WO2017170423A1 - Polyester terminé par un alcool et procédé de production de polyester - Google Patents

Polyester terminé par un alcool et procédé de production de polyester Download PDF

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WO2017170423A1
WO2017170423A1 PCT/JP2017/012431 JP2017012431W WO2017170423A1 WO 2017170423 A1 WO2017170423 A1 WO 2017170423A1 JP 2017012431 W JP2017012431 W JP 2017012431W WO 2017170423 A1 WO2017170423 A1 WO 2017170423A1
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polyester
acid
alcohol
lactic acid
molecular weight
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PCT/JP2017/012431
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English (en)
Japanese (ja)
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精一 田口
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国立大学法人北海道大学
株式会社Adeka
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Priority to JP2018508000A priority Critical patent/JP7149473B2/ja
Publication of WO2017170423A1 publication Critical patent/WO2017170423A1/fr

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    • 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
    • C08G63/91—Polymers modified by chemical after-treatment
    • 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

Definitions

  • the present invention relates to an alcohol-terminated polyester and a method for producing the polyester. According to the present invention, polyester produced by microorganisms can be efficiently secreted into the medium.
  • Patent Document 1 discloses a method for producing a target protein outside the cell by using a microorganism transformed with an expression vector containing a gene encoding the OmpF protein of E. coli.
  • Patent Document 2 discloses that the secretion of the target protein is enhanced in low-temperature culture by modifying the Gram-negative bacterium Type I secretion system used for secretion of hemolytic toxin ⁇ -hemolysin (HlyA) produced by uropathogenic E. coli. An efficient system is disclosed. However, the method of adding a signal sequence as in Patent Documents 1 and 2 is difficult to apply to other than proteins.
  • HlyA hemolytic toxin ⁇ -hemolysin
  • Patent Document 3 a method for producing shikimic acid using a microorganism belonging to a specific genus Citrobacter
  • Patent Document 4 a method for efficiently producing lipids containing unsaturated fatty acids using the microorganism
  • polyesters Furthermore, for polyesters, a gene encoding a protein having an activity of converting lactic acid into lactic acid CoA and a protein having an activity of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a substrate for a host microorganism
  • a method for producing an aliphatic polyester has been disclosed in which a recombinant microorganism obtained by introducing and is cultured and the aliphatic polyester is recovered from the medium (Patent Document 5).
  • Patent Documents 3 and 4 utilize specific strains that are secreted outside the cells, and the products are greatly limited. Further, in the method of Patent Document 5, the amount of the lactic acid polymer obtained is about 200 mg per liter, and the production amount was not sufficient.
  • an object of the present invention is to provide a method for producing polyester capable of secreting polyester outside the cells of microorganisms.
  • the present inventor surprisingly added an alcohol having a molecular weight of 300 or less to a culture medium for culturing a microorganism. It has been found that the polyester produced in the cells of the microorganism is efficiently secreted into the medium.
  • the present invention is based on these findings.
  • the present invention [1] An alcohol-terminated polyester having an alcohol residue with a molecular weight of 300 or less at the end, [2] The alcohol-terminated polyester according to [1], wherein the polyester is a polyester comprising ⁇ -hydroxy acid and / or ⁇ -hydroxy acid, [3] The alcohol-terminated polyester according to [2], wherein the ⁇ -hydroxy acid and / or ⁇ -hydroxy acid is lactic acid and / or 3-hydroxybutanoic acid, [4] The alcohol-terminated polyester according to any one of [1] to [3], wherein the average number of repeating units of the polyester is 2 to 12, [5] The alcohol-terminated polyester according to [3] or [4], wherein the average lactic acid content is 70 mol% to 100 mol%, [6] A method for producing polyester, comprising culturing a microorganism capable of producing polyester in a medium containing an alcohol having a molecular weight of 300 or less, [7] The method for producing a polyester according to [6], wherein
  • the polyester of the present invention it can be efficiently secreted outside the cells of microorganisms. Moreover, according to the manufacturing method of the polyester of this invention, the polyester containing lactic acid can be efficiently produced by microorganisms. Naturally, there are many L-form lactic acids, but when a specific polyhydroxyalkanoate synthase is used in the present invention, it is possible to secrete a polyester containing D-form lactic acid outside the cells, The obtained polyester is useful as a raw material for biodegradable materials.
  • the alcohol-terminated polyester obtained according to the present invention can be used as a raw material for food and cosmetics in addition to the alcohol-terminated polyester itself as a polymer raw material.
  • the alcohol-terminated polyester obtained by the present invention has a high lactic acid content
  • it can be suitably used as a lactide raw material for producing polylactic acid.
  • industrially producing high molecular weight polylactic acid it is generally carried out by a ring-opening polymerization reaction with a cyclic dimer (lactide) of lactic acid.
  • lactide cyclic dimer
  • it is necessary to synthesize lactide once, so that there are problems that the process becomes complicated and costs increase.
  • lactide By synthesizing lactide from an alcohol-terminated polyester having a high lactic acid content, it is possible to produce polylactic acid more simply and efficiently than in the past.
  • 3 is a graph showing the results of analyzing the molecular weight of the molecules in the supernatant of the medium obtained by the production method of the present invention by ESI-MS. It is a diagram showing the 1 H- 1 H COSY NMR (a ) and 1 H- 1 H DOSY NMR (b ) extracellular oligomer obtained in this invention. It is the figure which showed 1 H NMR (a), 13 C NMR (b), and 1 H- 13 C HMQC (c) of the extracellular oligomer obtained by the present invention. Is a diagram showing the 1 H NMR of lactide synthesized from the extracellular oligomer obtained in this invention.
  • Alcohol-terminated polyester has an alcohol residue having a molecular weight of 300 or less at the terminal. That is, when the alcohol is a polyhydric alcohol, the alcohol-terminated polyester of the present invention comprises a polyester unit composed of a repeating unit of A in the following general formula (1) and a hydrogen atom, and an alcohol residue composed of B and a hydroxy group.
  • l is not particularly limited, but is 2 to 1000 in some embodiments, 2 to 100 in some embodiments, 2 to 50 in some embodiments, and 2 in some embodiments. ⁇ 20.
  • polyester part is a polyester composed of lactic acid or 3-hydroxybutanoic acid, or a polyester composed of lactic acid and 3-hydroxybutanoic acid, and diethylene glycol, for example, as an alcohol is bonded to the terminal of the polyester part
  • the alcohol-terminated polyester of the invention has the general formula (2) It can be expressed as In the present specification, “polyester” includes oligomers.
  • the repeating unit of the polyester portion of the present invention is not particularly limited as long as it can constitute the polyester, but is preferably a hydroxy acid, more preferably an ⁇ -hydroxy acid and / or a ⁇ -hydroxy acid.
  • the hydroxy acid include 2-hydroxypropionic acid (lactic acid), 2-hydroxybutanoic acid, 2-hydroxypentanoic acid, 2-hydroxyhexanoic acid, 2-hydroxyheptanoic acid, 2-hydroxyoctanoic acid, and 2-hydroxynonanoic acid.
  • ⁇ -hydroxy acids such as 2-hydroxydecanoic acid, 3-hydroxypropionic acid, 3-hydroxybutanoic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3 ⁇ -hydroxy acids such as hydroxynonanoic acid and 3-hydroxydecanoic acid, 4-hydroxybutanoic acid, 4-hydroxypentanoic acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxy Nonanoic acid, 4-hydroxydecanoic acid, etc. be able to.
  • the repeating unit of the polyester part may be one type of repeating unit or a combination of two or more types of repeating units.
  • the repeating unit is referred to as, for example, a hydroxy acid residue, but for convenience, the hydroxy acid residue may be referred to as a “hydroxy acid”.
  • the polyester portion of the alcohol-terminated polyester of the present invention comprises a repeating unit and a terminal hydroxy group to which no alcohol is bonded.
  • a hydroxy acid is polymerized by a condensation reaction.
  • the polyester portion of the alcohol-terminated polyester of the present invention there can be mentioned a terminal hydroxy group to which a lactic acid residue and an alcohol are not bonded.
  • polyester portion there can be mentioned a hydroxy group at a terminal to which a lactic acid residue, a 3-hydroxybutanoic acid residue and an alcohol are not bonded. Furthermore, as another embodiment of the polyester portion, there can be mentioned a hydroxy group at a terminal to which a 3-hydroxybutanoic acid residue and an alcohol are not bonded.
  • lactic acid and lactic acid, lactic acid and 3-hydroxybutanoic acid, or 3-hydroxybutanoic acid and 3-hydroxybutanoic acid are polymerized by a condensation reaction.
  • the alcohol-terminated polyester of the present invention has the following formula (3)
  • the lactic acid represented by these can be included. More specifically, the alcohol-terminated polyester of the present invention is a lactic acid repeating unit (4).
  • the repeating unit is referred to as a lactic acid residue.
  • the lactic acid residue is sometimes referred to as “lactic acid” for convenience.
  • the alcohol-terminated polyester of the present invention has the following formula (5):
  • the 3-hydroxybutanoic acid represented by these can be included. More specifically, the alcohol-terminated polyester of the present invention contains 3-hydroxybutanoic acid repeating units (6)
  • the repeating unit is referred to as a 3-hydroxybutanoic acid residue.
  • the 3-hydroxybutanoic acid residue is sometimes referred to as “3-hydroxybutanoic acid” for convenience.
  • the number of repeating units of lactic acid and / or 3-hydroxybutanoic acid in the alcohol-terminated polyester of the present invention is not particularly limited, but is 2 to 1000 in some embodiments and 2 to 100 in some embodiments. In some embodiments, it is 2-50, in some embodiments 2-20, and in some embodiments 2-10. That is, “n + m” in the general formula (2) is not limited, but is 2 to 1000 in an embodiment, 2 to 100 in an embodiment, and 2 to 50 in an embodiment. Is from 2 to 20, and in some embodiments from 2 to 10.
  • n which is the number of repeating units of 3-hydroxybutanoic acid, is not limited, but in some embodiments, 0 to 1000, in some embodiments, 0 to 100, and in some embodiments, 0 to 50, In some embodiments, 0 to 20, and in some embodiments 0 to 10, but when the alcohol-terminated polyester consists only of 3-hydroxybutanoic acid, the lower limit of m is 2.
  • n which is the number of repeating units of lactic acid, is 0 to 1000 in an embodiment, 0 to 100 in an embodiment, 0 to 50 in an embodiment, and 0 to 20 in an embodiment. In this case, the lower limit of n is 2 when the alcohol-terminated polyester consists only of lactic acid.
  • the molecular weight of the alcohol-terminated polyester of the present invention is not particularly limited, but preferably the lower limit of the molecular weight is about 200 and the upper limit of the molecular weight is 100,000. Further, the upper limit of the more preferable molecular weight is 10,000, more preferably 5000, and most preferably 1000.
  • the ratio of lactic acid to 3-hydroxybutanoic acid in the alcohol-terminated polyester of the present invention is not particularly limited.
  • the alcohol-terminated polyester of the present invention is a polyester composed of lactic acid
  • the ratio of lactic acid is 100 mol%.
  • the alcohol-terminated polyester is a polyester composed of 3-hydroxybutanoic acid
  • the ratio of 3-hydroxybutanoic acid is 100 mol%.
  • the alcohol-terminated polyester of the present invention is a polyester comprising lactic acid and 3-hydroxybutanoic acid
  • the content of lactic acid is not particularly limited, but is preferably 70 mol% or more, more preferably 80 mol. % Or more, more preferably 90 mol% or more.
  • the alcohol-terminated polyester of the present invention can be suitably used as a raw material for biodegradable materials.
  • the lactic acid residue and the 3-hydroxybutanoic acid residue are represented as being arranged in a block form.
  • the sequence of these residues is a block form, a random form. Any combination of a block-like part and a random part may be used. That is, a 3-hydroxybutanoic acid residue may be present at the end of the polyester portion.
  • the lactic acid contained in the alcohol-terminated polyester may be D-form or L-form, but D-form is preferred.
  • Natural lactic acid existing in nature has many L-forms. When a biodegradable material is produced, the use of racemic polylactic acid may make the biodegradable material brittle. Therefore, it is preferable to use L-form polylactic acid and D-form polylactic acid to be co-crystallized and to use a stereocomplex type polymer. As mentioned above, since natural D-form polylactic acid is difficult to obtain, D-form polylactic acid is useful as a raw material for biodegradable materials.
  • an alcohol forms an ester bond with lactic acid by a condensation reaction, or an alcohol forms an ester bond with 3-hydroxybutanoic acid by a condensation reaction.
  • an alcohol residue an alcohol-derived group bonded to lactic acid or 3-hydroxybutanoic acid is referred to as an alcohol residue.
  • the alcohol residue means a residue obtained by removing a hydroxy group from the alcohol.
  • the alcohol residue is sometimes referred to as “alcohol” for convenience.
  • the alcohol capable of generating an alcohol residue contained in the alcohol-terminated polyester of the present invention is an alcohol having a molecular weight of 300 or less. Moreover, as a minimum of molecular weight, Preferably it is 50 or more, More preferably, it is 65 or more.
  • the alcohol having a molecular weight of 300 or less is not particularly limited as long as it has a hydroxy group and has a molecular weight of 300 or less, but is preferably a polyhydric alcohol or an alcohol having an ether bond, and more preferably. Is a polyhydric alcohol having an ether bond, more preferably a diol having an ether bond.
  • Another preferred embodiment is an aliphatic alcohol having a monovalent to trivalent hydroxy group.
  • Specific aliphatic alcohols include methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, glycidol , Methylcyclohexanol, 2-methyl-1-butanol, 3-methyl-2-butanol, 4-methyl-2-pentanol, isopropyl alcohol, 2-ethylbutanol, 2-ethylhexanol, 2-octanol, terpineol, dihydro Terpineol, 2-methoxyethanol, 2-ethoxyethanol, 2-n-butoxyethanol, carbitol, ethyl carbitol, n-butyl carbitol, diacetone alcohol, diethylene
  • the alcohol-terminated polyester of the present invention contains a monohydric alcohol such as methanol or ethanol
  • the alcohol-terminated polyester of the present invention is a polyester part composed of a repeating unit of A in the following general formula (7) and a hydrogen atom, and It consists of B alcohol residues.
  • the alcohol-terminated polyester of the present invention can be used as a polymer raw material, food, cosmetic raw material, etc., but can also be derivatized by further reacting with a terminal hydroxyl group.
  • the group that reacts with the hydroxyl group of the polyester include an isocyanate group, a carboxyl group, a carboxylic acid halide group, and an epoxy group.
  • examples of the low molecular weight compound having a group that reacts with the hydroxyl group of the polyester include isocyanate compounds, carboxylic acids, carboxylic acid halides, and epoxy compounds.
  • Examples of the monofunctional isocyanate compound include 2,6-diisopropylphenyl isocyanate.
  • Polyfunctional isocyanate compounds include tolylene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), xylylene diisocyanate (XDI), isophorone diisocyanate (IPDI), xylylene diisocyanate (XDI), isophorone diisocyanate (IPDI), methyl.
  • Cyclohexyl diisocyanate (H6TDI), 4,4′-dicyclohexylmethane diisocyanate (H12MDI), 1,3-bis (isocyanatomethyl) cyclohexane (H6XDI), tetramethylxylylene diisocyanate (TMXDI), 2,2,4-trimethylhexa Methylene diisocyanate (TMHDI), hexamethylene diisocyanate (HDI), norbornene diisocyanate (NBDI) 2,4,6-triisopropylphenyl diisocyanate (TIDI), 1,12-diisocyanate dodecane (DDI), 2,4, -bis- (8-isocyanate octyl) -1,3-dioctylcyclobutane (OCDI), or n -Pentane-1,4-diisocyanate.
  • H6TDI 4,4′-dicyclohexy
  • Monofunctional carboxylic acids include linear or branched saturated aliphatic monofunctional carboxylic acids such as acetic acid, propionic acid, butyric acid, and lauric acid, and linear or branched unsaturated groups such as acrylic acid and methacrylic acid. Mention may be made of aliphatic monofunctional carboxylic acids and aromatic monofunctional carboxylic acids such as benzoic acid and 2-phenoxybenzoic acid.
  • Examples of the bifunctional carboxylic acid include aliphatic dicarboxylic acids such as succinic acid, adipic acid, azelaic acid, sebacic acid, dodecanedicarboxylic acid, and eicosane diacid, 1,2-cyclohexanedicarboxylic acid, 1,3- Cyclohexanedicarboxylic acid, 1,4-cyclohexanedicarboxylic acid, perhydronaphthalenedicarboxylic acid, alicyclic dicarboxylic acid such as dimer acid, fumaric acid, maleic acid, and unsaturated dicarboxylic acid such as itaconic acid, tetrahydrophthalic acid, and Mention may be made of unsaturated alicyclic dicarboxylic acids such as cyclobutene dicarboxylic acid, p-hydroxyethyloxybenzoic acid, and oxyacids such as ⁇ -caprolactone. Further,
  • Monofunctional carboxylic acid halides include acetyl chloride, acetyl bromide, propanoyl chloride, propanoyl bromide, butanoyl chloride, butanoyl bromide, pentanoyl chloride, pentanoyl bromide, hexanoyl chloride, hexanoyl bromide, benzoyl chloride, and odor Benzoyl chloride.
  • Examples of polyfunctional carboxylic acid halides include dicarboxylic acid chlorides, dicarboxylic acid bromides, and dicarboxylic acid iodides. More specifically, polyfunctional carboxylic acid having two or more carboxylic acid halide groups.
  • biphenyl dicarboxylic acid dichloride biphenylene dicarboxylic acid dichloride, azobenzene dicarboxylic acid dichloride, terephthalic acid dichloride, isophthalic acid dichloride, naphthalene dicarboxylic acid dichloride, etc.
  • Aliphatic bifunctional carboxylic acid halides such as adipoyl dichloride, sebacoyl dichloride, cyclopentane dicarboxylic acid dichloride, cyclohexane dicarboxylic acid dichloride, tetrahydrofuran dicarbonate It can be mentioned alicyclic bifunctional carboxylic acid halides such as carbon acid dichloride.
  • examples of the trifunctional carboxylic acid halide include trimesic acid trichloride, 1,3,5-cyclohexane tricarboxylic acid trichloride, and 1,2,4-cyclobutane tricarboxylic acid trichloride.
  • an alcohol-terminated polyester derivative By using the isocyanate compound, carboxylic acid, or carboxylic acid halide, an alcohol-terminated polyester derivative can be obtained.
  • the isocyanate compound, carboxylic acid, or carboxylic acid halide-terminated polyester is obtained by replacing the alcohol with an isocyanate compound, carboxylic acid, or carboxylic acid halide. It can be carried out by replacing with an isocyanate compound, carboxylic acid, or carboxylic acid halide.
  • the epoxy compound include ethylene oxide, propylene oxide, and phenylene oxide.
  • the alcohol-terminated polyester of the present invention is not limited, but can be produced by, for example, microorganisms as described in the production method described later. As shown in FIG. 1, the alcohol-terminated polyester produced by a microorganism includes an extracellular alcohol-terminated polyester that is secreted outside the cells of the microorganism and an intracellular alcohol-terminated polyester that exists inside the cells of the microorganism.
  • the extracellular alcohol-terminated polyester can be recovered from the culture medium of the microorganism. Therefore, it is easy to collect and purify as compared with polyester accumulated in cells.
  • the number of repeating units of the extracellular alcohol-terminated polyester is not particularly limited, but is 2 to 12 in some embodiments, 2 to 10 in some embodiments, and 2 to 8 in some embodiments. Therefore, when the extracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid and lactic acid, “n + m” in the general formula (2) is not limited, but is 2 to 12 in some embodiments, and in some embodiments, 2 to 10, and in some embodiments 2 to 8. That is, the extracellular alcohol-terminated polyester contains a large amount of polyester generally called an oligomer.
  • m in the general formula (2) is not limited, but is 2 to 12 in some embodiments and 2 to 10 in some embodiments In some embodiments, it is 2-8.
  • n in the general formula (2) is not limited, but in some embodiments it is 2 to 12, in some embodiments 2 to 10, and in some embodiments 2-8.
  • the intracellular alcohol-terminated polyester has a larger number of repeating units than the extracellular alcohol-terminated polyester. Therefore, it is useful as a raw material for high molecular weight biodegradable materials.
  • the number of repeating units of the intracellular alcohol-terminated polyester is not particularly limited, but is 2 to 1000 in some embodiments, 2 to 100 in some embodiments, and 2 to 50 in some embodiments. That is, when the intracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid and lactic acid, “n + m” in the general formula (2) is not limited, but is 2 to 1000 in some embodiments, and in some embodiments, 2 to 100, and in some embodiments 2 to 50.
  • intracellular alcohol-terminated polyesters include high molecular weight polymers and low and medium molecular weight oligomers.
  • m in the general formula (2) is not limited, but is 2 to 12 in some embodiments and 2 to 10 in some embodiments In some embodiments, it is 2-8.
  • n in the general formula (2) is not limited, but in some embodiments it is 2 to 12, in some embodiments 2 to 10, and in some embodiments 2-8.
  • An alcohol-terminated polyester produced using Escherichia coli as a microorganism is represented by the following formula (8):
  • the D-form lactic acid residue represented by these can be included.
  • D-form polylactic acid produced using Escherichia coli as a host is useful as a raw material for biodegradable materials.
  • the method for producing a polyester of the present invention is characterized in that a microorganism capable of producing a polyester is cultured in a medium containing an alcohol having a molecular weight of 300 or less.
  • a microorganism capable of producing a polyester composed of lactic acid a microorganism capable of producing a polyester composed of 3-hydroxybutanoic acid, or a microorganism capable of producing a polyester composed of lactic acid and 3-hydroxybutanoic acid are mixed with an alcohol having a molecular weight of 300 or less.
  • Polyester can be obtained by culturing in a medium containing the same.
  • the microorganism used in the production method of the present invention is not particularly limited as long as it has the ability to produce polyester.
  • microorganisms having the ability to produce polyester comprising lactic acid and 3-hydroxybutanoic acid include bacteria such as Ralstonia and Eutropha.
  • a recombinant microorganism can also be used as a microorganism used for the production method of the present invention.
  • these transformed recombinant microorganisms the convenience of operation can be improved and the productivity can be improved.
  • Microorganisms that can be used as recombinant microorganisms are not particularly limited, but Escherichia coli is particularly preferable, and using tatB, tatE, and araf (deficient mutant) among E. coli can improve the amount of polyester produced per unit medium.
  • pfla and dld deletion mutants are preferable for improving the polyester production and increasing the content of lactic acid in the oligomer.
  • the recombinant microorganism for example, at least one gene selected from the group consisting of a polyhydroxyalkanoate synthase gene, a propionyl CoA transferase gene, a ⁇ -ketothiolase gene, and an acetoacetyl CoA reductase gene is introduced into the microorganism.
  • polyhydroxyalkanoate synthase The polyhydroxyalkanoate synthase, propionyl CoA transferase, ⁇ -ketothiolase, and acetoacetyl CoA reductase will be described.
  • the polyhydroxyalkanoic acid synthase is a protein having catalytic activity for a reaction of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a monomer.
  • Polyhydroxyalkanoic acid synthase catalyzes a reaction for synthesizing polylactic acid using lactyl CoA as a monomer.
  • polyhydroxyalkanoate synthase catalyzes a reaction for synthesizing a hydroxyalkanoic acid-lactic acid copolymer using hydroxyacyl CoA and lactyl CoA as monomers.
  • a preferred polyhydroxyalkanoate synthase gene to be introduced into the recombinant microorganism used in the present invention is derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera Particularly preferred is derived from Pseudomonas sp. 61-3, the base sequence thereof is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 2.
  • a gene encoding a mutant polyhydroxyalkanoate synthase can also be used as the polyhydroxyalkanoate synthase gene.
  • the 130th, 325th, 392rd, 477th and 481st amino acids of the amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence shown in SEQ ID NO: 1 are each independently substituted.
  • Propionyl CoA transferase is a protein having an activity of catalyzing a reaction in which CoA is transferred from an appropriate CoA substrate to propionic acid and / or lactic acid.
  • propionyl CoA transferase is described as “PCT”.
  • PCT genes in the present invention are those derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferred is derived from Megaphaphaela elsdenii, the base sequence thereof is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 4.
  • ⁇ -ketothiolase gene ⁇ -ketothiolase is a protein that catalyzes a reaction in which two molecules of acetyl CoA are condensed to form acetoacetyl CoA.
  • ⁇ -ketothiolase is referred to as “ ⁇ KT” in the present specification.
  • a preferred ⁇ KT gene in the present invention is derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferably, it is derived from Ralstonia eutropha, the base sequence thereof is shown in SEQ ID NO: 5, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 6.
  • Acetoacetyl-CoA reductase undergoes a reaction in which D (-)- ⁇ -hydroxybutyryl-CoA is formed by a reduction reaction that occurs in the presence of a coenzyme such as NADP of acetoacetyl-CoA. It is a protein that catalyzes.
  • acetoacetyl CoA reductase is referred to as “AACoA-R” in the present specification.
  • Preferred AACoA-R genes in the present invention are those derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferably, it is derived from Ralstonia eutropha, the base sequence thereof is shown in SEQ ID NO: 7, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 8.
  • the polyhydroxyalkanoate synthase gene, propionyl CoA transferase, ⁇ -ketothiolase, and acetoacetyl CoA reductase used in the present invention have the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. It may have 1 to several base deletions, substitutions, additions or insertions.
  • the term “several” means 1 to 40, preferably 1 to 20, more preferably 10 or less.
  • the polyhydroxyalkanoate synthase gene, propionyl CoA transferase, ⁇ -ketothiolase, and acetoacetyl CoA reductase are bases complementary to the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. It may be a base sequence of DNA that can hybridize with DNA comprising a sequence under stringent conditions.
  • the polyhydroxyalkanoic acid synthase gene is a gene encoding a protein having a catalytic activity for the reaction of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a monomer
  • propionyl CoA transferase gene is propionic acid and / or
  • the ⁇ -ketothiolase gene has an activity of catalyzing a reaction in which two molecules of acetyl CoA are condensed to form acetoacetyl CoA
  • the acetoacetyl CoA reductase gene is a gene encoding a protein having catalytic activity for the reduction reaction of acetoacetyl CoA.
  • the catalytic activity of the reaction in which CoA is transferred to propionic acid and / or lactic acid is, for example, A. E. It can be measured according to the method described in Hofmeister et al. (Eur. J. Biochem., 206, 547-552).
  • the catalytic activity of the reaction in which acetoacetyl CoA is formed from the two molecules of acetyl CoA is measured, for example, by the method described in Slater et al. (J. Bacteriology, 1998, 180, 1979-1987). Can do.
  • the catalytic activity of the reduction reaction of acetoacetyl CoA is, for example, G.M. W. It can be measured by the method described in Haywood et al. (FEMS Microbiology Letters, 1988, Vol. 52, pp. 259-264).
  • the medium used in the production method of the present invention can be appropriately selected depending on the microorganism used, but a medium containing a carbon source is usually used.
  • a medium containing a carbon source is usually used.
  • E. coli for example, LB medium, M9 medium, NZYM medium, SOB medium, 2 ⁇ YT medium, or terrific broth can be used.
  • the carbon source contained in the medium is not particularly limited as long as the polyester of the present invention can be produced.
  • glucose, xylose, fructose, cellobiose, raffinose, maltose, galactose, starch, starch hydrolysate, molasses examples include sugars such as waste molasses, and natural products such as wheat and rice.
  • xylose is preferable when obtaining a polyester having a high lactic acid content (lactic acid content).
  • Alcohol with a molecular weight of 300 or less is added to the medium. That is, the microorganism is brought into contact with an alcohol having a molecular weight of 300 or less.
  • the alcohol having a molecular weight of 300 or less the “alcohol having a molecular weight of 300 or less” described in the section “[1] Alcohol-terminated polyester” can be used without limitation.
  • a polyhydric alcohol or an ether bond is used. More preferably a polyhydric alcohol having an ether bond, and still more preferably a diol having an ether bond.
  • Another preferred embodiment is an aliphatic alcohol having a monovalent to trivalent hydroxy group.
  • diethylene glycol, butanediol, or polyethylene glycol is preferable.
  • polyethylene glycol polyethylene glycol having a molecular weight of 300 or less is used.
  • the molecular weight of polyethylene glycol is preferably 280 or less, more preferably 260 or less.
  • the lower limit of the molecular weight is preferably 50 or more, more preferably 65 or more.
  • the alcohol is presumed to be bonded to the end of the polyester portion by a condensation reaction to stop the polymerization of the polyester.
  • the alcohol concentration added to the medium is not particularly limited as long as the effects of the present invention can be obtained.
  • the concentration that does not inhibit the growth of microorganisms depends on the type of alcohol and the type of microorganism. It can be determined as appropriate.
  • the concentration of diethylene glycol is preferably 0.01 to 20% by volume, more preferably 0.1 to 10% by volume.
  • the concentration of polyethylene glycol is preferably 0.01 to 15% by volume, more preferably 0.1 to 10% by volume.
  • the polyester obtained is characterized by being not only accumulated in the cells of microorganisms but also secreted outside the cells. That is, the amount of extracellular polyester produced when alcohol having a molecular weight of 300 or less is contained in the medium is significantly higher than the amount of extracellular polyester produced when no alcohol having a molecular weight of 300 or less is contained in the medium. It has improved.
  • the polyester obtained by the method for producing a polyester of the present invention includes an alcohol-terminated polyester in which an alcohol residue having a molecular weight of 300 or less is bonded to the terminal of the polyester. That is, although not limited, the alcohol-terminated polyester described in the section “[1] Alcohol-terminated polyester” can be produced. Since the alcohol is bonded to the terminal of the polyester, the polyester can be efficiently secreted outside the cells of the microorganism.
  • Low molecular weight to high molecular weight polyesters can be produced by the polyester production method of the present invention.
  • the obtained polyester includes not only a polymer compound having a molecular weight of several thousand to several tens of thousands, but also an oligomer having about a dimer to a 10-mer and a copolymer composed of two or more types of monomer units. That is, in the present invention, it is usually possible to produce a polyester having a low molecular weight to a high molecular weight.
  • Many polyesters secreted extracellularly have a relatively small molecular weight. For example, the average number of repeating units of lactic acid and 3-hydroxybutanoic acid in a polyester is 2 to 12 in an embodiment, and 2 to 10 in an embodiment.
  • the molecular weight of the polyester in the cell is not particularly limited, but many of them have a relatively large molecular weight.
  • the average number of repeating units of lactic acid and 3-hydroxybutanoic acid in the polyester is 2 to 1000 in some embodiments. In some embodiments, it is 2 to 100, and in some embodiments, 2 to 50.
  • the recovered polyester can be recovered by a method known to those skilled in the art for recovering a copolyester from a microorganism.
  • the microorganism is collected from the culture solution by centrifugation, washed, dried, suspended in chloroform, and heated to extract the desired copolyester into the chloroform fraction. Methanol is added to precipitate the polyester, the supernatant is removed by filtration or centrifugation, and then dried to obtain a purified copolyester.
  • the composition of the recovered polyester may be confirmed by a usual method such as gas chromatography or nuclear magnetic resonance.
  • the polyester obtained by the production method of the present invention has an alcohol residue having a molecular weight of 300 or less bonded to the terminal of the polyester.
  • polyesters secreted extracellularly have a high proportion of bonded alcohol residues having a molecular weight of 300 or less.
  • the reason why the alcohol is bonded to the obtained polyester is not clear in detail, but can be estimated as follows. The added alcohol having a molecular weight of 300 or less seems to act on the termination of polyester biosynthesis after being taken into the cells. As a result, a polyester in which the alcohol is bonded to the terminal is obtained.
  • alcohol having a molecular weight of 300 or less acts as a chain transfer agent.
  • the chain transfer agent receives radicals from the polymer chain and stops the extension of the polymer, but the chain transfer agent can also attack the monomer to initiate polymerization. It is also possible that the alcohol bound to the end of the polyester has migrated out of the cell by playing a role like a signal sequence.
  • the present invention is not limited by the above description.
  • Example 1 Manufacture of polyesters The method described in Example 1 of WO2009 / 131186 DNA encoding propionyl CoA transferase from M. elsdenii, DNA encoding ⁇ -ketothiolase from R. eutropha, R. DNA encoding acetoacetyl-CoA reductase from R. eutropha, and Pseudomonas sp.
  • coli BW25113 was transformed by the calcium phosphate method, and a transformant in which propionyl CoA transferase, ⁇ -ketothiolase, acetoacetyl CoA reductase, and ST / QK mutant enzyme were expressed (hereinafter sometimes referred to as ST / QK E. coli).
  • ST / QK E. coli a transformant in which propionyl CoA transferase, ⁇ -ketothiolase, acetoacetyl CoA reductase, and ST / QK mutant enzyme
  • ST / QK E. coli transformants expressing propionyl CoA transferase, ⁇ -ketothiolase, acetoacetyl CoA reductase, and ST / FS / QK mutant enzyme (hereinafter sometimes referred to as ST / FS / QK E. coli) Got the body.
  • the obtained transformant was cultured at 30 ° C. for 48 hours while stirring at 180 rpm using LB medium containing 2 wt% glucose and ampicillin.
  • polyethylene glycol PEG 200, sometimes simply referred to as “polyethylene glycol” or “PEG” in the following examples
  • diethylene glycol in an amount of 1 to 5% by volume as a final concentration was added to LB medium in advance (hereinafter, alcohol was added to the medium). The same applies to the case of adding to the above).
  • the culture supernatant was collected, and lactic acid contained in the culture supernatant was measured with a quantification kit (International Ireland, Megazyme).
  • the amount of lactic acid is determined by measuring lactic acid (Free LA) using the culture supernatant as it is (not treated with hydrochloric acid), or adding 80 ⁇ L of 5N hydrochloric acid and 20 ⁇ L of water to 100 ⁇ L of the culture supernatant, and 100 ° C. The hydrolysis was carried out overnight. Thereafter, 200 ⁇ L of 2N NaOH was added for neutralization, and lactic acid (Total LA) in the sample was measured. Since Free LA is not treated with hydrochloric acid, monomeric lactic acid in the culture supernatant is measured.
  • Total LA measures the total amount of monomeric and oligomeric lactic acid because oligomeric lactic acid in the culture supernatant is decomposed by hydrochloric acid to become a monomer. Therefore, it is considered that the amount of lactic acid oligomer in the culture supernatant is obtained by subtracting the lactic acid amount of Free LA from the lactic acid amount of Total LA.
  • the results are shown in FIGS. The black bar indicates Free LA, and the white bar indicates Total LA.
  • polylactic acid is added to the culture supernatant by adding polyethylene glycol (PEG) or diethylene glycol (DEG).
  • PEG polyethylene glycol
  • DEG diethylene glycol
  • Example 2 In this example, the ratio of lactic acid to 3-hydroxybutanoic acid contained in the oligomer of polylactic acid secreted into the culture supernatant was measured.
  • the ST / FS / QK E. coli obtained in Example 1 was cultured in the same manner as in Example 1 in a medium to which diethylene glycol was added to obtain a culture supernatant.
  • a sample not treated with hydrochloric acid and a sample treated with hydrochloric acid were prepared from the obtained culture supernatant.
  • Example 3 In this example, alcohol was added to the oligomer of extracellular polylactic acid (FIG. 5A), the oligomer of intracellular polylactic acid (FIG. 5B), and the polymer of intracellular polylactic acid (FIG. 5C) using NMR. It was confirmed that they were bonded. (Separation and sample preparation of extracellular oligomers, intracellular oligomers, and polymers) Escherichia coli BW25113 having pTV118NpctphaC1ps (ST / FS / QK) AB was cultured at 30 ° C.
  • FIG. 9A shows 1 H- 1 H COSY NMR.
  • the crossover signal of 3.7 ppm / 4.3 ppm indicates that the 4.3 ppm resonance is derived from the proton of (B) of DEG, and therefore DEG was thought to be bound to the polyester at the carboxy terminus. .
  • FIG. 9 (b) shows 1 H- 1 H DOSY NMR, which shows that the diffusion coefficient of DEG is similar to that of D-lactic acid oligomer and is lower than that of unbound DEG. Therefore, the observed DEG was completely bound to the D-lactic acid oligomer.
  • FIG. 10 (a, b, c) shows 1 H NMR, 13 C NMR, and 1 H- 13 C HMQC. These data also show that the secretory oligomer obtained by adding DEG is D-lactic acid. It was supported to be an oligomeric DEG conjugate.
  • Example 4 the amount of intracellular and extracellular polyester was measured using a gene disruption mutant of E. coli.
  • a culture supernatant and Escherichia coli cells were obtained by repeating the procedure of Example 2 except that the gene disruption mutant Escherichia coli shown in FIG. 6 was used instead of Escherichia coli BW25113.
  • the amount of lactic acid was measured for the culture supernatant and the cells.
  • FIG. 6 an increase in the amount of polyester produced per unit medium amount was observed when deletion mutants such as tatB, tatE, and araf were used.
  • the upper diagram in FIG. 6 shows the amount of lactic acid oligomer produced per liter of the culture solution
  • the lower diagram shows the cell weight (Dry Cell Weight) per liter of the culture solution.
  • Example 5 The operation of Example 1 was repeated to measure Total LA and Free LA in the culture supernatant.
  • FIG. 7 shows the amount of oligomer produced by subtracting Free LA from Total LA.
  • PEG polyethylene glycol
  • DEG diethylene glycol
  • secretion of the oligomer increased depending on the volume of DEG.
  • ST / FS / QK E. coli when polyethylene glycol (PEG) was used as the alcohol, almost the same amount of oligomer was secreted in any of 1 to 5% by volume.
  • diethylene glycol (DEG) was used as the alcohol, secretion of the oligomer increased up to 5% by volume depending on the volume of DEG.
  • Example 6 molecules secreted into the culture supernatant were measured by ESI-MS using ST / FS / QK E. coli and 3% by volume of DEG. The operation of Example 2 was repeated to obtain a culture supernatant. The obtained culture supernatant was analyzed using ESI-MS (manufactured by Bruker). The results are shown in FIG.
  • Example 7 In this example, an extracellular polylactic acid oligomer, an intracellular polylactic acid oligomer produced by the production method of the present invention using ST / FS / QK E. coli and 1 to 5% by volume of DEG, and cells The amount of the polylactic acid polymer was measured. The operation of Example 3 was repeated to obtain an extracellular polylactic acid oligomer, an intracellular polylactic acid oligomer, and an intracellular polylactic acid polymer. As shown in Table 1, by culturing ST / FS / QK E. coli using DEG, oligomers secreted extracellularly increased remarkably.
  • Example 8 In this example, using the DEG-bonded polyester obtained by culturing in a medium containing 5% by volume DEG of Example 1, and the DEG non-bonded polyester obtained by culturing in a medium not containing DEG, Lactide was synthesized. 10 mg of dry DEG-bonded polyester or non-DEG-bonded polyester was mixed with 10 mg of zinc oxide (catalyst). The mixture was heated in a vacuum at 180 ° C. for 1 hour using an oven (GTO-350RD glass oven: manufactured by Shibata). The evaporated lactide was liquefied in a round bottom flask on ice and the crude lactide was recovered with chloroform. The crude lactide was analyzed by 1 H-NMR.
  • Example 9 In the same manner as in Example 1, R.I. DNA encoding ⁇ -ketothiolase from R. eutropha, R. DNA encoding acetoacetyl-CoA reductase from R. eutropha; A recombinant plasmid pTV118NphaCAB containing DNA encoding a polyhydroxyalkanoate synthase derived from R. eutropha was prepared. Subsequently, E. coli BW25113 was transformed by the calcium phosphate method to obtain a transformant expressing ⁇ -ketothiolase, acetoacetyl CoA reductase, and polyhydroxyalkanoate synthase.
  • the obtained transformant was cultured at 30 ° C. for 48 hours while stirring at 180 rpm using LB medium containing 2 wt% glucose and ampicillin. Any one of compounds A to C in an amount of 3% by volume as a final concentration was previously added to the LB medium.
  • the culture supernatant was collected and a sample treated with hydrochloric acid was prepared.
  • the amount of 3-hydroxybutanoic acid in the sample was measured with a quantification kit (Wako Pure Chemical Industries, AUTOKIT 3-HB). The results are shown in Table 3.
  • Example 10 the effect of adding a carbon source to the LB medium was examined.
  • E. coli BW25113 was transformed by the calcium phosphate method, and a transformant (ST / FS / QK) in which propionyl CoA transferase, ⁇ -ketothiolase, acetoacetyl CoA reductase, ST / FS / QK mutant enzyme was expressed.
  • E. coli was obtained.
  • LB medium containing 100 mg / L ampicillin was prepared, to which diethylene glycol was added to a final concentration of 5% by volume and glucose or xylose was added to a concentration of 20 g / L.
  • the obtained ST / FS / QK E. coli was cultured at 30 ° C. for 48 hours while stirring at 180 rpm in the above-mentioned two types of media supplemented with glucose or xylose. After completion of the culture, the culture supernatant was collected and analyzed in the same manner as in Examples 1 and 2. The results are shown in Table 4.
  • lactic acid contained in the oligomer increased to 97 mol%.
  • Example 11 culture was performed using two gene-deficient ( ⁇ pfla and ⁇ dld) mutants JWMB1 as E. coli. The procedure of Example 10 was repeated except that JWMB1 strain was used as E. coli. The results are shown in Table 5.
  • the alcohol-terminated polyester of the present invention and the polyester obtained by the method for producing the polyester of the present invention can be used as raw materials for biodegradable materials.
  • this invention was demonstrated along the specific aspect, the modification and improvement obvious to those skilled in the art are contained in the scope of the present invention.

Abstract

L'objectif de la présente invention est de fournir un procédé de production d'un polyester entraînant la sécrétion du polyester à l'extérieur d'une cellule de micro-organisme. Ce problème peut être résolu par ce polyester terminé par un alcool ayant un résidu alcool d'un poids moléculaire de 300 ou moins à une terminaison de ce dernier. De plus, le problème peut être résolu par un procédé de production de polyester caractérisé par la culture d'un micro-organisme capable de produire ce polyester dans un milieu de culture contenant de l'alcool d'un poids moléculaire de 300 ou moins.
PCT/JP2017/012431 2016-03-29 2017-03-27 Polyester terminé par un alcool et procédé de production de polyester WO2017170423A1 (fr)

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WO1997007153A1 (fr) * 1995-08-14 1997-02-27 University Of Massachusetts Medical Center Procedes pour reguler des structures de polyester microbiennes
US5994478A (en) * 1997-04-21 1999-11-30 Monsanto Company Hydroxy-terminated polyhydroxyalkanoates
JP2001247482A (ja) * 1999-01-28 2001-09-11 Mitsui Chemicals Inc ヒドロキシ酸系オリゴマー薬剤組成物
US20060280721A1 (en) * 2003-06-03 2006-12-14 The Gov Of Usa Represented By Secretary Of Dpt Of Nutritional supplements and therapeutic compositions comprising (r)-3- hydroxybutyrate derivatives
JP2009207420A (ja) * 2008-03-04 2009-09-17 Tokyo Institute Of Technology メタノールを原料としたポリヒドロキシアルカン酸共重合の製造法
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JPH06284892A (ja) * 1992-05-14 1994-10-11 Yoshiharu Doi ポリエステルの製造方法
JPH07316273A (ja) * 1994-05-24 1995-12-05 Toyobo Co Ltd 酸末端封鎖ポリ乳酸
WO1997007153A1 (fr) * 1995-08-14 1997-02-27 University Of Massachusetts Medical Center Procedes pour reguler des structures de polyester microbiennes
US5994478A (en) * 1997-04-21 1999-11-30 Monsanto Company Hydroxy-terminated polyhydroxyalkanoates
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US20060280721A1 (en) * 2003-06-03 2006-12-14 The Gov Of Usa Represented By Secretary Of Dpt Of Nutritional supplements and therapeutic compositions comprising (r)-3- hydroxybutyrate derivatives
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Publication number Priority date Publication date Assignee Title
JP7383264B2 (ja) 2018-09-28 2023-11-20 株式会社カネカ 樹脂組成物およびその成形体

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