented by the chemical formula (1), and a unit represented by the chemical formula (2) or an ⁇ -cyclohexylalkanoic acid unit represented by the chemical formula (3) in the molecule.
• the method for producing precursor alkoxycarbonyl PHA of the present invention may comprise the steps of: culturing a production microorganism under the aforementioned conditions, and recovering from the cells a polyhydroxy alkanoate copolymer produced by the microorganism which at least contains a 3-hydroxy- ⁇ -alkoxycarbonylalkanoic acid unit represented by the chemical formula (48), and a unit represented by the chemical formula (2) or an ⁇ -cyclohexylalkanoic acid unit represented by the chemical formula (3) in the molecule.
• the object PHA can be recovered from the cells by an ordinarily employed method.
• an extraction with an organic solvent such as chloroform, dichloromethane, ethyl acetate or acetone is most simple, but there may also be employed dioxane, tetrahydrofuran or acetonitrile.
• a surfactant such as SDS, chemicals such as hypochlorous acid and EDTA, or with an enzyme such as lysozynae, or by ultrasonic disruption, homogenizer disruption, pressure disruption, beads impulse, grinding or pounding or freeze-and-thawing, to remove cell components other than PHA and recover PHA.
• a production microorganism to be employed in the production method of the present invention can be any microorganisms having an ability meeting the aforementioned conditions, but there are preferred those belonging to the Pseudomonas genus, and more preferably Pseudomonas cichorii , Pseudomonas putida , Pseudomonas fluorecense, Pseudomonas oleovorans, Pseudomonas aeruginosa , Pseudomonas stutzeri or Pseudomonas jessenii .
• More specific examples include Pseudomonas cichorii YN2 (FERM BP-7375) , Pseudomonas cichorii H45 (FERM BP-7374), Pseudomonas jessenii P161 (FERM BP-7376), and Pseudomonas putida P91 (FERM BP-7373) .
• composition of an inorganic salt M9 culture medium employed in the method of the present invention.
• the above-mentioned inorganic culture medium has to be replenished with the essential trace elements by adding the following trace component solution by about 0.3 % '(v/v).
• a polyhydroxy alkanoate copolymer including a unit represented by the chemical formula (1) and a unit represented by the chemical formula (2) or a unit represented by the chemical formula (3) can be oxidized at the carbon- carbon double bond portion to give a polyhydroxy alkanoate copolymer including a unit represented by the chemical formula (19), and a unit represented by the chemical formula (2) or a unit represented by the chemical formula (3) .
• a carboxylic acid by oxidizing a carbon-carbon double bond with an oxidant there are known, for example, a method of utilizing a permanganate salt (J. Chem. Soc Perkin. Trans.
• the oxidant to be employed in the present invention is preferably a permanganate salt.
• Such permanganate salt to be employed as the oxidant is usually potassium permanganate. Since the oxidation reaction is a stoichiometric reaction, the amount of the permanganate salt is usually 1 molar equivalent or more with respect to 1 mole of the unit represented by the chemical formula (1), preferably 2 to 10 molar equivalents .
• an inorganic acid such as sulfuric acid, hydrochloric acid, acetic acid or nitric acid, or an organic acid.
• acetic acid An amount of acid is usually within a range of 0.2 to 2000 molar equivalents per 1 mole of the unit represented by the chemical formula (1), preferably 0.4 to 1000 molar equivalents. An amount less than 0.2 molar equivalents results in a low yield, while an amount exceeding 2000 molar equivalents generates by- products by decomposition with acid. Also a crown ether may be employed for the purpose of accelerating the reaction.
• crown ether and permanganate salt form a complex, thereby providing an effect of increasing the reaction activity.
• the crown ether there is generally employed dibenzo- 18-crown-6-ether, dicyclo-18-crown-6-ether, or 18- crown-6-ether .
• An amount of crown ether is generally within a range of 0.005 to 2.0 molar equivalents per 1 mole of permanganate salt, preferably 0.05 to 1.5 molar equivalents.
• a solvent to be employed in the oxidation reaction of the present invention there may be employed any solvent inert to the reaction without particular limitation, for example water, acetone; an ether such as tetrahydrofuran or dioxane; an aromatic hydrocarbon such as benzene; an aliphatic hydrocarbon such as hexane or heptane; or a halogenated hydrocarbon such as methyl chloride, dichloromethane or chloroform.
• a halogenated hydrocarbon such as methyl chloride, dichloromethane or chloroform, or acetone is preferred.
• a precursor vinyl PHA, a permanganate salt and an acid may be introduced into a solvent at a time from the beginning and reacted together, or they may be added to the reaction system one by one continuously or intermittently to be , ⁇ ⁇ und 4/044213
• a permanganate alone is dissolved or suspended in a solvent, followed by continuous or intermittent addition of a polyhydroxyalkanoate and an acid to the reaction system, or first a • polyhydroxyalkanoate alone is dissolved or suspended in a solvent, followed by continuous or intermittent addition of a permanganate and an acid to the reaction system.
• a reaction temperature is selected generally within a range from -40 to 40°C, preferably -10 to 30°C.
• a reaction time depends on a stoichiometric ratio of the unit represented by the chemical formula (1) and permanganate salt and the reaction temperature, but is generally selected within a range of 2 to 48 hours.
• R 2 in the unit represented by the chemical formula (2) is a residue represented by the chemical formula (11)
• a sulfide bond therein may be converted into a sulfoxide or a sulfone.
• the producing method of the precursor ester PHA of the present invention employing, as a starting material, a polyhydroxy alkanoate copolymer including a unit represented by the chemical formula (48), and a unit represented by a chemical formula (2) or a unit represented by a chemical formula (3) .
• a precursor ester PHA synthesized can provide the carboxyl PHA by hydrolysis in the presence of an acid or an alkali or hydrogenolysis including catalytic reduction of an ester bond portion shown in the chemical formula (48) .
• Such method of hydrolysis in the presence of an acid or an alkali can be carried out by employing, in a water-miscible organic solvent such as methanol, ethanol, tetrahydrofuran, dioxane, dimethylformamide or dimethylsulfoxide, an aqueous solution or an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid; an organic acid such as trifluoroacetic acid, trichloroacetic acid, p- toluenesulfonic acid or methanesulfonic acid; an aqueous caustic alkali such as sodium hydroxide or potassium hydroxide; an aqueous solution or an alkali carbonate such as sodium carbonate or potassium carbonate
• the reaction temperature is selected ordinarily from 0 to 40°C, preferably from 0 to 30°C.
• the reaction period is ordinarily selected from 0.5 to 48 hours.
• a hydrolysis with an acid or an alkali may also cause a cleavage of an ester bonding of the main molecular chain, thereby resulting in a decrease in the molecular weight.
• the method of obtaining a carboxylic acid by hydrogenolysis including catalytic reduction is carried out in the following manner.
• Catalytic reduction is carried out in a suitable solvent and within a temperature range from -20°C to the boiling point of the used solvent, preferably from 0 to 50°C, by reacting hydrogen under a normal pressure or an elevated pressure in the presence of a reducing catalyst.
• the usable solvent include water, methanol, ethanol, propanol, ethyl acetate, diethyl ether, tetrahydrofuran, dioxane, benzene, toluene, dimethylformamide and pyridine.
• tetrahydrofuran, toluene or dimethylformamide is particularly preferable.
• the reducing catalyst there can be employed palladium, platinum or rhodium either singly or held on a carrier, or Raney nickel.
• the catalytic reduction may also cause cleavage of an ester bonding of the main molecular chain to decrease the molecular weight.
• the cells were collected by centrifugation, washed with methanol and dried.
• the dried cells after weighing, were put in chloroform ' and stirred for 72 hours at 35°C to extract a polymer, The chloroform extract was filtered, then concentrated on an evaporator, and a solid precipitate formed by an addition of cold methanol was collected and dried under a reduced pressure to obtain a desired polymer.
• Fig. 1 shows a 1 H-NMR spectrum of the obtained polymer.
• the molecular weight of the obtained polymer was measured by gel permeation chromatography (GPC) (Toso HLC-8220 GPC, column: Toso TSK-GEL Super HM-H, solvent: chloroform, molecular weight converted into polystyrene) .
• GPC gel permeation chromatography
• the obtained polymer dry weight (PDW) was 0.19 g/L and the number-averaged molecular weight was 30,000.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• a desired polymer was obtained in the same manner as in Example 1, except that 5-phenoxyvaleric acid employed in Example 1 was changed to 4- cyclohexylbutyric acid.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• Example 4 A desired polymer was obtained in the same manner as in Example 3, except that polypeptone employed in Example 3 was changed to yeast extract.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• the obtained polymer weighed (PDW) 0.41 g/L and the number-averaged molecular weight was 164,000.
• a polymer was obtained in the same manner as in Example 3, except that the strain YN2 employed in Example 3 was replaced by Pseudomonas cichorii H45 and polypeptone was changed to sodium pyruvate .
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• the weight of the obtained polymer (PDW) was 0.28 g/L and the number-averaged molecular weight was 156,000.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• the weight of the obtained polymer (PDW) was 0.38 g/L and the number-averaged molecular weight of 145,000.
• a polymer was obtained in the same manner as in Example 3, except that the strain YN2 employed in Example 3 was replaced by Pseudomonas jessenii P161 and 0.5% polypeptone was changed to 0.1% of nonanic acid.
• the molecular weight of the obtained polymer was measured by GPC as in Example 1.
• the dried cells after weighing, were put in chloroform and stirred for 72 hours at 35°C to extract a polymer, The chloroform extract was filtered, then concentrated on an evaporator, and a solid precipitate formed by an addition of cold methanol was collected and dried under a reduced pressure to obtain a desired polymer.
• the obtained PHA polymer weighed 1528 mg (dry weight) in the present example.
• the structure of the obtained polymer was determined by 1 H-NMR and 13 C-NMR as in Example 1.
• a polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenoxyvaleric acid represented by the following chemical formula (53), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5), 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) .
• the proportion of such units confirmed by X H-NMR was: 69 mol% of 3-hydroxy-5-phenoxyvaleric acid, 23 mol% of three units of 3-hydroxy-10-undecenoic acid, 3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid in total, and 8 mol% of others (linear 3- hydroxyalkanoic acids of 4 to 12 carbon atoms and 3- hydroxyalk-5-enoic acids with 10 or 12 carbon atoms).
• the polyhydroxy alkanoate thus obtained was utilized in the following reaction.
• a structure determination of the obtained polymer carried out by 1 H-NMR and 13 C-NMR as in Example 1 confirmed a polyhydroxy alkanoate copolymer including, as monomer units, 3-hydroxy-5- phenoxyvaleric acid represented by the following chemical formula (53) , 3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54), 3- - 55
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• 50 mg of the object PHA were charged in a 100- ml eggplant-shaped flask and were dissolved by adding 3.5 ml of chloroform and 0.7 ml of methanol. The solution was added with 2 ml of a 0.63 mol/L solution of trimethylsilyldiazomethane in hexane (supplied by Tokyo Kasei Kogyo Co.) and was agitated for 30 minutes at the room temperature. After the reaction, the solvent was distilled off in an evaporator to recover a polymer. The polymer was recovered by washing with 50 ml of methanol. A drying under a reduced pressure provided 49 mg of PHA.
• Example 10 NMR analysis as in Example 1 confirmed a proportion of the units in which 3-hydroxy-5- phenoxyvaleric acid was present by 83 mol%, a sum of three units of 3-hydroxy-9-carboxynonanoic acid, 3- hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5- carboxyvaleric acid by 8 mol%, and others (linear 3- hydroxyalkanoic acid of 4 to 12 carbon atoms and 3- hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 9 mol%.
• Example 10 Example 10
• a structure of the obtained PHA was determined by a NMR analysis as in Example 1.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-cyclohexylbutyric acid represented by the following chemical formula (57), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5) , 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7).
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 11 A NMR analysis was carried out as in Example 1. As a result, there was confirmed a proportion of the units in which a sum of three units of 3-hydroxy-9- carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid was present by 9 mol%, and 3-hydroxy-4-cyclohexyl butyric acid and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 91 mol%. [Example 11]
• a structure of the obtained PHA was determined by a NMR analysis as in Example 1. An obtained X H-NMR spectrum is shown in Fig. 3.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (phenylsulfanyl) valeric acid represented by the following chemical formula (58), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5) 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) .
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (phenylsulfonyl) valeric acid represented by the following chemical formula (59) , 3-hydroxy-9- carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 12 There were prepared three 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of 5-phenylvaleric acid, and 1.5 mmol/L of 10-undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature.
• polypeptone supplied by Wako Pure Chemical Co.
• 6 mmol/L of 5-phenylvaleric acid 6 mmol/L of 5-phenylvaleric acid
• 10-undecenoic acid 10-undecenoic acid
• a structure of the obtained PHA was determined by a NMR analysis as in Example 1.
• a polymer was recovered by extraction followed by distilling off of the solvent.
• the polymer was recovered by washing with 300 ml of purified water, then with 200 ml of methanol, three times with 200 ml of purified water and finally with 200 ml of methanol.
• the obtained polymer was dissolved in 10 ml of tetrahydrofuran and dialyzed for 1 day with a dialysis film (manufactured by Spectrum Inc., Stectra/Por Standard Regenerated Cellulose Dialysis Membrane 3) , in a 1-L beaker containing 500 ml of methanol.
• the polymer present in the dialysis film was recovered and dried under a reduced pressure to obtain 953 mg of a desired PHA.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenylvaleric acid represented by the following chemical formula (60), 3-hydroxy-9- carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 13 A NMR analysis was carried out as in Example 1. As a result, " " ⁇ -NMR spectrum confirmed a proportion of the units in which 3-hydroxy-5-phenylvaleric acid was present by 86 mol%, a sum of three units of 3- hydroxy-9-carboxynonanoic acid, 3-hydroxy-7- carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by 9 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 5 mol%. [Example 13]
• polyhydroxy alkanoate copolymer including 3-hydroxy-5-phenylvaleric acid represented by the following chemical formula (60), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5) , 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) as monomer units used for the bacterial production in Example 12 were changed in a 500-ml three-necked flask, and were suspended by adding 150 ml of distilled water containing 50 ppm of hydrogen peroxide. Ozone was blown in with a rate of 50 g/hr and the mixture was agitated for 3 hours at the room temperature.
• reaction liquid was filtered to recover a polymer.
• the polymer was resuspended in distilled water, and centrifuged to wash off remaining hydrogen peroxide.
• the obtained polymer was further dissolved in 5 ml of tetrahydrofuran and dialyzed for 1 day with a dialysis film (manufactured by Spectrum Inc.,
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenylvaleric acid represented by the following chemical formula (60), 3-hydroxy-9- carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 2 A NMR analysis was carried out as in Example 1. As a result, there was confirmed a proportion of the units in which 3-hydroxy-5-phenylvaleric acid was present by 85 mol%, a sum of three units of 3- hydroxy-9-carboxynonanoic acid, 3-hydroxy-7- carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by 10 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 5 mol%.
• Example 14 There were prepared five 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of 5- (4- vinylphenyl) valeric acid, and 1 mmol/L of 10- undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature.
• polypeptone supplied by Wako Pure Chemical Co.
• a structure of the obtained PHA was determined by a NMR analysis as in Example 1.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (4-vinylphenyl) valeric acid represented by the following chemical formula (61), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5), 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) .
• a structure of the obtained PHA was determined by a NMR analysis as in Example 1.
• a polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-benzoylvaleric acid represented by the following chemical formula (62), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5), 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) .
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-benzoylvaleric acid represented by the following chemical formula (62), 3-hydroxy-9- carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 16 There were prepared ten 2000-ml shake flasks, and, in each, 0.5 wt .
• % of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of 5- [ (phenylmethyl) sulfanyl] valeric acid, and 1.5 mmol/L of 10-undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature. Then 10 ml of a culture liquid of Pseudomonas cichorii YN2 strain, shake cultured in advance in an M9 culture medium containing 0.5% of polypeptone for 8 hours, was added to each prepared culture medium, and culture was conducted for 60 hours at 30°C.
• the culture liquids were united, and the cells were recovered by centrifuging, rinsed with methanol and dried.
• the dried cells after weighing, were agitated with chloroform for 72 hours at 25°C to extract a polymer.
• the chloroform extract was filtered with a 0.45 ⁇ m membrane filter, then concentrated in an evaporator, and the polymer was recovered by a reprecipitation in cold methanol.
• a desired polymer was then obtained by drying under a reduced pressure. According to a weighing of the obtained polymer, 1714 mg (dry weight) of PHA were obtained in the present example.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (2-thienyl) valeric acid represented by the following chemical formula (64), 3-hydroxy-9- carboxynonanoic acid represented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy- 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 18 A NMR analysis was carried out as in Example 1. As a result, 1 H-NMR spectrum confirmed a proportion of the units in which 3-hydroxy-5- (2-thienyl) valeric acid was present by 85 mol%, a sum of three units of 3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7- carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by 10 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 5 mol%. [Example 18]
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (2-thienylsulfanyl) valeric acid represented by the following chemical formula (65), 3-hydroxy-10- undecenoic acid represented by a chemical formula (5) 3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7) .
• Example 19 There were prepared ten 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of 5- (2- thienylcarbonyl) valeric acid, and 1 mmol/L of 10- undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature.
• polypeptone supplied by Wako Pure Chemical Co.
• 6- thienylcarbonyl valeric acid 6 mmol/L of 5- (2- thienylcarbonyl) valeric acid
• 1 mmol/L of 10- undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature.
• 5-carboxyvaleric acid represented by a chemical formula (56) .
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 2 A NMR analysis was carried out as in Example 1. As a result, 1 H-NMR spectrum confirmed a proportion of the units in which 3-hydroxy-5- (2- thienylcarbonyl) valeric acid was present by 83 mol%, a sum of three units of 3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoic acid and 3- hydroxy-5-carboxyvaleric acid by 7 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 10 mol%.
• Example 20 There were prepared fifteen 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of 5- [ (phenylmethyl) oxy] valeric acid, and 1 mmol/L of 10- undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature .
• polypeptone supplied by Wako Pure Chemical Co.
• 6 mmol/L of 5- [ (phenylmethyl) oxy] valeric acid 6 mmol/L of 5- [ (phenylmethyl) oxy] valeric acid
• 1 mmol/L of 10- undecenoic acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled
• the polymer present in the dialysis film was recovered and dried under a reduced pressure to obtain 940 mg of a desired PHA.
• An average molecular weight of the obtained PHA was measured by gel permeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GEL Super HM-H, solvent: chloroform, converted to polystyrene) .
• GPC gel permeation chromatography
• a proportion of the units of the obtained PHA was calculated by a methylesterification, utilizing trimethylsilyldiazomethane, of a carboxyl group at an end of a side chain of the PHA.
• Example 2 A NMR analysis was carried out as in Example 1. As a result, 1 H-NMR spectrum confirmed a proportion of the units in which 3-hydroxy-5- [ (phenylmethyl) oxy] valeric acid was present by 84 mol%, a sum of three units of 3-hydroxy-9-carboxynonanoic acid, 3-hydroxy- 7-carboxyheptanoic acid and 3-hydroxy-5- carboxyvaleric acid by 8 mol%, and others (linear 3- hydroxyalkanoic acid of 4 to 12 carbon atoms and 3- hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol%.
• Example 21 There were prepared five 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 3 mmol/L of 5-phenoxyvaleric acid, 3 mmol/L of 5-cyclohexylvaleric acid and 1 mmol/L of 10-undecenoic. acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature.
• polypeptone supplied by Wako Pure Chemical Co.
• 3 mmol/L of 5-phenoxyvaleric acid 3 mmol/L of 5-cyclohexylvaleric acid
• 1 mmol/L of 10-undecenoic. acid were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenoxyvaleric acid represented by the following chemical formula (53) , 3-hydroxy-5- cyclohexylvaleric acid represented by the following chemical formula (68), 3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54), 3- hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55) and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula (56) .
• Example 22 There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 4 mmol/L of 5-phenylvaleric acid, and 1 mmol/L of dodecanedioic acid monoethyl ester were dissolved in 1000 ml of an aforementioned M9 culture medium, which was placed in a 2000 ml shake flask, then sterilized in an autoclave and cooled to the room temperature .
• polypeptone supplied by Wako Pure Chemical Co.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenylvaleric acid represented by the following chemical formula (60) by 78 mol%, three units of 3-hydroxy-ll-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70), and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 14 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to '12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or carbon atoms) by 8 mol%
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenyl aleric acid represented by the following chemical formula (60) by 75 mol%, three units of 3-hydroxy-ll-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70) , and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 15 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or carbon atoms) by 10 mol'
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenylvaleric acid represented by the following chemical formula (60) by 78 mol%, three units of 3-hydroxy-ll-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70) , and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 14 mol%, and others (linear 3-hydroxyalkanoic acid, of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or carbon atoms) by 8 mol%
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenoxyvaleric acid represented by the following chemical formula (53) by 74 mol%, three units of 3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70) , and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 17 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or carbon atoms) by 9 mol%
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-4-cyclohexylbutyric acid represented by the following chemical formula (57) by 76 mol%, three units of 3-hydroxy-ll-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70), and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 16 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 135
• the cells were recovered by centrifuging, rinsed with methanol and lyophilized.
• the dried cells after weighing, were agitated with chloroform for 48 hours at 50°C to extract a polymer.
• the chloroform extract was filtered, then concentrated in an evaporator, and a solid precipitate formed with cold methanol was collected and dried under a reduced pressure to obtain a desired polymer. According to a weighing of the obtained polymer, 890 mg (dry weight) of PHA were obtained in the present example.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (phenylsulfanyl) valeric acid represented by the following chemical formula (58) by 80 mol%, three units of 3-hydroxy-ll-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70), and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 14 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms ) by 6 mol% .
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-benzoylvaleric acid represented by the following chemical formula (62) by 69 mol%, three units of 3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the following chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acid represented by a chemical formula (70) , and 3-hydroxy-7- ethoxycarbonylheptanoic acid represented by a chemical formula (71) collectively by 18 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or carbon atoms) by 13 mol'
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (4-cyanophenoxy) aleric acid represented by the following chemical formula (72) by 34 mol%, two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acid represented by a chemical formula (74) collectively by 16 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 50 mol%.
• Example 30 There were prepared two 2000-ml shake flasks, and, in each, 0.1 wt.% of n-nonanoic acid (supplied 4/044213
• the cells were recovered by centrifuging, rinsed with methanol and lyophilized.
• the dried cells after weighing, were agitated with chloroform for 48- hours at 50°C to extract a polymer.
• the chloroform extract was filtered, then concentrated in an evaporator, and a solid precipitate formed with cold methanol was collected and dried under a reduced pressure to obtain a desired polymer. According to a weighing of the obtained polymer, 170 mg (dry weight) of PHA were obtained in the present example.
• Example 2 For specifying the structure of the obtained PHA, a NMR analysis was conducted under same conditions as in Example 1. As a result, there was confirmed a polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- (4-nitrophenyl) valeric acid represented by the following chemical formula (75) by 8 mol%, two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acid represented by a chemical formula (74) collectively by 18 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 74 mol%.
• 3- hydroxy-5- (4-nitrophenyl) valeric acid represented by the following chemical formula (75) by 8 mol%
• two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73)
• Example 2 For specifying the structure of the obtained PHA, a NMR analysis was conducted under same conditions as in Example 1. As a result, there was confirmed a polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5- [ (phenylmethyl) oxy] aleric acid represented by the following chemical formula (68) by 81 mol%, two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acid represented by a chemical formula (74) collectively by 13 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 6 mol%.
• 3- hydroxy-5- [ (phenylmethyl) oxy] aleric acid represented by the following chemical formula (68) by 81 mol%
• two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenylvaleric acid represented by the following chemical formula (60) by 77 mol%, two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acid represented by a chemical formula (74) collectively by 19 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 4 mol%.
• the synthesized polyhydroxy alkanoate was formed into a film, and 500 mg of such film was placed on a Petri dish and was let to stand for 5 hours in 100 ml of a 0. IN aqueous solution of sodium hydroxide. After the reaction, the sodium hydroxide solution was removed, and the polymer was washed three times with 100 ml of distilled water. Then the polymer was dissolved in 200 ml of ethyl acetate, and, after an addition of 100 ml of a 1. ON aqueous solution of hydrochloric acid, the solution was agitated for 1 hour at the room temperature. Then the polymer was extracted, washed with distilled water and the solvent was distilled off to recover the polymer. Thereafter, a drying under a reduced pressure was carried out to obtain 350 mg of a desired polymer.
• polyhydroxy alkanoate copolymer including, as monomer units, 3- hydroxy-5-phenoxyvaleric acid represented by the following chemical formula (53) by 74 mol%, two units of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the following chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acid represented by a chemical formula (74) collectively by 18 mol%, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol%.
• the synthesized polyhydroxy alkanoate was formed into a film, and 500 mg of such film was placed on a Petri dish and was let to stand for 5 hours in 100 ml of a 0.1N aqueous solution of sodium hydroxide. After the reaction, the sodium hydroxide solution was removed, and the polymer was washed three times with 100 ml of distilled water. Then the polymer was dissolved in 200 ml of ethyl acetate, and, after an addition of 100 ml of a 1. ON aqueous solution of hydrochloric acid, the solution was agitated for 1 hour at the room temperature . Then the polymer was extracted, washed with distilled water and the solvent was distilled off to recover the polymer.
• the cells were recovered by centrifuging, rinsed with methanol and lyophilized.
• the dried cells after weighing, were agitated with chloroform for 48 hours at 50°C to extract a polymer.
• the chloroform extract was filtered, then concentrated in an evaporator, and a solid precipitate formed with cold methanol was collected and dried under a reduced pressure to obtain a desired polymer.
• a structure determination of the obtained polymer was carried out by a methynolysis-GC/MS method to be explained in the following. 5 mg of the polymer were dissolved in 2 mL of chloroform, then added with 2 mL of a 3% methanol solution of sulfuric acid and refluxed for 3.5 hours at 100°C. After the reaction, the reaction mixture was cooled to the room temperature and separated by adding 10 mL of deionized water under agitation.
• the molecular weight of the polymer was measured by gel permeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GEL Super HM-H, solvent: chloroform, converted to polystyrene) .
• Table 1 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, The molecular weight and The molecular weight distribution- of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number-averaged molecular weight
• ' Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.
• a desired polymer was obtained in the same manner as in Example 34, except that the YN2 strain employed in Example 34 was replaced by Pseudomonas jessenii P161 strain.
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 2 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, the molecular weight and the molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• a desired polymer was obtained in the same manner as in Example 34, except that the YN2 strain employed in Example 34 was replaced by Pseudomonas cichorii H45 strain and polypeptone was replaced by yeast extract (DIFCO) .
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 3 shows weights of the obtained cells and the obtained polymer, polymer weight ratio per cell, the molecular weight and the molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number- averaged molecular weight
• Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.
• a desired polymer was obtained in the same manner as in Example 34, except that the YN2 strain employed in Example 34 was replaced by Pseudomonas ⁇ putida P91 strain and polypeptone was replaced by n- nonanoic acid (Kishida Kagaku, concentration: 0.1%).
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 4 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, a molecular weight and a molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number- averaged molecular weight
• Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 5 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, a molecular weight and a molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number- averaged molecular weight
• Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.
• a desired polymer was obtained in the same manner as in Example 34, except that polypeptone was replaced by sodium piruvate (Kishida Kagaku) .
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 6 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, a molecular weight and a molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number- averaged molecular weight
• Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.
• a desired polymer was obtained in the same manner as in Example 34, except that polypeptone employed in Example 34 was replaced by sodium L- glutamate (Kishida Kagaku) and sebacic acid monomethyl ester, which is one of substrates for polymer synthesis was replaced by suberic acid monomethyl ester.
• the molecular weight of the polymer was measured by gel permeation chromatography as in Example 34.
• Table 7 shows weights of the obtained cells and the obtained polymer, a polymer weight ratio per cell, a molecular weight and a molecular weight distribution of the obtained polymer.
• CDW cell dry weight
• PDW polymer dry weight
• P/C cell dry weight/polymer dry weight
• Mn number- averaged molecular weight
• Mw weight-averaged molecular weight
• Mw/Mn molecular weight distribution.

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