WO2014017469A1 - Method for producing d-lactic acid, method for producing polymer, and polymer - Google Patents

Method for producing d-lactic acid, method for producing polymer, and polymer Download PDF

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WO2014017469A1
WO2014017469A1 PCT/JP2013/069876 JP2013069876W WO2014017469A1 WO 2014017469 A1 WO2014017469 A1 WO 2014017469A1 JP 2013069876 W JP2013069876 W JP 2013069876W WO 2014017469 A1 WO2014017469 A1 WO 2014017469A1
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lactic acid
producing
inorganic salt
salt medium
culture
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PCT/JP2013/069876
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French (fr)
Japanese (ja)
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大輔 宮澤
智美 酒井
高橋　均
大垣　弘毅
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三井化学株式会社
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Priority to CN201380038822.2A priority Critical patent/CN104487584B/en
Priority to JP2014526927A priority patent/JPWO2014017469A1/en
Publication of WO2014017469A1 publication Critical patent/WO2014017469A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

Definitions

  • the present invention relates to a method for producing D-lactic acid, a method for producing a polymer, and a polymer.
  • Lactic acid is a useful substance that has recently attracted attention as an intermediate for polymer raw materials, agricultural chemicals, and pharmaceuticals.
  • microorganisms that efficiently produce lactic acid such as lactic acid bacteria and filamentous fungi.
  • Lactic acid production methods using these microorganisms include Lactobacillus delbrueckii as a microorganism that can produce L-lactic acid efficiently, and D-lactic acid efficiently.
  • a method using a microorganism of the genus Sporolactobacillus is known.
  • D-lactic acid has recently attracted attention as a raw material and a pharmaceutical intermediate of stereocomplex polylactic acid with L-lactic acid.
  • the raw material D-lactic acid is required to have high optical purity.
  • D-lactic acid is generally produced industrially by fermentation using microorganisms such as E. coli. Since the fermentation process is a process in which culturing of microorganisms and production of substances using the grown microorganisms as a catalyst are performed, a nutrient source is required for the growth of microorganisms. As a nutrient source, corn steep liquor obtained in the process of corn is rich in amino acids and has a high nutritional value and is inexpensive, so it is used as a fermentation raw material for D-lactic acid.
  • Patent Document 1 discusses rapidly decomposing L-lactic acid contained in a raw material for the purpose of efficiently producing lactic acid and improving the optical purity of the product. Yes.
  • Patent Document 2 discloses a method for fermentative production of D-lactic acid using a medium in which sugar is added to a medium mainly composed of the following inorganic salts. Has been.
  • Patent Document 3 a medium mainly composed of an inorganic salt for culturing Escherichia coli is also described in, for example, Patent Document 3 and Non-Patent Document 1.
  • Those described in Patent Documents 2 and 3 and Non-Patent Document 1 are media containing thiamine and further transition metals such as cobalt, zinc and copper in addition to several inorganic salts. These thiamines and transition metal ions play an important role as cofactors in biochemical reactions by enzymes in microbial cells, and are therefore considered to be very important components.
  • Patent Documents 2 and 3 also describe that betaine is appropriately added to a medium containing an inorganic salt as a main component.
  • the present invention is to provide a method for producing D-lactic acid more efficiently at low cost.
  • the present inventors have found that a high-quality polymer can be obtained when polymerized by culturing D-lactic acid-producing Escherichia coli in an inorganic salt medium.
  • D-lactic acid can be produced more efficiently and efficiently by minimizing the components in the inorganic salt medium. That is, the present invention is as described in [1] to [17] below.
  • the content of the inorganic electrolyte in the inorganic salt medium is 11 g / L or less at the start of culture.
  • the content of thiamine in the inorganic salt medium is 0.1 mg / L or less.
  • D) The content of transition metal ions in the inorganic salt medium is 10 mg / L or less.
  • [2] The method for producing D-lactic acid according to [1], wherein the inorganic salt medium satisfies at least one of (c) and (d).
  • [3] The method for producing D-lactic acid according to [1] or [2] for producing D-lactic acid as a polymer raw material.
  • [4] The method for producing D-lactic acid according to any one of [1] to [3], wherein the inorganic salt medium at the start of culture further satisfies the following (e): (E) The content of the transition metal ion in the inorganic salt medium is 0.85 mg / L or less.
  • [5] The method for producing D-lactic acid according to any one of [1] to [4], wherein the carbon source is sugar.
  • the sugar is glucose, fructose, xylose, sucrose, glycerin, arabinose, melibiose, trehalose, maltose, melibionic acid, lactose, maltotriose, ribose, galactose, galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol
  • the method for producing D-lactic acid according to [5] comprising one or more compounds selected from the group consisting of mannose, saccharic acid, sorbitol, fucose, rhamnose, allose, and N-acetylglucosamine.
  • the inorganic electrolyte includes one or more ions selected from the group consisting of potassium ions, phosphate ions, ammonium ions, sulfate ions, and magnesium ions as a constituent component.
  • the inorganic electrolyte contains potassium ions, The method for producing D-lactic acid according to any one of [1] to [7], wherein in the inorganic salt medium at the start of culture, the concentration of the potassium ion is 5.8 mmol / L or more and 73 mmol / L or less. .
  • [14] Obtaining an alkali metal salt of D-lactic acid in the step of obtaining the D-lactate, and desalting the alkali metal salt of D-lactic acid by electrodialysis in the step of obtaining the D-lactic acid.
  • the method further comprises a purification step of purifying D-lactic acid after the step of desalting the D-lactate to obtain D-lactic acid, The method for producing D-lactic acid according to any one of [12] to [14], wherein distillation is performed while hydrolysis in the purification step.
  • [16] A method for producing a polymer, wherein a polymerization reaction is performed using D-lactic acid obtained by the method for producing D-lactic acid according to any one of [1] to [15].
  • [17] A polymer obtained by the method for producing a polymer according to [16].
  • a method for producing D-lactic acid at a lower cost and more efficiently is provided.
  • the method for producing D-lactic acid includes an inorganic electrolyte and a carbon source, and satisfies at least one selected from the group consisting of (a), (b), (c) and (d) below.
  • D-lactic acid is produced by culturing D-lactic acid-producing Escherichia coli in an inorganic salt medium. As a result, it is possible to produce D-lactic acid at a lower cost and more efficiently.
  • the content of organic substances other than the carbon source in the inorganic salt medium is 10 g / L or less at the start of culture.
  • the content of the inorganic electrolyte in the inorganic salt medium is 11 g / L or less at the start of culture.
  • the content of thiamine in the inorganic salt medium is 0.1 mg / L or less.
  • D) The content of transition metal ions in the inorganic salt medium is 10 mg / L or less.
  • the inorganic salt medium satisfies one or more selected from the group consisting of the above (a), (b), (c) and (d), preferably Satisfies at least two selected from the group consisting of (a), (b), (c) and (d), more preferably (a), (b), (c) and (d) Satisfying at least three selected from the group consisting of: (a), (b), (c) and (d).
  • D-lactic acid as a polymer raw material can be produced.
  • D-lactic acid suitable for the use of the polymer raw material can be obtained.
  • the method for producing D-lactic acid according to the present embodiment will be described in detail.
  • the carbon source is a fermentable sugar that can be used when D-lactic acid-producing Escherichia coli produces D-lactic acid, and more specifically, glucose, fructose, xylose, sucrose, glycerin.
  • a starch hydrolyzate, a herbaceous degradation product, a cellulose hydrolyzate, etc. as a carbon source.
  • an enzyme gene that is necessary or a gene that improves the utilization efficiency may be introduced into Escherichia coli. Specific examples include D-lactic acid production from sucrose by E. coli into which invertase has been introduced, as described in the Examples of the present application.
  • the inorganic salt medium according to the present embodiment includes organic substances other than the carbon source.
  • organic substances other than a carbon source are organic substances other than the fermentable sugar mentioned above, for example, an organic nitrogen source is mentioned. More specific examples of organic substances other than carbon sources include oil cakes, soybean hydrolysates, casein digests, amino acids, corn steep liquor, yeast and yeast extract, meat extracts, peptides such as peptone, malt extract, Examples include whey, various fermented bacterial cells and hydrolysates thereof, or organic components contained therein. Vitamins are also examples of organic substances other than carbon sources.
  • organic substances that serve as buffering agents added for the purpose of enhancing the pH buffering action of the inorganic salt medium are listed as examples of organic substances other than the carbon source. For example, MOPS (3- (N-morpholino) propanesulphonic acid), Tris ( tris (hydroxymethyl) aminomethane), EDTA (ethylenediamineacetic acid), and the like.
  • the purification process for obtaining D-lactic acid becomes complicated, Since inconveniences such as adversely affecting the color tone may occur, it is preferable to limit the amount of organic substances other than the carbon source contained in the inorganic salt medium.
  • the content of organic substances other than the carbon source in the inorganic salt medium is preferably 10 g / L or less at the start of culture, and more preferably 2 g / L or less from the viewpoint of the color of the polymer. More preferably, it is 0.6 g / L or less.
  • the content of organic substances other than the carbon source is preferably in the range of 0 to 10 g / L, more preferably 0 to 2 g / L, and still more preferably 0 to 0.6 g. / L.
  • the lower limit is preferably 0 g / L or more from the viewpoint of the color tone of the polymer.
  • “at the start of culture” means immediately before inoculating D-lactic acid-producing Escherichia coli into the medium.
  • the “inorganic salt medium” in the D-lactic acid production method according to the present embodiment refers to an inorganic element that contains an element excluding a carbon source as a constituent component among elements required by a microorganism in lactic acid fermentation using a microorganism.
  • a liquid medium prepared by dissolving or suspending an electrolyte in water.
  • the inorganic electrolyte includes potassium ions (K + ), phosphate ions (PO 4 3 ⁇ ), ammonium ions (NH 4 + ), sulfate ions (SO 4 2 ⁇ ). And at least one ion selected from the group consisting of magnesium ions (Mg 2+ ).
  • the total content of the inorganic electrolyte is preferably 11 g / L or less, and preferably in the range of 0 to 11 g / L. Further, the lower limit is preferably 0 g / L or more.
  • the inorganic electrolyte used is not particularly limited.
  • dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ) , Potassium hydroxide (KOH), tripotassium phosphate (K 3 PO 4 ), and at least one selected from the group consisting of potassium sulfate (K 2 SO 4 ) can be selected and used.
  • the K + content in the inorganic salt medium is preferably 5 to 50 mmol / L, more preferably 15 to 25 mmol / L.
  • the K + content in the inorganic salt medium at the start of the culture is preferably 5 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and is 5.8 mmol / L or more. More preferably, it is more preferably 15 mmol / L or more.
  • the upper limit value of the K + content in the inorganic salt medium at the start of the culture is preferably 73 mmol / L or less, more preferably 50 mmol / L or less, from the viewpoint of more efficiently producing D-lactic acid. And more preferably 25 mmol / L or less.
  • the inorganic electrolyte to be used is not particularly limited.
  • the content of PO 4 3 ⁇ in the inorganic salt medium is 2 to 40 mmol / L, more preferably 10 to 30 mmol / L.
  • the PO 4 3 ⁇ content in the inorganic salt medium at the start of the culture is preferably 2 mmol / L or more, and more preferably 10 mmol / L or more from the viewpoint of producing D-lactic acid more efficiently.
  • the upper limit of the content of PO 4 3 ⁇ in the inorganic salt medium at the start of the culture is preferably 40 mmol / L or less, more preferably 30 mmol / L or less, from the viewpoint of more efficiently producing D-lactic acid. Is more preferable.
  • the inorganic electrolyte to be used is not particularly limited.
  • ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ), diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), ammonium phosphate ((NH 4 ) 3 PO 4 ), ammonium magnesium phosphate (MgNH 4 PO 4 ), ammonium sulfate ((NH 4 ) 2 SO 4 ) and potassium ammonium hydrogen phosphate (NH 4 KHPO) It is possible to select and use at least one selected from the group consisting of 4 ).
  • the NH 4 + content in the inorganic salt medium is preferably 20 to 120 mmol / L, more preferably 40 to 80 mmol / L.
  • the NH 4 + content in the inorganic salt medium at the start of the culture is preferably 20 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and more preferably 40 mmol / L or more. preferable.
  • the upper limit of the NH 4 + content in the inorganic salt medium at the start of the culture is preferably 120 mmol / L or less, more preferably 80 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid. More preferably.
  • the inorganic electrolyte to be used is not particularly limited.
  • potassium sulfate (K 2 SO 4 ) ammonium sulfate ((NH 4 ) 2 SO 4 ) and sulfuric acid
  • MgSO 4 magnesium
  • the SO 4 2 ⁇ content in the inorganic salt medium is preferably 10 to 50 mmol / L, more preferably 20 to 40 mmol / L.
  • the SO 4 2 ⁇ content in the inorganic salt medium at the start of the culture is preferably 10 mmol / L or more, more preferably 20 mmol / L or more, from the viewpoint of more efficiently producing D-lactic acid. Further preferred.
  • the upper limit of the SO 4 2 ⁇ content in the inorganic salt medium at the start of culture is preferably 50 mmol / L or less, more preferably 40 mmol / L or less from the viewpoint of producing D-lactic acid more efficiently. Is more preferable.
  • the inorganic electrolyte used is not particularly limited.
  • magnesium hydrogen phosphate (MgHPO 4 ), magnesium dihydrogen phosphate (Mg (H 2 PO 4 ) 2 ), Magnesium phosphate (Mg 3 (PO 4 ) 2 ), ammonium magnesium phosphate (MgNH 4 PO 4 ) and magnesium sulfate (MgSO 4 ) can be selected and used.
  • the Mg 2+ content in the inorganic salt medium is preferably 0.2 to 2 mmol / L, more preferably 0.5 to 1.4 mmol / L.
  • the Mg 2+ content in the inorganic salt medium at the start of the culture is preferably 0.2 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and is 0.5 mmol / L or more. More preferably.
  • the upper limit of the Mg 2+ content in the inorganic salt medium at the start of the culture is preferably 2 mmol / L or less, more preferably 1.4 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid. Is more preferable.
  • the “inorganic salt medium” in the present embodiment may further contain thiamine or transition metal ions.
  • thiamine or transition metal ions are contained in the inorganic salt medium, at least one of the above (c) and (d) is satisfied from the viewpoint of inexpensive and efficient production of D-lactic acid It is preferable to satisfy both of the above (c) and (d) from the viewpoint of simplifying the purification.
  • the thiamine content is preferably 0.1 mg / L or less, and more preferably 0.02 mg / L or less. Further, it is more preferable to use an inorganic salt medium that does not contain thiamine. For this reason, the thiamine content in the inorganic salt medium at the start of culture is preferably 0 to 0.1 mg / L, more preferably 0 to 0.02 mg / L. Further, the lower limit is preferably 0 mg / L or more.
  • the transition metal ion is not particularly limited, and for example, at least one ion selected from the group consisting of iron ions, cobalt ions, copper ions, zinc ions, and molybdate ions can be used.
  • the transition metal ion content in the inorganic salt medium at the start of the culture is preferably 10 mg / L or less, and more preferably 0.85 mg / L or less.
  • the D-lactic acid according to this embodiment In the production method, it is preferable that the inorganic salt medium at the start of the culture further satisfies the following (e). In this way, a higher quality polymer can be obtained, and D-lactic acid can be produced at a lower cost and more efficiently.
  • the content of transition metal ions in the inorganic salt medium is 0.85 mg / L or less.
  • the “inorganic salt medium” in the present embodiment is preferably a liquid prepared by dissolving or suspending an inorganic electrolyte in water.
  • D-lactic acid can be produced by fermentation using the carbon source as a raw material.
  • fermentable sugar used as a carbon source is not particularly limited, for example, glucose, fructose, xylose, sucrose, glycerin, arabinose, melibiose, trehalose, maltose, melbionic acid, lactose, maltotriose, ribose, galactose, Examples thereof include one or more compounds selected from the group consisting of galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol, mannose, saccharic acid, sorbitol, fucose, rhamnose, allose, and N-acetylglucosamine.
  • starch hydrolyzate, vegetative degradation product, cellulose hydrolyzate and the like may be used as fermentable sugar (carbon source).
  • the sugar content in the inorganic salt medium at the start of the culture is 200 g / L or less in terms of glucose with respect to the total mass of the inorganic salt medium in consideration of the fact that a high concentration of sugar inhibits the growth of microorganisms. It is preferable, and it is further more preferable in it being 150 g / L or less.
  • the lower limit of the sugar content in the inorganic salt medium at the start of culture is preferably 20 g / L or more, and more preferably 50 g / L or more. Based on the above, the sugar content in the inorganic salt medium at the start of culture is preferably 20 to 200 g / L, more preferably 50 to 150 g / L.
  • the “inorganic salt medium” in the present embodiment may further contain betaine.
  • betaine in the present embodiment is a compound having CAS registration number 107-43-7, which is also known as trimethylglycine or glycine betaine. It is preferable to use an inorganic salt medium further containing betaine as a constituent component because D-lactic acid-producing Escherichia coli can perform excellent fermentation of D-lactic acid.
  • the content thereof is preferably 0.01 to 5 mmol / L, more preferably 0.3 to 2 mmol / L in the inorganic salt medium.
  • the betaine content is preferably 0.01 mmol / L or more, more preferably 0.3 mmol / L or more, from the viewpoint of more efficiently producing D-lactic acid. More preferably.
  • the upper limit of the betaine content in the inorganic salt medium at the start of the culture is preferably 5 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid, and is 2 mmol / L or less. Further preferred.
  • the “inorganic salt medium” in the present embodiment may include an antifoaming agent.
  • Any known antifoaming agent can be suitably used. Examples thereof include Adecanol LG126 (trade name, manufactured by ADEKA).
  • the content of the antifoaming agent is preferably 0.01 to 1 g / L, more preferably 0.05 to 0.5 g / L with respect to the total mass of the inorganic salt medium.
  • the content of the antifoaming agent in the inorganic salt medium at the start of the culture is preferably 0.01 g / L or more from the viewpoint of producing D-lactic acid more efficiently, and 0.05 g / L More preferably, the above is true.
  • the upper limit of the content of the antifoaming agent in the inorganic salt medium at the start of the culture is preferably 1 g / L or less from the viewpoint of more efficiently producing D-lactic acid, and preferably 0.5 g / L. More preferably, it is L or less.
  • the cells cultured in an inorganic salt medium are D-lactic acid-producing Escherichia coli capable of fermentatively producing D-lactic acid from a carbon source, preferably sugar.
  • D-lactic acid production activity and / or D-lactic acid optical purity by enhancing, inactivating, reducing or a combination of enzyme activities involved in lactic acid production activity of the target Escherichia coli by genetic recombination It is preferable to use recombinant Escherichia coli having improved D as a D-lactic acid producing Escherichia coli.
  • the term “by genetic recombination” in the present embodiment means that a change in the base sequence is caused by the insertion of another DNA into the base sequence of the native gene, or the substitution, deletion or combination of a part of the gene. It may be included, for example, as long as it is obtained as a result of mutation.
  • Examples of recombinant Escherichia coli having improved D-lactic acid production activity and / or D-lactic acid optical purity include those described in WO2005 / 033324, WO2010 / 032697, and WO2010 / 032698.
  • the D-lactic acid-producing E. coli is preferably a recombinant E. coli containing one or more genetic recombination selected from the group consisting of the following (i) to (v): (i) to (iii) It is further preferred that the recombinant E. coli contains all of the genetic recombination.
  • the recombinant Escherichia coli obtained by genetic recombination in this way has an increased amount of accumulated D-lactic acid compared to the one in which the expression of ldhA is not enhanced when producing D-lactic acid under aeration conditions. It is possible to improve the optical purity of D-lactic acid while reducing the concentration of impurity pyruvic acid.
  • Dld means a general term for enzymes that catalyze the reaction of producing pyruvic acid from D-lactic acid in the presence of oxidized flavin adenine dinucleotide as a coenzyme.
  • Pfl refers to an enzyme classified as enzyme number 2.3.54 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee and also called formate acetyltransferase. This enzyme is a general term for enzymes that reversibly catalyze the reaction of producing formic acid from pyruvic acid.
  • LdhA refers to an enzyme derived from E. coli that produces D-lactic acid and NAD from pyruvic acid and NADH.
  • Miv of (iv) is classified into enzyme number 1.1.1.17 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee, from malic acid to oxidized nicotine which is a coenzyme.
  • AspA refers to an enzyme classified as enzyme number 4.3.1.1 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee and also called aspartase. This enzyme is a general term for enzymes that reversibly catalyze the reaction of producing fumaric acid from L-aspartic acid.
  • the gene encoding LdhA is incorporated into an expression plasmid in a state of being linked to the promoter of a gene that controls the expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.
  • LdhA From the gene encoding LdhA derived from Escherichia coli, which is present in the genome of Escherichia coli, and the promoter of the gene responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis
  • the method of expressing LdhA can be mentioned by using the gene, which is responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis from genes encoding LdhA present in the genome of E. coli.
  • a method of expressing LdhA using a promoter is preferred.
  • the promoter of the gene responsible for the expression of the protein involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis is a strong promoter that constantly functions in bacteria, preferably in E. coli, and A promoter that is less susceptible to expression suppression even in the presence of glucose.
  • promoters for genes that are responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis include glyceraldehyde 3-phosphate dehydrogenase promoter and serine hydroxymethyltransferase (glyA) promoter. Is mentioned.
  • sucrose non-PTS gene group and FruK may be strengthened, but both the sucrose non-PTS gene group and FruK may be strengthened.
  • Sucrose hydrolase invertase, CscA
  • CscA Sucrose hydrolase
  • I.U.B International Biochemical Union
  • the enhancement of CscA can be realized, for example, by introducing a DNA having a base sequence of a gene encoding CscA derived from E. coli into E. coli.
  • FruK Fructose-1-phosphate kinase
  • I.B. International Biochemical Union
  • phosphofructokinase 1 Refers to an enzyme. Enhancement of FruK can be realized, for example, by introducing DNA having the base sequence of a gene encoding FruK derived from E. coli into E. coli.
  • the production method of D-lactic acid includes producing D-lactic acid from a carbon source, preferably sugar, using D-lactic acid-producing Escherichia coli. That is, it includes a step of contacting D-lactic acid-producing Escherichia coli with a carbon source and a step of recovering D-lactic acid obtained by the contact.
  • the method for producing D-lactic acid includes a step of contacting D-lactic acid-producing Escherichia coli with a carbon source in an inorganic salt medium to obtain D-lactate, and D-lactate. Removing D-lactic acid-producing Escherichia coli from the inorganic salt medium and then desalting D-lactate to obtain D-lactic acid.
  • the alkaline earth metal salt of D-lactic acid is obtained, and then in the step of obtaining D-lactic acid, D-lactic acid is obtained.
  • An inorganic salt containing an alkaline earth metal may be precipitated by desalting the alkaline earth metal salt of lactic acid.
  • an alkali metal salt of D-lactic acid is obtained, and then In the step of obtaining D-lactic acid, the alkali metal salt of D-lactic acid may be desalted by electrodialysis.
  • the method for producing D-lactic acid according to the present embodiment further includes a purification step of purifying D-lactic acid after the step of desalting D-lactate to obtain D-lactic acid, It is preferred to carry out the distillation while hydrolysis. By doing so, it is possible to further increase the D-lactic acid recovery rate in the distillation step, and to produce D-lactic acid more efficiently at a lower cost.
  • the contact between the D-lactic acid-producing Escherichia coli and the carbon source is carried out by culturing the D-lactic acid-producing Escherichia coli in the above-described inorganic salt medium, whereby a fermentation broth containing D-lactic acid can be obtained.
  • the culture temperature is preferably 20 ° C. or higher and 40 ° C. or lower, more preferably 25 ° C. or higher and 35 ° C. or lower. .
  • the pressure in the culture tank is preferably normal pressure (0.1 MPa) or more and 0.5 MPa or less, and more preferably normal pressure (0.1 MPa) or more and 0.2 MPa or less at the time of culture.
  • the pH of the culture is preferably 4.0 or more and 9.0 or less from the viewpoint of enhancing the fermentation productivity of Escherichia coli, preferably 6.0 or more and 8.0 or less, and more preferably 7.0 or more and 7. More preferably, it is 8 or less.
  • the pH is preferably adjusted while adding an inorganic base to the culture tank while producing D-lactic acid.
  • the produced D-lactic acid can be neutralized with an inorganic base to obtain a lactate.
  • inorganic bases examples include alkali metal salts such as lithium hydroxide, sodium hydroxide, potassium hydroxide and rubidium hydroxide, alkaline earth metal salts such as magnesium hydroxide, calcium hydroxide and barium hydroxide, and ammonia. What is necessary is just to use at least 1 sort (s) selected from the group which consists of.
  • counter ions of lactate corresponding to D-lactic acid produced by selecting the kind of inorganic base include lithium ion, sodium ion, potassium ion, rubidium ion, magnesium ion, calcium ion, barium ion, ammonium ion, etc. It can be any cation.
  • the culture time is not particularly limited as long as it is necessary for the bacterial cells to grow sufficiently and to produce D-lactic acid.
  • a culture tank capable of controlling temperature, pH, aeration conditions, stirring speed, and pressure.
  • the culturing according to the present embodiment is not limited to using a culture tank.
  • it is necessary to inoculate E. coli in the culture medium in the culture tank but there is no particular limitation on the amount to be inoculated, and colonies are formed on the agar medium for the E. coli used. Colonies may be directly inoculated into the culture tank using platinum ears or the like.
  • the culture solution previously cultured with containers, such as a flask and another culture tank may be prepared, and this may be seed
  • the required amount of the culture solution at this time is not particularly limited, but may be an amount that allows even one cell of E. coli to be inoculated into the medium in the culture tank.
  • the amount of the culture solution is preferably 0.01% to 40% equivalent to the amount of the medium solution in the culture tank. More preferably, the amount is equivalent to 0.1% to 10%.
  • the necessary amount of the culture solution is preferably 0.01% or more, more preferably 0.1% or more, from the viewpoint of producing D-lactic acid more efficiently.
  • the upper limit of the required amount of the culture broth is preferably 40% or less, more preferably 10% or less, from the viewpoint of more efficiently producing D-lactic acid.
  • the aeration amount in the fermentation process may be 0 vvm or more, preferably 0.001 vvm or more, and more preferably 0.01 vvm or more from the viewpoint of lactic acid production efficiency.
  • the amount of aeration in the fermentation process may be 5 vvm or less from the viewpoint of lactic acid production efficiency, preferably 2 vvm or less, and more preferably 1 vvm or less.
  • the notation “vvm” may be used as the air flow rate.
  • “vvm” indicates how many times the liquid volume is aerated in 1 minute. For example, 5 vvm aeration is performed on 10 L of fermentation broth. This means that 50 L was ventilated per minute.
  • the rate of oxygen dissolution into the liquid varies depending on the combination of the internal pressure, the position of the stirring blade, the shape of the stirring blade, and the stirring speed, so the productivity of D-lactic acid and other than D-lactic acid Various conditions can be set by using the amount of organic acid as an index. In addition, it is not necessary to carry out the set aeration conditions consistently from the beginning of fermentation to completion
  • the oxygen uptake rate (OUR) is preferably 0.0 mmol / L / hr or more, and more preferably 1.0 mmol / L / hr or more, from the viewpoint of lactic acid productivity.
  • the upper limit of the oxygen uptake rate (OUR) is preferably 100.0 mmol / L / hr or less, more preferably 50.0 mmol / L / hr or less from the viewpoint of lactic acid productivity, It is preferably 10.0 mmol / L / hr or less, and more preferably 5.0 mmol / L / hr or less.
  • the OUR may be any one measured during the production period of D-lactic acid.
  • the production phase of D-lactic acid refers to the time when E. coli starts to grow and produce D-lactic acid through the adaptation phase of E. coli culture after the start of fermentation. Moreover, when carrying out culture
  • the oxygen uptake rate (OUR) is the oxygen transfer rate per unit volume of the fermentation broth, that is, it can be said that the oxygen uptake rate of E. coli. OUR obtained from the following formula 1 by exhaust gas analysis is used.
  • the measured values at one place may be applied if the air flow rate, air pressure, and absolute temperature are not negligible at the air inlet and outlet.
  • the pressure and air pressure referred to in the present embodiment refer to absolute pressure.
  • OUR varies depending on aeration amount, stirring rotation speed, temperature, pressure, pH, and the like. Therefore, in order to adjust the OUR within the above-described range, the aeration flow rate, the stirring rotation speed, and the like may be adjusted as appropriate.
  • the OUR can be converted into another index.
  • examples of such other indicators include a liquid film capacity coefficient (k L a).
  • Ekisakaimaku capacity coefficient (k L a) is a function of the aeration rate and stirring speed, the following relationship is known (Equation 2, Richards, J. W. 1961, Prog.Ind.Micro.3, 143-172)
  • FIG. 1 shows an example of a fermentation apparatus used for culturing D-lactic acid-producing E. coli.
  • the fermentation apparatus 10 is provided with a fermentation tank 12. Air is supplied to the fermenter 12 from the air inlet via the mass flow meter 14 (arrow A), while the air in the tank is discharged from the exhaust port via the condenser 16. (Arrow B).
  • An in-tank pressure gauge 18 and an exhaust gas analyzer 20 are connected between the condenser 16 and the exhaust port, and the pressure in the tank and the oxygen partial pressure at the outlet can be measured.
  • a temperature sensor 22, a DO sensor 24, and a pH sensor 26 are arranged, respectively, so that the temperature, DO (dissolved oxygen) and pH in the reaction solution in the fermenter 12 can be measured.
  • the fermenter 12 is provided with a disc turbine blade 28 as a stirrer, and the disc turbine blade 28 is stirred and controlled by a motor 44.
  • the periphery of the fermenter 12 is double and can be heated or cooled with warm water.
  • a pH adjusting unit 34 filled with a pH adjusting agent is provided outside the fermenter 12.
  • the pH adjuster 34 can supply a pH adjuster to the fermenter 12 via the pump 36.
  • the pH adjuster the aforementioned inorganic base can be used.
  • the fermenter 12 is provided with a controller 38 that controls the whole, and is connected to the temperature sensor 22, the DO sensor 24, and the pH sensor 26 so that information from each sensor can be input. Further, the controller 38 adjusts the temperature of the jacket 32 according to information from various sensors to control the solution temperature in the fermenter, and controls the pH by operating the pump 36.
  • D-lactic acid accumulated in the fermenter 12 can be recovered by separation and purification from the inorganic salt medium. Specifically, it is a process of removing solids such as cells from the inorganic salt medium, desalting, concentrating and purifying, but the method and order thereof are not particularly limited, but it is determined from the inorganic salt medium after fermentation. -It is preferable to obtain D-lactic acid by desalting D-lactate after removing lactic acid-producing Escherichia coli.
  • methods such as centrifugation and filtration can be used to remove solids such as bacterial cells.
  • Examples of methods for converting D-lactate to D-lactic acid in the purification step include ion exchange columns and electrodialysis. It may be desalted to D-lactic acid by adding an inorganic acid.
  • an inorganic acid For example, when the counter ion of D-lactate is an alkaline earth metal ion, an inorganic salt containing an alkaline earth metal can be precipitated by desalting D-lactate.
  • the counter ion of D-lactate is calcium ion, it can be desalted by adding sulfuric acid and precipitating calcium sulfate in the solution to obtain D-lactic acid.
  • D-lactate when the counter ion of D-lactate is an alkali metal ion, preferably sodium ion or potassium ion, it is preferable to desalinate D-lactate by electrodialysis.
  • a water splitting electrodialyzer can be used for the electrodialysis treatment.
  • the apparatus is an electrodialysis apparatus in which bipolar membranes and cation exchange membranes are alternately arranged to form an acid chamber and a base chamber.
  • a typical example is a “two-chamber water-splitting electrodialysis apparatus”.
  • the electrodialysis operation using the “water splitting electrodialysis apparatus” is defined as “water splitting electrodialysis step”.
  • a more specific process is typically exemplified by a “two-chamber water-splitting electrodialysis process”.
  • the obtained fermentation broth containing D-lactate is subjected to a water-splitting electrodialysis step using a water-splitting electrodialyzer, and D-lactic acid and alkali are recovered. That is, a water-splitting electrodialysis apparatus using a bipolar membrane and a cation exchange membrane is supplied with a D-lactate solution to carry out a water-splitting electrodialysis step, and D-lactic acid from the acid chamber and alkali from the base chamber, respectively. obtain.
  • bipolar membrane As the bipolar membrane, a conventionally known bipolar membrane, that is, a known bipolar membrane having a structure in which a cation exchange membrane and an anion exchange membrane are bonded together can be used. Moreover, a well-known cation exchange membrane can be used as a cation exchange membrane of a water-splitting electrodialysis apparatus.
  • FIG. 2 shows a schematic diagram of a typical aspect of the water-splitting electrodialysis apparatus used in the present embodiment. That is, in FIG. 2, the water-splitting electrodialysis apparatus has two types of membranes, a bipolar membrane B and a cation exchange membrane C, arranged alternately between a positive electrode 3 and a negative electrode 4, and an acid chamber 8 and a base Two chambers of the chamber 7 are formed.
  • the gap between the cation exchange membrane C and the positive electrode 3 (anode chamber 5) and the gap between the cation exchange membrane C and the negative electrode 4 (cathode chamber 6) are filled with an electrode solution.
  • the chamber between the anion exchanger side of the bipolar membrane B and the cation exchange membrane C is the base chamber 7
  • the chamber between the cation exchanger side of the bipolar membrane B and the cation exchange membrane C is the acid chamber 8. .
  • the water-splitting electrodialysis step using the water-splitting electrodialysis apparatus includes an external tank for liquid to be supplied to each of the acid chamber 8 and the base chamber 7 (not shown). ), And a method of performing electrodialysis while circulating the liquid between the respective chambers and the external tank is preferably employed.
  • the D-lactate in the acid chamber 8 is converted to D-lactic acid with energization. That is, the cation of D-lactate introduced into the acid chamber 8 permeates through the cation exchange membrane C and moves to the base chamber 7. At this time, it binds to OH ions generated from the bipolar membrane B. Become a base. In the acid chamber 8, the proton generated from the bipolar membrane B and the D-lactic acid anion are combined to form non-dissociable D-lactic acid, and remains in the acid chamber 8 as it is.
  • the temperature of various liquids during the water-splitting electrodialysis step is usually 5 ° C. or higher and 70 ° C. or lower, and preferably 20 ° C. or higher and 50 ° C. or lower.
  • D-lactic acid can be decomposed and D-lactic acid and alkali can be separated by the water-splitting electrodialysis step to recover D-lactic acid.
  • the separated alkali can be reused as a neutralizing agent in organic acid fermentation such as D-lactic acid.
  • D-lactic acid with high purity can be obtained by the water-splitting electrodialysis step.
  • concentration and purification process for example, activated carbon treatment or NF membrane treatment is used to remove impurities such as proteins and by-product organic acids, and ion exchange columns, anion / cation exchange membrane electrodialysis, and evaporation are used to remove ionic substances.
  • Concentration, purification and recovery of lactic acid include desalting and direct distillation, steam distillation, lactide formation and distillation, alcohol and catalyst esterification followed by distillation, in organic solvents
  • Examples of the method include extraction, chromatography column separation, ion exchange column separation, anion / cation exchange membrane electrodialysis concentration and recovery, and crystallization by crystallization. Moreover, you may combine these methods suitably in arbitrary orders.
  • the coloring component can be removed, and D-lactic acid suitable for polymerizing use can be obtained.
  • the activated carbon treatment is performed by adding, for example, 0.2% by weight or more and 2% by weight or less of activated carbon to the weight of the D-lactic acid solution, stirring the mixture as necessary, and then removing the activated carbon by filtration. Can do.
  • the light transmittance at 400 nm of the D-lactic acid solution after the activated carbon treatment can be 80% or more and 99% or less when the light transmittance at 400 nm of water is 100%.
  • the D-lactic acid solution obtained after desalting is distilled batchwise or continuously, it is preferable to distill while adding water to perform hydrolysis. Thereby, superposition
  • the polymerization degree n is preferably 1 or more and 4 or less, more preferably 1 or more and 2 or less, and even more preferably 1 or more and 1.5 or less.
  • the distillation yield can be 70 wt% or more and 99.5 wt% or less.
  • the distillation yield here is the recovery rate of the lactic acid component in the distillation step, and the lactic acid component is lactic acid and a lactic acid oligomer.
  • the polymerization proceeds in the evaporation and concentration step, it is also effective to improve the distillation yield by performing hydrolysis before the distillation to lower the polymerization degree n of lactic acid.
  • the polymer production method according to the present embodiment is a polymerization reaction using D-lactic acid obtained by the D-lactic acid production method according to the present embodiment. The details will be described below.
  • the polylactic acid referred to here includes a stereocomplex polymer of D-lactic acid and L-lactic acid and a homopolymer of D-lactic acid.
  • L-lactic acid which is one of the raw materials for the stereocomplex polymer of D-lactic acid and L-lactic acid, known ones can be preferably used.
  • the method for producing L-lactic acid is not particularly limited, and examples thereof include fermentation methods and organic synthesis methods.
  • polylactic acid and lactic acid copolymer are processed by various molding methods, for example, injection molding, film molding, sheet molding, extrusion molding, blow molding, foam molding, etc., and are used for various applications.
  • Applications include, for example, the properties and heat resistance of biodegradable plastics, packaging containers, cushioning materials, various electronic and electrical equipment, OA equipment, vehicle parts, machine parts, other agricultural materials, fishery materials, transport containers, Examples include play equipment and miscellaneous goods.
  • the polymerization method is not particularly limited, and a known method can be adopted.
  • ring-opening polymerization via lactic acid lactide, condensation polymerization in which lactic acid or lactic acid ester is directly polymerized, and oligomer in which lactic acid or lactic acid ester is directly polymerized are chain-extended by amide bond or urethane bond with polyisocyanate.
  • Examples of the polymerization method include solid phase polymerization and melt polymerization.
  • the arrangement order and the polymerization method are not particularly limited, and a known method can be employed.
  • the sequence order includes random copolymerization, alternating copolymerization, block copolymerization, graft copolymerization, and the like
  • the polymerization method includes radical copolymerization, ionic copolymerization, cocondensation, and the like.
  • D-lactic acid obtained by the method according to this embodiment can be used for the production of polylactic acid.
  • the weight average molecular weight (Mw) of the obtained polylactic acid is preferably 50,000 or more and 400,000 or less, and more preferably 80,000 or more and 300,000 or less.
  • the molecular weight distribution of the polymer is usually expressed by Mw / Mn.
  • the molecular weight distribution approaches 2 when the reaction approaches completion (Seizo Okamura) Introduction to Polymer Chemistry (2nd edition, p.210).
  • the molecular weight distribution greatly affects the fluidity and mechanical properties of the polymer. If the molecular weight distribution is wide, that is, if the value of Mw / Mn increases, the non-Newtonian characteristics increase and the fluidity improves. It is known that the resin can be easily molded in this process.
  • the polylactic acid or lactic acid copolymer produced from D-lactic acid obtained by the method according to this embodiment has a wider molecular weight distribution than a general polycondensation polymer, and the range is preferably 3 or more and 20 or less, More preferably, it is 5 or more and 15 or less, and further preferably 8 or more and 12 or less. Furthermore, D-lactic acid obtained by the method according to the present embodiment can produce polylactic acid and lactic acid copolymer having a high molecular weight distribution without going through a large amount of activated carbon treatment and complicated purification steps.
  • a polylactic acid or a lactic acid copolymer having a wide molecular weight distribution can be produced even by treating with a small amount of activated carbon of 0.2 wt% or more and 2 wt% or less.
  • the coloration degree (YI value) of polylactic acid or lactic acid copolymer produced from D-lactic acid obtained by the method according to this embodiment is preferably YI value of 10 or less, more preferably YI value of 5 or less. be able to.
  • the YI value here indicates the distance from this white, assuming that the ideal white color (complete diffuse reflection surface) is 0, and the color difference from white to yellow is plus, and the color difference in blue is minus. It is represented by Therefore, when the negative value is increased, the bluish color is increased, and when the positive value is increased, the yellow color is increased, which causes deterioration of the hue.
  • X is a value that feels red
  • Y is a value that feels green
  • Z is a value that feels blue.
  • tristimulus values X, Y, Z are measured using a visible spectrometer UV-2400PC) and calculated according to equation 3.
  • YI value 100 ⁇ (1.28X ⁇ 1.06Z) / Y (Formula 3)
  • a D-lactic acid monomer that can be polymerized into a D-lactic acid polymer suitable for the quality required for the D-lactic acid polymer such as hue and molecular weight is produced at low cost and efficiently.
  • a method can be provided.
  • the optical purity of the obtained D-lactic acid was 100% ee.
  • the optical purity was measured by HPLC according to a conventional method (column: SUMICHILARIROA-5000 (manufactured by Sumika Chemical Analysis)), eluent: aqueous solution consisting of 10 g / L of isopropyl alcohol ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ and 319.2 mg / L of anhydrous copper (II) sulfate, detection Wavelength: 254 nm).
  • An E. coli strain, MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain / GAPldhA genome insertion strain was prepared in the same manner as described in Example 23 of WO2005 / 033324. Specifically, an E. coli strain, MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain / GAPldhA genome insertion strain was prepared by the following method.
  • ⁇ Production 1 Cloning of the vicinity region of the Escherichia coli dld gene>
  • the entire base sequence of the genomic DNA of Escherichia coli is known (GenBank accession number U00096), and the base sequence of a gene encoding the FAD-dependent D-lactate dehydrogenase of Escherichia coli (hereinafter sometimes abbreviated as dld) is also reported. (Genbank accession number M10038).
  • the genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), and the obtained genomic DNA and the gene information of the region near the dld gene of the genomic DNA derived from MG1655 strain were used.
  • PCR was performed using the prepared CAACACCAAGCTTTCGCG (SEQ ID NO: 1) and TTCCACTCCCTTGTGGTGCC (SEQ ID NO: 2), AACTGCAGAAATTACGGATGGCAGAG (SEQ ID NO: 3) and TGTTCTAGAAAGTTCTTTGAC (SEQ ID NO: 4).
  • the obtained fragments were digested with restriction enzymes HindIII and PstI, and PstI and XbaI, respectively, to obtain fragments of about 1140 bp.
  • This fragment was mixed with a fragment obtained by digesting the temperature sensitive plasmid pTH18cs1 (Hashimoto-Gotoh, T., et.al., Gene, Vol.
  • ⁇ Production 2 Production of Escherichia coli MG1655 strain dld gene deletion strain>
  • the plasmid pTH ⁇ dld obtained in Production 1 was transformed into the MG1655 strain, and a transformant that grew on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol at 30 ° C. was obtained.
  • the obtained transformant was inoculated into an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol and cultured at 30 ° C. overnight. Next, these cultured cells were applied to an LB agar plate containing 10 ⁇ g / ml of chloramphenicol to obtain colonies that grew at 42 ° C.
  • the operation of obtaining a single colony growing at 42 ° C. was repeated once more.
  • the clones were inoculated into LB liquid medium (3 ml / tube) and cultured with shaking at 30 ° C. for 3 to 4 hours. This was appropriately diluted (about 10 ⁇ 2 to 10 ⁇ 6 ) so that a single colony was obtained, applied to an LB agar plate, and cultured overnight at 42 ° C. to obtain a colony. 100 colonies were picked up randomly from the colonies that appeared, and each was grown on an LB agar plate and an LB agar plate containing 10 ⁇ g / ml chloramphenicol. I picked a clone.
  • a strain lacking the dld gene region was selected from the chromosomal DNA of these target clones by PCR, a clone satisfying the above was designated as a dld-deficient strain, and the resulting strain was designated as MG1655 ⁇ dld strain.
  • Escherichia coli MG1655 strain can be obtained from the American Type Culture Collection, which is a cell / microorganism / gene bank.
  • ⁇ Production 3 Cloning of the vicinity region of the Escherichia coli pfl gene>
  • the entire nucleotide sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the nucleotide sequence of a gene encoding Escherichia coli pyruvate formate lyase (hereinafter sometimes referred to as pfl) has also been reported. (Genbank accession number AE000192).
  • a gene encoding Escherichia coli pyruvate formate lyase hereinafter sometimes referred to as pfl
  • Genbank accession number AE000192 In order to clone the region near the base sequence of the gene encoding pfl, four oligonucleotide primers shown in SEQ ID NOs: 5, 6, 7 and 8 were synthesized.
  • the primers of SEQ ID NOs: 6 and 7 have a SphI recognition site on the 5 ′ end side.
  • a genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), the obtained genomic DNA, a primer having the base sequence of SEQ ID NO: 5, and the base sequence of SEQ ID NO: 6 And a primer having the nucleotide sequence of SEQ ID NO: 7 and a primer having the nucleotide sequence of SEQ ID NO: 8, and performing PCR under normal conditions, it is about 1.8 kbp (hereinafter referred to as pflB-L fragment) And a DNA fragment of about 1.3 kbp (hereinafter sometimes referred to as a pflB-R fragment) was amplified.
  • the digested product was reacted with T4 DNA ligase, transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd., DNA-903), and cultured at 30 ° C. on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol. Thus, transformants that grow were obtained.
  • the obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol, and the plasmid was recovered from the obtained cells.
  • the insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target pflB were inserted.
  • This plasmid was named pTH ⁇ pfl.
  • MG1655 pfl, dld gene deletion strain preparation The plasmid pTH ⁇ pfl obtained in Production 3 was transformed into the MG1655 ⁇ dld strain to obtain a transformant that grew at 30 ° C. on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol. The obtained transformant was cultured and selected in the same manner as in Production 2 above, and finally a strain lacking the pfl gene was selected to obtain the MG1655 ⁇ dld strain in which the pfl gene was disrupted. This strain was designated as MG1655 ⁇ pfl ⁇ dld strain.
  • the primer having the base sequence of SEQ ID NO: 9 has a KpnI recognition site on the 5 ′ end side
  • the primer having the base sequences of SEQ ID NOS: 10 and 11 has a BamHI recognition site on the 5 ′ end side
  • Each primer has an XbaI recognition site on the 5 ′ end side.
  • Genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), and the resulting genomic DNA, SEQ ID NO: 9 and SEQ ID NO: 10, and combinations of SEQ ID NO: 11 and SEQ ID NO: 12
  • DNA fragments of about 800 bp hereinafter sometimes referred to as mdh-L fragment
  • about 1,000 bp hereinafter also referred to as mdh-R fragment
  • This DNA fragment was separated and collected by agarose electrophoresis, and the mdh-L fragment was digested with KpnI and BamHI, and the mdh-R fragment was digested with BamHI and XbaI, respectively.
  • Two of these digested fragments and KpnI and XbaI of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610) (Hashimoto-Gotoh, T., et., Gene, Vol. 241 (1), pp185-191 (2000)).
  • the digested product was reacted with T4 DNA ligase, transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd., DNA-903), and cultured at 30 ° C. on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol. Thus, transformants that grow were obtained.
  • the obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol, and the plasmid was recovered from the obtained cells.
  • the insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target mdh were inserted.
  • This plasmid was named pTH ⁇ mdh.
  • the plasmid pTH ⁇ mdh is transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld strain, cultured and selected in the same manner as in Production 2 above, and finally the mdh gene is disrupted by selecting a strain lacking the mdh gene.
  • MG1655 ⁇ pfl ⁇ dld strain was obtained. This strain was designated as MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain.
  • About 910 bp (hereinafter sometimes referred to as an aspAL fragment) by performing PCR under the normal conditions with a combination of the genomic DNA of Escherichia coli MG1655 strain, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 15 and SEQ ID NO: 16.
  • a DNA fragment of about 1,100 bp (hereinafter sometimes referred to as an aspA-R fragment) was amplified. This DNA fragment was separated and collected by agarose electrophoresis. Since SEQ ID NO: 14 and SEQ ID NO: 15 contain complementary regions, the aspA-L fragment and the aspA-R fragment are mixed with the aspA-L fragment and subjected to a PCR reaction without using a primer.
  • the aspA-L fragment and the aspA-R fragment were ligated and separated and recovered by agarose electrophoresis.
  • the resulting DNA fragment was blunted with DNA Blunting Kit (Takara Bio), and then phosphorylated at the 5 'end using T4 polynucleotide kinase by a conventional method.
  • the temperature sensitive plasmid pTH18cs1 was digested with SmaI and then dephosphorylated with alkaline phosphatase.
  • the above two phosphorylated fragments and the dephosphorylated plasmid were reacted with T4 DNA ligase, and transformed into Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. DNA-903), and chloramphenicol. Culture was performed at 30 ° C. on an LB agar plate containing 10 ⁇ g / ml to obtain a transformant that grew. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol, and the plasmid was recovered from the obtained cells.
  • the insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target aspA were inserted.
  • This plasmid was designated as pTH ⁇ asp.
  • the plasmid pTH ⁇ asp is transformed into the Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain, cultured and selected in the same manner as in Production 2 above, and finally, the strain lacking the aspA gene is selected to destroy the aspA gene.
  • MG1655 ⁇ pfl ⁇ dld ⁇ mdh strain was obtained. This strain was designated as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain.
  • GAPDH glyceride 3-phosphate dehydrogenase
  • the DNA fragment was digested with restriction enzymes EcoRI and HindIII to obtain an about 1.0 kbp D-lactate dehydrogenase (ldhA) gene fragment.
  • the above two DNA fragments and the fragment obtained by digesting plasmid pUC18 with restriction enzymes EcoRI and HindIII were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ competent cell (Toyobo Co., Ltd. DNA-903). And a transformant that grows on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colony was cultured overnight at 30 ° C.
  • plasmid was recovered from the obtained cells.
  • the inserted sequence into the plasmid was confirmed by sequencing, and the target GAPDH promoter and the gene encoding ldhA were inserted, and a plasmid predicted to express ldhA by the GAPDH promoter was obtained.
  • This plasmid was named pGAP-ldhA.
  • AAGGTACCACCACCAGAGCGTTCTCAAGC SEQ ID NO: 21
  • GCTTCTAGATTCTCCAGTGATGTTTGAATCAC SEQ ID NO: 22
  • GTCTAGAGCAATGATCTACACGATTCG (SEQ ID NO: 23) prepared based on the sequence information of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter of Escherichia coli MG1655 strain and the sequence of the ldhA gene of Escherichia coli MG1655 strain.
  • PCR was performed using the previously prepared vector pGAP-ldhA as a template using AACTGCAGGTTCGTTCTCATACACGTCC (SEQ ID NO: 24) to obtain a DNA fragment of about 850 bp consisting of the GAPDH promoter and the region near the start codon of the ldhA gene.
  • the fragments obtained above were digested with restriction enzymes KpnI and XbaI, XbaI and PstI, this fragment was mixed with a fragment obtained by digesting temperature sensitive plasmid pTH18cs1 with KpnI and PstI, and ligated using ligase. Thereafter, the cells were transformed into DH5 ⁇ competent cells (DNA-903, Toyobo Co., Ltd.) to obtain transformants that grew at 30 ° C. on LB agar plates containing 10 ⁇ g / ml of chloramphenicol. The obtained colony was cultured overnight at 30 ° C.
  • Plasmid pTHGAPldhA was transformed into MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain, and cultured on an LB agar plate containing 10 ⁇ g / ml of chloramphenicol at 30 ° C.
  • the obtained transformant was inoculated into an LB liquid medium containing 10 ⁇ g / ml of chloramphenicol and cultured at 30 ° C. overnight. Next, these cells were applied to an LB agar plate containing 10 ⁇ g / ml of chloramphenicol to obtain colonies that grew at 42 ° C. The obtained colonies were cultured overnight at 30 ° C. in an LB liquid medium, and further applied to an LB agar plate to obtain colonies that grew at 42 ° C.
  • GAPldhA genome insertion strain 100 colonies were picked up randomly from the appearing colonies and grown on LB agar plates and LB agar plates containing 10 ⁇ g / ml of chloramphenicol, and chloramphenicol sensitive clones were selected. Furthermore, an approximately 800 bp fragment containing the GAPDH promoter and the ldhA gene is amplified from the chromosomal DNA of these target clones by PCR, a strain in which the ldhA promoter region is replaced by the GAPDH promoter is selected, and a clone satisfying the above is selected as MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / It was named GAPldhA genome insertion strain.
  • Example 1 Production of D-lactic acid in inorganic salt medium by MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain / GAPldhA-inserted strain 100 mg of LB Broth, Miller culture solution (Difco244620d) in erlenmeyer flask as a pre-culture was prepared in MG1655 ⁇ pfl ⁇ dhl ⁇ dhlAdplAdhdAPdhdAPdhlAdpl The strain was inoculated and stirred and cultured overnight at 120 rpm.
  • a 1 L culture tank (culture device BMJ-01 manufactured by ABLE) containing 475 g of the inorganic salt medium having the composition shown in Table 1 was sterilized by autoclave, and 25 ml of the preculture was transferred to the culture vessel. Cultivation was performed under atmospheric pressure until glucose was depleted at an aeration rate of 0.5 vvm, a stirring speed of 300 rpm, a culture temperature of 35 ° C., and a pH of 7.5 (pH adjustment with a slurry of 20 wt% calcium hydroxide and 80 wt% pure water).
  • the fermentation end time was 18 hours, and the D-lactic acid accumulation concentration was 92.7 g / L.
  • the fermentation end time is the time when all the added sugar is consumed, and the productivity can be evaluated by the length.
  • Example 2 Crude purification of D-lactic acid produced in an inorganic salt medium
  • D-lactic acid fermentation broth obtained in Example 1 it was centrifuged at 8000 xg.
  • 95% by weight sulfuric acid was added until the pH of the supernatant reached 2.5.
  • suction filtration was performed using a filter paper (Watman 42).
  • activated carbon corresponding to 0.5% by weight of the filtrate was added and stirred.
  • 25 ml of the preculture was transferred to a 1 L culture tank (culture device BMJ-01 manufactured by ABLE) containing 475 g of corn steep liquor medium having the composition shown in Table 2 and cultured. Cultivation was carried out under atmospheric pressure at an aeration rate of 0.5 vvm, a stirring speed of 300 rpm, a culture temperature of 35 ° C., pH 7.5 (adjusted with an aqueous calcium hydroxide solution) until glucose was depleted. After completion of the culture, the amount of D-lactic acid accumulated in the obtained culture broth was measured by HPLC according to a conventional method. The D-lactic acid accumulation concentration was 90.7 g / L in 20 hours.
  • Example 3 Production of D-lactic acid in inorganic salt medium supplemented with transition metal ions Under the culture conditions of Example 1, 0.1% by volume of a transition metal-containing aqueous solution having the composition shown in Table 3 was added to the medium. The fermentation end time and D-lactic acid accumulation concentration were the same as when no transition metal-containing aqueous solution was added.
  • Example 4 Production of D-lactic acid in an inorganic salt medium supplemented with calcium chloride and thiamine Under the culture conditions of Example 1, both calcium chloride and thiamine were added to the medium of Example 1 to a concentration of 10 mg / L. Culture was performed using the prepared medium. The fermentation end time and D-lactic acid accumulation concentration were the same as when calcium chloride and thiamine were not added.
  • Example 5 Production of D-lactic acid and crude purification of D-lactic acid in an inorganic salt medium supplemented with transition metal ions, calcium chloride and thiamine Table 3 was further added to the medium of Example 1 under the culture conditions of Example 1. Culturing was carried out using a medium containing 0.1% by volume of a transition metal-containing solution having the composition shown in FIG. 5 and further adding calcium chloride and thiamine to 10 mg / L. The fermentation end time and D-lactic acid accumulation concentration were the same as in Example 1. In order to remove the cells from the obtained D-lactic acid fermentation broth, it was centrifuged at 8000 ⁇ g.
  • the transmittance of 400 nm wavelength of pure water in the spectrophotometer is 100%
  • the transmittance of 400 nm wavelength of the filtrate after removal of activated carbon is 93.7% and 1.0 wt. It was 98.1% for% activated carbon.
  • Example 6 Production of D-lactic acid in inorganic salt medium with varying betaine content Under the culture conditions of Example 1, betaine added to the medium was added at a content of 0 to 6 g / L. Table 4 shows the fermentation end time under each condition. The D-lactic acid accumulation concentration was the same in all test groups. In order to perform decolorization and crude purification, activated carbon corresponding to 0.5% by weight of the filtrate was added and stirred. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 ⁇ m membrane filter.
  • the transmittance of 400 nm wavelength of pure water in the spectrophotometer is 100%
  • the transmittance of 400 nm wavelength of the filtrate after removal of activated carbon is 98. It was 0% or more.
  • Example 7 Production of D-lactic acid in an inorganic salt medium having a reduced ammonium sulfate content Under the culture conditions of Example 1, the ammonium sulfate content in the inorganic salt medium was set to 2 g / L. The fermentation end time was 34 hours. The D-lactic acid accumulation concentration was equivalent.
  • Example 8 Production of D-lactic acid in inorganic salt medium with reduced magnesium sulfate content Under the culture conditions of Example 1, magnesium sulfate heptahydrate in inorganic salt medium was 0.1 g / L. did. The fermentation end time was 34 hours. The D-lactic acid accumulation concentration was equivalent.
  • Example 9 Production of D-lactic acid in an inorganic salt medium in which dipotassium hydrogen phosphate is replaced with potassium dihydrogen phosphate. Under the same culture conditions as in Example 1, the same content of dipotassium hydrogen phosphate in the inorganic salt medium is contained. The culture was carried out by substituting with potassium dihydrogen phosphate in an amount (g / L). It was revealed that the fermentation end time and D-lactic acid accumulation concentration were the same, and dipotassium hydrogen phosphate may be replaced with potassium dihydrogen phosphate.
  • Example 10 Production of D-lactic acid in an inorganic salt medium with a reduced content of dipotassium hydrogen phosphate Under the culture conditions of Example 1, 0.5 g / L of dipotassium hydrogen phosphate in the inorganic salt medium was used. It was. The fermentation end time was 27 hours. The D-lactic acid accumulation concentration was equivalent.
  • Example 2 In the same manner as in Example 9 of WO2010 / 032698, an E. coli strain, MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-cscA-fruK strain was prepared. Specifically, an E. coli strain, MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP-cscA-fruK strain was prepared by the following method.
  • ⁇ Production 8 Escherichia coli O157-derived invertase gene, Escherichia coli MG1655-derived fructose-1-phosphate kinase gene expression vector and construction of the expression vector transformant>
  • the amino acid sequence of Escherichia coli O157 invertase and the nucleotide sequence of the gene have already been reported. That is, the gene (cscA) encoding invertase is described in 3274383 to 3275816 of the Escherichia coli O157 strain genomic sequence described in GenBank accession number AE005174.
  • GAPDH glyceraldehyde 3-phosphate dehydrogenase derived from Escherichia coli described in 397 to 440 in the base sequence information of GenBank accession number X02662 Promoter sequences
  • GAPDH promoter the genomic DNA of Escherichia coli MG1655 strain was used as a template, and the DNA fragment obtained was amplified by PCR using CGAGCTCACATATGCAATGATTGACACGATTCCG (SEQ ID NO: 25) and TCTAGAGCTATTTTGTTAGTGAAATAAAGG (SEQ ID NO: 26).
  • the DNA fragment corresponding to the GAPDH promoter of about 110 bp was obtained by digestion.
  • the obtained DNA fragment was mixed with the plasmid pBR322 (GenBank accession number J01749) digested with restriction enzymes NdeI and PvuII, ligated with ligase, Escherichia coli DH5 ⁇ strain competent cell (Toyo Spinning Co., Ltd. DNA-903) was transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 ⁇ g / mL.
  • the obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid was recovered from the obtained cells.
  • the insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that the target GAPDH promoter sequence was correctly inserted.
  • This plasmid was designated as pBRgapP.
  • Escherichia coli O157 genomic DNA SIGMA-ALDRICH: IRMM449
  • GATCTAGACGGGAGAAGTCTTATGACGCAATCTCGATTTGCATTG SEQ ID NO: 27
  • ATGGTACCTTAACCCAGTTGCAGGTG was obtained by PCR method.
  • the obtained DNA fragment was digested with the restriction enzyme XbaI to obtain an invertase gene fragment of about 1.4 kbp.
  • the obtained DNA fragment and the previously prepared plasmid pBRgapP were digested with restriction enzymes XbaI and PshAI, mixed together using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd.) Company DNA-903) was transformed to obtain transformants that grew on LB agar plates containing 50 ⁇ g / mL of ampicillin.
  • the obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid was recovered from the obtained cells.
  • the insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that the target cscA was correctly inserted.
  • This plasmid was named pGAP-cscA.
  • the amino acid sequence of the fructose-1-phosphate kinase of Escherichia coli MG1655 and the nucleotide sequence of the gene have already been reported. That is, the gene (fruK) encoding fructose-1-phosphate kinase is described in 2260387 to 2259449 of the Escherichia coli MG1655 strain genome sequence described in GenBank accession number U00096.
  • fructose-1-phosphate kinase gene In order to obtain the fructose-1-phosphate kinase gene, using the genomic DNA of Escherichia coli MG1655 as a template, ATGGTACCGGAGAAAGTCTTATGGAGCAGACGTGTTCCTAC (SEQ ID NO: 29) and TCGGATCCTTATGCCTCTCCCTGCTGTCAG (SEQ ID NO: 30) were obtained by PCR. The obtained DNA fragment was digested with the restriction enzyme KpnI to obtain a fructose-1-phosphate kinase gene fragment of about 1.0 kbp.
  • the obtained DNA fragment was mixed with the previously prepared plasmid pGAP-cscA by digestion with restriction enzymes KpnI and EcoRV, ligated with ligase, and then Escherichia coli DH5 ⁇ strain competent cells (Toyo Spinning Co., Ltd. DNA-903) was transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 ⁇ g / mL.
  • the obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid was recovered from the obtained cells.
  • the inserted sequence into the plasmid was confirmed by sequencing, and it was confirmed that the desired fruK was correctly inserted.
  • This plasmid was designated as pGAP-cscA-fruK.
  • This plasmid pGAP-cscA-fruK was transformed into the MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain prepared in Preparation 7 above, and cultured overnight at 37 ° C. on LB Broth, Miller agar plates containing ampicillin 50 ⁇ g / mL. A genome inserted strain / pGAP-cscA-fruK strain was obtained.
  • Example 11 Production of D-lactic acid using sucrose as a raw material Escherichia coli uses the MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp / GAPldhA genome insertion strain / pGAP18-cscA-fruK strain prepared in Production Example 2 under the culture conditions of Example 1, The content of dipotassium hydrogen phosphate and magnesium sulfate was changed from glucose to sucrose. Table 5 shows the contents of dipotassium hydrogen phosphate and magnesium sulfate added to the medium and the fermentation end time. The D-lactic acid accumulation concentration was 85 to 90 g / L, which was almost the same as the test using glucose.
  • Example 12 ⁇ Seed culture> LB medium (Difco TM LB Broth Miller) was placed in an Erlenmeyer flask in an amount 1/5 of the flask volume, and autoclaved at 121 ° C. for 15 minutes. The autoclave-sterilized medium was inoculated with 0.1% by volume of the Escherichia coli MG1655 ⁇ pfl ⁇ dld ⁇ mdh ⁇ asp strain / GAPldhA genome insertion strain prepared in Production Example 1. Shaking culture was performed in a thermostatic chamber at 35 ° C. for 3 hours to grow seed cells.
  • BML-01PI BML-01PI
  • inoculated 820 mL of the above preculture solution.
  • the fermentation apparatus 10 shown in FIG. 1 was used.
  • the air flow rate at the air inlet and outlet was 1.6 L / min
  • the air pressure at the air inlet and outlet was 0.12 MPa
  • the temperature of the air inlet and outlet was 35 ° C.
  • the oxygen mole fraction at the air inlet was 0.21
  • the measured oxygen mole fraction (0.21 to 0.18) was used as the oxygen mole fraction at the outlet.
  • the average value after 5 hours of culture of the value calculated according to the above-mentioned formula 1 was defined as OUR.
  • the dissolved oxygen concentration (DO) in the tank was almost 0 ppm after 3 hours from the start of fermentation.
  • the value of the mass flow meter 14 was adopted as the air flow rate at the air inlet, and the value of the mass flow meter 14 was adopted as the range in which the decrease due to oxygen consumption was negligible.
  • the values of the pressure gauge 18 in the tank were adopted for the air pressure at the air inlet and outlet.
  • the absolute temperature at the air inlet and outlet is the value of the temperature sensor 22 in the tank.
  • the oxygen mole fraction at the air inlet was 0.21, and the value of the exhaust gas analyzer 20 was adopted as the oxygen mole fraction at the outlet.
  • the precipitated Ca content in the obtained fermentation broth was measured as follows. 200 g of the fermentation broth is weighed into a 250 mL centrifuge tube, and centrifuged at 100 G for 2 minutes. Remove the floating supernatant of the cells. Centrifuge at 2,400 G for 5 minutes. Remove the supernatant and weigh. The water content of this solid content was measured, and the solid content per 1 kg of the fermentation broth was defined as calcium content (precipitated Ca content). The results after 24 hours fermentation are shown in Table 7.
  • Example 13 Fermentation was carried out in the same manner as in Example 12 except that stirring was performed at a rotational speed of 220 rpm, an air flow rate of 0.013 vvm, and a nitrogen flow rate of 0.287 vvm to obtain the target product. The results are shown in Table 7.
  • Example 14 Fermentation was carried out in the same manner as in Example 13 except that the stirring was performed at a rotational speed of 350 rpm to obtain the target product. The results are shown in Table 7.
  • Example 15 Bacteria were separated from 21.6 kg of the culture solution obtained in Example 12 using a centrifuge, and the bacteria remaining in the supernatant were passed through a filter to be completely sterilized. Sulfuric acid was added to the supernatant until the sulfuric acid pH was 2.5 or less to precipitate calcium sulfate. The precipitated calcium sulfate was filtered, and activated carbon corresponding to 0.5% by weight of the filtrate was added to the obtained separated liquid. The slurry was stirred overnight and then filtered again to remove the activated carbon.
  • calcium ions (Ca 2+ ) and sulfate ions (SO 4 2 ⁇ ) are respectively removed to 50 ppm or less through an anion exchange column and a cation exchange column, and a crude lactic acid aqueous solution 18 containing 8.8% D-lactic acid is obtained. 4 kg was obtained. This solution was purified by distillation to obtain 1.14 kg of purified D-lactic acid having a lactic acid concentration of 90% by weight.
  • Example 16 20 g of D-lactic acid and 0.083 g of tin oxide obtained in Example 15 were placed in a 200 mL reaction flask, and a stirring blade, a thermometer, and a reflux tube with a Dean-Stark tube were attached. From the upper part of the reflux tube, it was connected to a vacuum pump and a nitrogen introduction line. Depressurization and nitrogen release were repeated to remove oxygen, and then the pressure was reduced to 1 kPa and heated to 140 ° C. The water was removed under reduced pressure by holding at 140 ° C. for 3 hours. After removing water, the temperature was returned to normal temperature and normal pressure.
  • ODCB o-dichlorobenzene
  • the obtained polylactic acid composition was dissolved in chloroform and the molecular weight was measured by GPC (column: Shodex GPC LF-804, eluent: chloroform).
  • the molecular weight after the polymerization was a number average molecular weight (Mn) of 21,000.
  • the weight average molecular weight (Mw) was 200,000 when calculated as a polystyrene equivalent value.
  • a polymer having a molecular weight distribution (Mw / Mn) of 9.5 and having a wide molecular weight distribution that has a favorable influence on moldability and the like was obtained.
  • the YI value was measured with a Shimadzu UV-visible spectrometer UV-2400PC.
  • the polylactic acid composition was dissolved in dichloromethane to 1% by weight and used for measurement.
  • the YI value calculated by the above-described formula 3 was 0.6.
  • Example 17 Purification of fermentation broth The culture was conducted in the same manner as in Example 1 except that a sodium hydroxide aqueous solution was used as a pH adjuster. Centrifuged. Activated charcoal having a filtrate weight of 0.5% by weight was added to the centrifuged supernatant, and the mixture was stirred at 25 ° C. for 1 hour. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 ⁇ m membrane filter. When the transmittance at a wavelength of 400 nm of pure water in the spectrophotometer was 100%, the transmittance at a wavelength of 400 nm of the filtrate after removal of activated carbon was 98.0%.
  • the obtained filtrate was concentrated under reduced pressure at 10 to 20 hpa and 50 ° C. using a rotary vacuum evaporator (manufactured by EYELA) to obtain a sodium lactate solution having a sodium lactate concentration of 19.7% by weight.
  • Sodium lactate was desalted using the two-chamber water splitting electrodialysis apparatus shown in FIG.
  • 10 cation exchange membranes (trade name: Neocepta CMX, manufactured by Astom Co., Ltd.) and 10 bipolar membranes (Neoceptor BP-1) are arranged in order (total effective membrane area is 550 cm 2 ).
  • a filter press type electrodialyzer in which an acid chamber and a base chamber were formed was used.
  • a tank corresponding to 1000 g of the sodium lactate solution after the activated carbon treatment was provided in the acid chamber, and 1000 g of pure water was provided in the base chamber and supplied and circulated.
  • 1000 g of 5% sulfuric acid aqueous solution was used as the electrode solution.
  • electrodialysis was performed at 30 ° C. and a constant voltage of 30V.
  • 920 g of a lactic acid solution having a lactic acid concentration of 20.3% by weight was obtained from the acid chamber.
  • the obtained lactic acid solution was concentrated to 90.0% by weight with a rotary vacuum evaporator in the same manner as described above.
  • Example 18 Polymerization Polymerization was carried out in the same manner as in Example 16 using the lactic acid solution obtained in Example 17.
  • the molecular weight of the obtained polymer was a number average molecular weight (Mn) of 21,000 and a weight average molecular weight (Mw) of 210,000.
  • Mn number average molecular weight
  • Mw weight average molecular weight
  • the YI value was 0.5.
  • Example 19 Using E. coli and the medium used for D-lactic acid production in Examples 3 to 4 and 6 to 11, the other conditions were set in the same manner as in Example 12 to perform D-lactic acid fermentation production. It was. The obtained D-lactate was purified in the same manner as in Example 15. In addition, E. coli and medium used for D-lactic acid production in Example 5 were used, and other conditions were set in the same manner as in Example 12 and cultured. The obtained D-lactate was purified in the same manner as in Example 15 except that the amount of activated carbon used for purification was 1% by weight of the filtrate. The resulting D-lactic acid was polymerized in the same manner as in Example 16.
  • the number average molecular weight (Mn) was 190000-21,000
  • the weight average molecular weight (Mw) 180,000-250,000 was 190000-21,000
  • the molecular weight distribution was 190000-21,000
  • a polymer having 8.5 to 12 and a YI value of 0.2 to 2 was obtained, and a good polymer was obtained in any test section.
  • the number average molecular weight (Mn) was 44,000
  • the weight average molecular weight (Mw) was 80000
  • the molecular weight distribution (Mw / Mn) was 1.8
  • a polymer having a YI value of 15 was obtained.

Abstract

D-lactic acid is produced by cultivation of a D-lactic acid-producing Escherichia coli in an inorganic salt medium comprising an inorganic electrolyte and carbon source and satisfying at least one selected from the following (a), (b), (c), and (d): (a) the content of the organic matter other than the carbon source in the inorganic salt medium is 10 g/L or less when culture starts, (b) the content of the inorganic electrolyte in the inorganic salt medium is 11 g/L or less when culture starts, (c) the content of thiamine in the inorganic salt medium is 0.1 mg/L or less, and (d) the content of transition metal ions in the inorganic salt medium is 10 mg/L or less.

Description

Ｄ－乳酸の生産方法、ポリマーの生産方法およびポリマーD-lactic acid production method, polymer production method and polymer
　本発明は、Ｄ－乳酸の生産方法、ポリマーの生産方法およびポリマーに関する。 The present invention relates to a method for producing D-lactic acid, a method for producing a polymer, and a polymer.
　乳酸は、ポリマー原料や農薬、医薬の中間体として近年注目が集まりつつある有用な物質である。自然界には乳酸菌や糸状菌など乳酸を効率良く生産する微生物が存在し、それらを用いた乳酸製造法には、Ｌ－乳酸を効率良く生産させる微生物としてＬａｃｔｂａｃｉｌｌｕｓ　ｄｅｌｂｒｕｅｃｋｉｉ等を、またＤ－乳酸を効率良く生産させる微生物としてＳｐｏｒｏｌａｃｔｏｂａｃｉｌｌｕｓ属の微生物等を用いた方法が知られている。 Lactic acid is a useful substance that has recently attracted attention as an intermediate for polymer raw materials, agricultural chemicals, and pharmaceuticals. In nature, there are microorganisms that efficiently produce lactic acid such as lactic acid bacteria and filamentous fungi. Lactic acid production methods using these microorganisms include Lactobacillus delbrueckii as a microorganism that can produce L-lactic acid efficiently, and D-lactic acid efficiently. As a well-produced microorganism, a method using a microorganism of the genus Sporolactobacillus is known.
　Ｄ－乳酸は、Ｌ－乳酸とのステレオコンプレックス型ポリ乳酸の原料や医薬中間体として近年注目を集めている。上記いずれの用途においても原料であるＤ－乳酸には高い光学純度が求められる。 D-lactic acid has recently attracted attention as a raw material and a pharmaceutical intermediate of stereocomplex polylactic acid with L-lactic acid. In any of the above applications, the raw material D-lactic acid is required to have high optical purity.
　Ｄ－乳酸は、一般的に大腸菌をはじめとする微生物を利用した発酵法により工業的に製造されている。発酵プロセスは、微生物の培養と、増殖した微生物を触媒とした物質生産とが行われる工程であるので、微生物の増殖には栄養源が必要となる。こうした栄養源としてとうもろこしの加工過程で得られるコーンスティープリカーが、アミノ酸などを多く含み栄養価が高いこと、さらには安価であることから、Ｄ－乳酸の発酵原料に用いられている。 D-lactic acid is generally produced industrially by fermentation using microorganisms such as E. coli. Since the fermentation process is a process in which culturing of microorganisms and production of substances using the grown microorganisms as a catalyst are performed, a nutrient source is required for the growth of microorganisms. As a nutrient source, corn steep liquor obtained in the process of corn is rich in amino acids and has a high nutritional value and is inexpensive, so it is used as a fermentation raw material for D-lactic acid.
　このコーンスティープリカーはＬ－乳酸を含むため、得られるＤ－乳酸光学純度の低下を招く原因の一つとなっている。このような観点から、例えば特許文献１では、効率的に乳酸を製造すると共に製品の光学純度を向上させることを目的として、原料中に含まれるＬ－乳酸を速やかに分解することが検討されている。 Since this corn steep liquor contains L-lactic acid, it is one of the causes of the resulting decrease in optical purity of D-lactic acid. From this point of view, for example, Patent Document 1 discusses rapidly decomposing L-lactic acid contained in a raw material for the purpose of efficiently producing lactic acid and improving the optical purity of the product. Yes.
　こうした取り組みとは別に、コーンスティープリカーのようなＬ－乳酸を含む原料を用いることなく、Ｄ－乳酸を効率的に製造する検討がなされている。特に大腸菌を用いたＤ－乳酸製造法に関して言えば、特許文献２においては、下記のような無機塩を主成分とする培地に糖を加えたものを用いたＤ－乳酸の発酵生産方法が開示されている。
　リン酸二水素カリウム　　　　　　３．５　ｇ／Ｌ
　リン酸水素二カリウム　　　　　　５．０　ｇ／Ｌ
　硫酸アンモニウム　　　　　　　　３．５　ｇ／Ｌ
　硫酸マグネシウム・７水和物　　　０．２５ｇ／Ｌ
　塩化カルシウム・２水和物　　　　　１５　ｍｇ／Ｌ
　チアミン　　　　　　　　　　　　０．５　ｍｇ／Ｌ
　塩化鉄（ＩＩＩ）　　　　　　　　１．６　ｍｇ／Ｌ
　塩化コバルト（ＩＩ）・６水和物　０．２　ｍｇ／Ｌ
　塩化銅（ＩＩ）　　　　　　　　　０．１　ｍｇ／Ｌ
　塩化亜鉛（ＩＩ）・４水和物　　　０．２　ｍｇ／Ｌ
　酸化モリブデンナトリウム　　　　０．２　ｍｇ／Ｌ
　ホウ酸　　　　　　　　　　　　０．０５　ｍｇ／Ｌ Apart from such efforts, studies have been made to efficiently produce D-lactic acid without using a raw material containing L-lactic acid such as corn steep liquor. In particular, with regard to a method for producing D-lactic acid using E. coli, Patent Document 2 discloses a method for fermentative production of D-lactic acid using a medium in which sugar is added to a medium mainly composed of the following inorganic salts. Has been.
Potassium dihydrogen phosphate 3.5 g / L
Dipotassium hydrogen phosphate 5.0 g / L
Ammonium sulfate 3.5 g / L
Magnesium sulfate heptahydrate 0.25g / L
Calcium chloride dihydrate 15 mg / L
Thiamine 0.5 mg / L
Iron (III) chloride 1.6 mg / L
Cobalt (II) chloride hexahydrate 0.2 mg / L
Copper (II) chloride 0.1 mg / L
Zinc (II) chloride tetrahydrate 0.2 mg / L
Sodium molybdenum oxide 0.2 mg / L
Boric acid 0.05 mg / L
　また、大腸菌を培養するための無機塩を主成分とする培地は、例えば特許文献３及び非特許文献１にも記載されている。特許文献２、３及び非特許文献１記載のものは、数種の無機塩に加え、チアミン、更にはコバルトや亜鉛、銅などの遷移金属などを含んだ培地である。これらチアミンや遷移金属イオンは微生物細胞内での酵素による生化学反応に補因子として重要な役割を果たすことから、非常に重要な成分であると考えられる。なお、特許文献２、３には、無機塩を主成分とする培地に、適宜ベタインを添加することも記載されている。 Further, a medium mainly composed of an inorganic salt for culturing Escherichia coli is also described in, for example, Patent Document 3 and Non-Patent Document 1. Those described in Patent Documents 2 and 3 and Non-Patent Document 1 are media containing thiamine and further transition metals such as cobalt, zinc and copper in addition to several inorganic salts. These thiamines and transition metal ions play an important role as cofactors in biochemical reactions by enzymes in microbial cells, and are therefore considered to be very important components. Patent Documents 2 and 3 also describe that betaine is appropriately added to a medium containing an inorganic salt as a main component.
国際公開第２０１０／０３２６９７号パンフレットInternational Publication No. 2010/032697 Pamphlet 特開２０１２－１０７１５号公報JP 2012-10715 A 特開２００９－２６１３６０号公報JP 2009-261360 A
　しかし、上記文献には、得られたＤ－乳酸から、どのようなＤ－乳酸ポリマーが得られるかについての知見は記載されていない。 However, the above document does not describe what kind of D-lactic acid polymer can be obtained from the obtained D-lactic acid.
　また、特許文献２、３記載の培地は多くの成分が必要であるため、培地調整に手間がかかるのみならず、多数の成分をある一定量使用することにより培地コストの上昇を招く結果となる。 In addition, since the culture medium described in Patent Documents 2 and 3 requires many components, it takes time to adjust the culture medium, and the use of a certain amount of a large number of components results in an increase in culture medium cost. .
　本発明は、より安価に、かつ、効率よくＤ－乳酸を生産する方法を提供することにある。 The present invention is to provide a method for producing D-lactic acid more efficiently at low cost.
　本発明者らは上記課題を解決するために鋭意検討を重ねた結果、Ｄ－乳酸生産大腸菌を無機塩培地で培養することにより、重合化したとき高品質のポリマーが得られることを見出した。また、無機塩培地中の成分を最小限にすることで、より安価に、かつ、効率よくＤ－乳酸を生産できることを見出した。
　即ち、本発明は以下の［１］から［１７］に記載のとおりである。 As a result of intensive studies to solve the above problems, the present inventors have found that a high-quality polymer can be obtained when polymerized by culturing D-lactic acid-producing Escherichia coli in an inorganic salt medium. In addition, it has been found that D-lactic acid can be produced more efficiently and efficiently by minimizing the components in the inorganic salt medium.
That is, the present invention is as described in [1] to [17] below.
［１］無機電解質と炭素源とを含み、下記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される少なくとも１つを満たす無機塩培地でＤ－乳酸生産大腸菌を培養することによりＤ－乳酸を生産する、Ｄ－乳酸の生産方法。
（ａ）前記無機塩培地中の前記炭素源以外の有機物の含有量が、培養開始時に１０ｇ／Ｌ以下である。
（ｂ）前記無機塩培地中の前記無機電解質の含有量が、培養開始時に１１ｇ／Ｌ以下である。
（ｃ）前記無機塩培地中のチアミンの含有量が０．１ｍｇ／Ｌ以下である。
（ｄ）前記無機塩培地中の遷移金属イオンの含有量が１０ｍｇ／Ｌ以下である。 [1] D-lactic acid-producing Escherichia coli in an inorganic salt medium containing an inorganic electrolyte and a carbon source and satisfying at least one selected from the group consisting of the following (a), (b), (c) and (d) A method for producing D-lactic acid, wherein D-lactic acid is produced by culturing.
(A) Content of organic substances other than the carbon source in the inorganic salt medium is 10 g / L or less at the start of culture.
(B) The content of the inorganic electrolyte in the inorganic salt medium is 11 g / L or less at the start of culture.
(C) The content of thiamine in the inorganic salt medium is 0.1 mg / L or less.
(D) The content of transition metal ions in the inorganic salt medium is 10 mg / L or less.
［２］前記無機塩培地は、少なくとも前記（ｃ）または前記（ｄ）のいずれかを満たす、［１］に記載のＤ－乳酸の生産方法。
［３］ポリマー原料となるＤ－乳酸を生産するための、［１］または［２］に記載のＤ－乳酸の生産方法。
［４］培養開始時の前記無機塩培地がさらに下記（ｅ）を満たす、［１］乃至［３］のいずれか一つに記載のＤ－乳酸の生産方法。
（ｅ）前記無機塩培地中の前記遷移金属イオンの含有量が０．８５ｍｇ／Ｌ以下である。
［５］前記炭素源が糖である、［１］乃至［４］のいずれか一つに記載のＤ－乳酸の生産方法。
［６］前記糖は、グルコース、フルクトース、キシロース、スクロース、グリセリン、アラビノース、メリビオース、トレハロース、マルトース、メリビオン酸、ラクトース、マルトトリオース、リボース、ガラクトース、ガラクツロン酸、グルコン酸、グルコサミン、グルクロン酸、マンニトール、マンノース、サッカリン酸、ソルビトール、フコース、ラムノース、アロース、及びＮ-アセチルグルコサミンからなる群より選択される１以上の化合物を含む、［５］に記載のＤ－乳酸の生産方法。
［７］前記無機電解質が、カリウムイオン、リン酸イオン、アンモニウムイオン、硫酸イオン及びマグネシウムイオンからなる群より選択される１以上のイオンを構成成分として含む、［１］乃至［６］のいずれか一つに記載のＤ－乳酸の生産方法。
［８］前記無機電解質が、カリウムイオンを含み、
　培養開始時の前記無機塩培地において、前記カリウムイオンの濃度が５．８ｍｍｏｌ／Ｌ以上７３ｍｍｏｌ／Ｌ以下である、［１］乃至［７］のいずれか一つに記載のＤ－乳酸の生産方法。
［９］前記無機塩培地が、前記無機電解質を水に溶解又は懸濁して調製された液体である、［１］乃至［８］のいずれか一つに記載のＤ－乳酸の生産方法。
［１０］前記無機塩培地が、さらにベタインを含む、［１］乃至［９］のいずれか一つに記載のＤ－乳酸の生産方法。
［１１］前記Ｄ－乳酸生産大腸菌が、組換え大腸菌である、［１］乃至［１０］のいずれか一つに記載のＤ－乳酸の生産方法。
［１２］前記無機塩培地において前記Ｄ－乳酸生産大腸菌と前記炭素源とを接触させてＤ－乳酸塩を得る工程と、
　前記Ｄ－乳酸塩を含む前記無機塩培地から前記Ｄ－乳酸生産大腸菌を除去した後に前記Ｄ－乳酸塩を脱塩してＤ－乳酸を得る工程と、
を含む、［１］乃至［１１］のいずれか一つに記載のＤ－乳酸の生産方法。
［１３］前記Ｄ－乳酸塩を得る前記工程において、Ｄ－乳酸のアルカリ土類金属塩を得て、
　前記Ｄ－乳酸を得る前記工程において、Ｄ－乳酸の前記アルカリ土類金属塩を脱塩することでアルカリ土類金属を含む無機塩を析出させる［１２］に記載のＤ－乳酸の生産方法。
［１４］前記Ｄ－乳酸塩を得る前記工程において、前記Ｄ－乳酸のアルカリ金属塩を得て、　前記Ｄ－乳酸を得る前記工程において、電解透析処理によりＤ－乳酸のアルカリ金属塩を脱塩する、［１２］に記載のＤ－乳酸の生産方法。
［１５］前記Ｄ－乳酸塩を脱塩してＤ－乳酸を得る前記工程の後にＤ－乳酸の精製を行う精製工程を更に含み、
　前記精製工程において、加水分解しながら蒸留を行う、［１２］乃至［１４］のいずれか一つに記載のＤ－乳酸の生産方法。
［１６］［１］乃至［１５］のいずれか一に記載のＤ－乳酸の生産方法により得られたＤ－乳酸を用いて重合反応を行う、ポリマーの生産方法。
［１７］［１６］記載のポリマーの生産方法で得られるポリマー。 [2] The method for producing D-lactic acid according to [1], wherein the inorganic salt medium satisfies at least one of (c) and (d).
[3] The method for producing D-lactic acid according to [1] or [2] for producing D-lactic acid as a polymer raw material.
[4] The method for producing D-lactic acid according to any one of [1] to [3], wherein the inorganic salt medium at the start of culture further satisfies the following (e):
(E) The content of the transition metal ion in the inorganic salt medium is 0.85 mg / L or less.
[5] The method for producing D-lactic acid according to any one of [1] to [4], wherein the carbon source is sugar.
[6] The sugar is glucose, fructose, xylose, sucrose, glycerin, arabinose, melibiose, trehalose, maltose, melibionic acid, lactose, maltotriose, ribose, galactose, galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol The method for producing D-lactic acid according to [5], comprising one or more compounds selected from the group consisting of mannose, saccharic acid, sorbitol, fucose, rhamnose, allose, and N-acetylglucosamine.
[7] Any of [1] to [6], wherein the inorganic electrolyte includes one or more ions selected from the group consisting of potassium ions, phosphate ions, ammonium ions, sulfate ions, and magnesium ions as a constituent component. The method for producing D-lactic acid according to one.
[8] The inorganic electrolyte contains potassium ions,
The method for producing D-lactic acid according to any one of [1] to [7], wherein in the inorganic salt medium at the start of culture, the concentration of the potassium ion is 5.8 mmol / L or more and 73 mmol / L or less. .
[9] The method for producing D-lactic acid according to any one of [1] to [8], wherein the inorganic salt medium is a liquid prepared by dissolving or suspending the inorganic electrolyte in water.
[10] The method for producing D-lactic acid according to any one of [1] to [9], wherein the inorganic salt medium further contains betaine.
[11] The method for producing D-lactic acid according to any one of [1] to [10], wherein the D-lactic acid-producing E. coli is a recombinant E. coli.
[12] contacting the D-lactic acid-producing Escherichia coli with the carbon source in the inorganic salt medium to obtain D-lactate;
Removing D-lactic acid-producing Escherichia coli from the inorganic salt medium containing the D-lactate and then desalting the D-lactate to obtain D-lactic acid;
The method for producing D-lactic acid according to any one of [1] to [11], comprising:
[13] In the step of obtaining the D-lactate, an alkaline earth metal salt of D-lactic acid is obtained,
The method for producing D-lactic acid according to [12], wherein, in the step of obtaining D-lactic acid, an inorganic salt containing an alkaline earth metal is precipitated by desalting the alkaline earth metal salt of D-lactic acid.
[14] Obtaining an alkali metal salt of D-lactic acid in the step of obtaining the D-lactate, and desalting the alkali metal salt of D-lactic acid by electrodialysis in the step of obtaining the D-lactic acid. The method for producing D-lactic acid according to [12].
[15] The method further comprises a purification step of purifying D-lactic acid after the step of desalting the D-lactate to obtain D-lactic acid,
The method for producing D-lactic acid according to any one of [12] to [14], wherein distillation is performed while hydrolysis in the purification step.
[16] A method for producing a polymer, wherein a polymerization reaction is performed using D-lactic acid obtained by the method for producing D-lactic acid according to any one of [1] to [15].
[17] A polymer obtained by the method for producing a polymer according to [16].
　本発明によれば、より安価に、かつ、効率よくＤ－乳酸を生産する方法が提供される。 According to the present invention, a method for producing D-lactic acid at a lower cost and more efficiently is provided.
　上述した目的、およびその他の目的、特徴および利点は、以下に述べる好適な実施の形態、およびそれに付随する以下の図面によってさらに明らかになる。 The above-described object and other objects, features, and advantages will be further clarified by a preferred embodiment described below and the following drawings attached thereto.
本実施形態に係るＤ－乳酸の生産方法に用いられる発酵装置の一例を示す図である。It is a figure which shows an example of the fermenter used for the production method of D-lactic acid concerning this embodiment. 本実施形態に係るＤ－乳酸の生産方法に用いられる電気透析装置の一例を示す図である。It is a figure which shows an example of the electrodialysis apparatus used for the production method of D-lactic acid concerning this embodiment.
　本実施形態に係るＤ－乳酸の生産方法は、無機電解質と炭素源とを含み、下記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される少なくとも１つを満たす無機塩培地でＤ－乳酸生産大腸菌を培養することによりＤ－乳酸を生産するものである。これにより、より安価に、かつ、効率よくＤ－乳酸を生産することができる。
（ａ）無機塩培地中の炭素源以外の有機物の含有量が、培養開始時に１０ｇ／Ｌ以下である。
（ｂ）無機塩培地中の無機電解質の含有量が、培養開始時に１１ｇ／Ｌ以下である。
（ｃ）無機塩培地中のチアミンの含有量が０．１ｍｇ／Ｌ以下である。
（ｄ）無機塩培地中の遷移金属イオンの含有量が１０ｍｇ／Ｌ以下である。 The method for producing D-lactic acid according to this embodiment includes an inorganic electrolyte and a carbon source, and satisfies at least one selected from the group consisting of (a), (b), (c) and (d) below. D-lactic acid is produced by culturing D-lactic acid-producing Escherichia coli in an inorganic salt medium. As a result, it is possible to produce D-lactic acid at a lower cost and more efficiently.
(A) The content of organic substances other than the carbon source in the inorganic salt medium is 10 g / L or less at the start of culture.
(B) The content of the inorganic electrolyte in the inorganic salt medium is 11 g / L or less at the start of culture.
(C) The content of thiamine in the inorganic salt medium is 0.1 mg / L or less.
(D) The content of transition metal ions in the inorganic salt medium is 10 mg / L or less.
　本実施形態に係るＤ－乳酸の生産方法において無機塩培地は、上記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される１つ以上を満たすものであり、好ましくは、上記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される２つ以上を満たし、より好ましくは上記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される３つ以上を満たし、更に好ましくは上記（ａ）、（ｂ）、（ｃ）及び（ｄ）の全てを満たすものである。これにより、ポリマー原料となるＤ－乳酸を生産することができる。また、本実施形態に係るＤ－乳酸の生産方法によれば、ポリマー原料の用途に適した、Ｄ－乳酸を得ることができる。
　以下、本実施形態に係るＤ－乳酸の生産方法について詳しく説明する。 In the method for producing D-lactic acid according to this embodiment, the inorganic salt medium satisfies one or more selected from the group consisting of the above (a), (b), (c) and (d), preferably Satisfies at least two selected from the group consisting of (a), (b), (c) and (d), more preferably (a), (b), (c) and (d) Satisfying at least three selected from the group consisting of: (a), (b), (c) and (d). As a result, D-lactic acid as a polymer raw material can be produced. Further, according to the production method of D-lactic acid according to this embodiment, D-lactic acid suitable for the use of the polymer raw material can be obtained.
Hereinafter, the method for producing D-lactic acid according to the present embodiment will be described in detail.
　本実施形態において、炭素源とは、Ｄ－乳酸生産大腸菌がＤ－乳酸の生産を行う上で利用可能な発酵性の糖であり、より具体的には、グルコース、フルクトース、キシロース、スクロース、グリセリン、アラビノース、メリビオース、トレハロース、マルトース、メリビオン酸、ラクトース、マルトトリオース、リボース、ガラクトース、ガラクツロン酸、グルコン酸、グルコサミン、グルクロン酸、マンニトール、マンノース、サッカリン酸、ソルビトール、フコース、ラムノース、アロース、及びＮ-アセチルグルコサミンからなる群より選択される１以上の化合物を含むことが好ましい。また、デンプン加水分解物、草木質分解産物やセルロース加水分解物などを炭素源として用いてもよい。
　なおＤ－乳酸の生産を行う上で上記の糖を利用するために、必要となる酵素遺伝子、または、利用効率を向上させるような遺伝子を大腸菌に導入してもよい。具体的な例としては、本願実施例記載の通り、インベルターゼを導入した大腸菌によるスクロースからのＤ－乳酸生産が例示される。 In the present embodiment, the carbon source is a fermentable sugar that can be used when D-lactic acid-producing Escherichia coli produces D-lactic acid, and more specifically, glucose, fructose, xylose, sucrose, glycerin. Arabinose, melibiose, trehalose, maltose, melbionic acid, lactose, maltotriose, ribose, galactose, galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol, mannose, saccharic acid, sorbitol, fucose, rhamnose, allose, and N -Preferably it contains one or more compounds selected from the group consisting of acetylglucosamine. Moreover, you may use a starch hydrolyzate, a herbaceous degradation product, a cellulose hydrolyzate, etc. as a carbon source.
In addition, in order to use the above-mentioned sugar for producing D-lactic acid, an enzyme gene that is necessary or a gene that improves the utilization efficiency may be introduced into Escherichia coli. Specific examples include D-lactic acid production from sucrose by E. coli into which invertase has been introduced, as described in the Examples of the present application.
　本実施形態に係る無機塩培地には、炭素源以外の有機物も含まれている。ここで、炭素源以外の有機物とは、上述した発酵性の糖以外の有機物であり、たとえば、有機窒素源が挙げられる。炭素源以外の有機物に関するより具体的な例としては、油粕類、大豆加水分解物、カゼイン分解物、アミノ酸類、コーンスティープリカー、酵母および酵母エキス、肉エキス、ペプトン等のペプチド類、麦芽エキス、ホエー、各種発酵菌体およびその加水分解物、またはそれらに含まれる有機物成分等が挙げられる。また、ビタミン類も炭素源以外の有機物の一例である。さらに、無機塩培地のｐＨ緩衝作用を高める目的で加えられる緩衝剤となる有機物が、炭素源以外の有機物の例として挙げられ、たとえば、ＭＯＰＳ（３－（Ｎ－ｍｏｒｐｈｏｌｉｎｏ）ｐｒｏｐａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ）、Ｔｒｉｓ（ｔｒｉｓ（ｈｙｄｒｏｘｙｍｅｔｈｙｌ）ａｍｉｎｏｍｅｔｈａｎｅ）、ＥＤＴＡ（ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅｔｅｔｒａａｃｅｔｉｃ　ａｃｉｄ）等が挙げられる。 The inorganic salt medium according to the present embodiment includes organic substances other than the carbon source. Here, organic substances other than a carbon source are organic substances other than the fermentable sugar mentioned above, for example, an organic nitrogen source is mentioned. More specific examples of organic substances other than carbon sources include oil cakes, soybean hydrolysates, casein digests, amino acids, corn steep liquor, yeast and yeast extract, meat extracts, peptides such as peptone, malt extract, Examples include whey, various fermented bacterial cells and hydrolysates thereof, or organic components contained therein. Vitamins are also examples of organic substances other than carbon sources. Furthermore, organic substances that serve as buffering agents added for the purpose of enhancing the pH buffering action of the inorganic salt medium are listed as examples of organic substances other than the carbon source. For example, MOPS (3- (N-morpholino) propanesulphonic acid), Tris ( tris (hydroxymethyl) aminomethane), EDTA (ethylenediamineacetic acid), and the like.
　本実施形態における無機電解質と炭素源とを含む無機塩培地に、上述した炭素源以外の有機物が大量に含まれている場合、Ｄ－乳酸を得るための精製工程が煩雑になったり、ポリマーの色調に悪影響を及ぼしたりするといった不都合が生じる可能性があるため、無機塩培地中に含有させる炭素源以外の有機物量を制限しておくことが好ましい。 When the inorganic salt medium containing the inorganic electrolyte and the carbon source in the present embodiment contains a large amount of organic substances other than the above-described carbon source, the purification process for obtaining D-lactic acid becomes complicated, Since inconveniences such as adversely affecting the color tone may occur, it is preferable to limit the amount of organic substances other than the carbon source contained in the inorganic salt medium.
　本実施形態において、無機塩培地中の炭素源以外の有機物の含有量は、培養開始時に、１０ｇ／Ｌ以下であることが好ましく、ポリマーの色調の観点から、２ｇ／Ｌ以下であるとより好ましく、０．６ｇ／Ｌ以下であるとさらに好ましい。例えば、培養開始時の無機塩培地において、炭素源以外の有機物の含有量は、０～１０ｇ／Ｌの範囲が好ましく、より好ましくは０～２ｇ／Ｌであり、さらに好ましくは０～０．６ｇ／Ｌある。また、下限値については、ポリマーの色調の観点から、０ｇ／Ｌ以上であることが好ましい。
　なお、本実施形態に係るＤ－乳酸の生産方法において、「培養開始時」とは、Ｄ－乳酸生産大腸菌を培地に接種する直前のことをいう。 In the present embodiment, the content of organic substances other than the carbon source in the inorganic salt medium is preferably 10 g / L or less at the start of culture, and more preferably 2 g / L or less from the viewpoint of the color of the polymer. More preferably, it is 0.6 g / L or less. For example, in the inorganic salt medium at the start of culture, the content of organic substances other than the carbon source is preferably in the range of 0 to 10 g / L, more preferably 0 to 2 g / L, and still more preferably 0 to 0.6 g. / L. The lower limit is preferably 0 g / L or more from the viewpoint of the color tone of the polymer.
In the method for producing D-lactic acid according to the present embodiment, “at the start of culture” means immediately before inoculating D-lactic acid-producing Escherichia coli into the medium.
　また、本実施形態に係るＤ－乳酸の生産方法における「無機塩培地」とは、微生物を用いた乳酸発酵において、微生物が必要とする元素のうち、炭素源を除く元素を構成成分として含む無機電解質を水に溶解又は懸濁して調製された液体の培地である。 In addition, the “inorganic salt medium” in the D-lactic acid production method according to the present embodiment refers to an inorganic element that contains an element excluding a carbon source as a constituent component among elements required by a microorganism in lactic acid fermentation using a microorganism. A liquid medium prepared by dissolving or suspending an electrolyte in water.
　また、本実施形態に係るＤ－乳酸の生産方法において無機電解質は、カリウムイオン（Ｋ＋）、リン酸イオン（ＰＯ４ ３－）、アンモニウムイオン（ＮＨ４ ＋）、硫酸イオン（ＳＯ４ ２－）及びマグネシウムイオン（Ｍｇ２＋）からなる群より選択される１以上のイオンを構成成分として含むことが好ましい。こうすることで、高品質のポリマーを得ることができ、より安価に、かつ、効率よくＤ－乳酸を生産することができる。なお、培養開始時の無機塩培地中において、上記無機電解質の含有量は、合計で１１ｇ／Ｌ以下であることが好ましく、０～１１ｇ／Ｌの範囲が好ましい。また、下限値については、０ｇ／Ｌ以上であることが好ましい。 In addition, in the method for producing D-lactic acid according to the present embodiment, the inorganic electrolyte includes potassium ions (K + ), phosphate ions (PO 4 3− ), ammonium ions (NH 4 + ), sulfate ions (SO 4 2−). And at least one ion selected from the group consisting of magnesium ions (Mg 2+ ). By doing so, a high-quality polymer can be obtained, and D-lactic acid can be produced more efficiently at low cost. In the inorganic salt medium at the start of culture, the total content of the inorganic electrolyte is preferably 11 g / L or less, and preferably in the range of 0 to 11 g / L. Further, the lower limit is preferably 0 g / L or more.
　無機塩培地中にＫ＋を構成成分として含ませる場合、用いる無機電解質は、特に限定されないが、たとえば、リン酸水素二カリウム（Ｋ２ＨＰＯ４）、リン酸二水素カリウム（ＫＨ２ＰＯ４）、水酸化カリウム（ＫＯＨ）、リン酸三カリウム（Ｋ３ＰＯ４）、及び、硫酸カリウム（Ｋ２ＳＯ４）からなる群より選択される少なくとも１以上を選択して用いることができる。また、無機塩培地中のＫ＋含有量は、好ましくは５～５０ｍｍｏｌ／Ｌ、より好ましくは１５～２５ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中においてＫ＋含有量は、より一層効率よくＤ－乳酸を生産するという観点から、５ｍｍｏｌ／Ｌ以上であることが好ましく、５．８ｍｍｏｌ／Ｌ以上であるとさらに好ましく、１５ｍｍｏｌ／Ｌ以上であるとより一層好ましい。一方、培養開始時の無機塩培地中においてＫ＋含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、７３ｍｍｏｌ／Ｌ以下であることが好ましく、５０ｍｍｏｌ／Ｌ以下であるとさらに好ましく、２５ｍｍｏｌ／Ｌ以下であるとより一層好ましい。 In the case where K + is included as a constituent in the inorganic salt medium, the inorganic electrolyte used is not particularly limited. For example, dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ) , Potassium hydroxide (KOH), tripotassium phosphate (K 3 PO 4 ), and at least one selected from the group consisting of potassium sulfate (K 2 SO 4 ) can be selected and used. The K + content in the inorganic salt medium is preferably 5 to 50 mmol / L, more preferably 15 to 25 mmol / L. Further, the K + content in the inorganic salt medium at the start of the culture is preferably 5 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and is 5.8 mmol / L or more. More preferably, it is more preferably 15 mmol / L or more. On the other hand, the upper limit value of the K + content in the inorganic salt medium at the start of the culture is preferably 73 mmol / L or less, more preferably 50 mmol / L or less, from the viewpoint of more efficiently producing D-lactic acid. And more preferably 25 mmol / L or less.
　無機塩培地中にＰＯ４ ３－を構成成分として含ませる場合、用いる無機電解質は、特に限定されないが、たとえば、リン酸（Ｈ３ＰＯ４）、リン酸二水素アンモニウム（ＮＨ４Ｈ２ＰＯ４）、リン酸水素二アンモニウム（（ＮＨ４）２ＨＰＯ４）、リン酸三アンモニウム（（ＮＨ４）３ＰＯ４）、リン酸一水素マグネシウム（ＭｇＨＰＯ４）、リン酸マグネシウム（Ｍｇ３（ＰＯ４）２）、リン酸二水素カリウム（ＫＨ２ＰＯ４）、リン酸水素二カリウム（Ｋ２ＨＰＯ４）、リン酸三カリウム（Ｋ３ＰＯ４）、リン酸アンモニウムマグネシウム（ＭｇＮＨ４ＰＯ４）、リン酸二水素マグネシウム（Ｍｇ（Ｈ２ＰＯ４）２）、リン酸一水素マグネシウム（ＭｇＨＰＯ４）及びリン酸マグネシウム（Ｍｇ３（ＰＯ４）２）からなる群より選択される少なくとも１以上を選択して用いることができる。無機塩培地中のＰＯ４ ３－含有量は、２～４０ｍｍｏｌ／Ｌ、より好ましくは１０～３０ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中においてＰＯ４ ３－含有量は、より一層効率よくＤ－乳酸を生産するという観点から、２ｍｍｏｌ／Ｌ以上であることが好ましく、１０ｍｍｏｌ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中においてＰＯ４ ３－含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、４０ｍｍｏｌ／Ｌ以下であることが好ましく、３０ｍｍｏｌ／Ｌ以下であるとさらに好ましい。 When PO 4 3- is included as a constituent in the inorganic salt medium, the inorganic electrolyte to be used is not particularly limited. For example, phosphoric acid (H 3 PO 4 ), ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ), Diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), triammonium phosphate ((NH 4 ) 3 PO 4 ), magnesium monohydrogen phosphate (MgHPO 4 ), magnesium phosphate (Mg 3 (PO 4 2 ), potassium dihydrogen phosphate (KH 2 PO 4 ), dipotassium hydrogen phosphate (K 2 HPO 4 ), tripotassium phosphate (K 3 PO 4 ), ammonium magnesium phosphate (MgNH 4 PO 4 ), magnesium dihydrogen phosphate (Mg (H 2 PO 4) 2), monohydrogen magnesium phosphate (MgHPO 4) and magnesium phosphate (Mg (PO 4) 2) is selected from the group consisting of can be selected at least one or more. The content of PO 4 3− in the inorganic salt medium is 2 to 40 mmol / L, more preferably 10 to 30 mmol / L. Further, the PO 4 3− content in the inorganic salt medium at the start of the culture is preferably 2 mmol / L or more, and more preferably 10 mmol / L or more from the viewpoint of producing D-lactic acid more efficiently. Further preferred. On the other hand, the upper limit of the content of PO 4 3− in the inorganic salt medium at the start of the culture is preferably 40 mmol / L or less, more preferably 30 mmol / L or less, from the viewpoint of more efficiently producing D-lactic acid. Is more preferable.
　無機塩培地中にＮＨ４ ＋を構成成分として含ませる場合、用いる無機電解質は、特に限定されないが、たとえば、リン酸二水素アンモニウム（ＮＨ４Ｈ２ＰＯ４）、リン酸水素二アンモニウム（（ＮＨ４）２ＨＰＯ４）、リン酸アンモニウム（（ＮＨ４）３ＰＯ４）、リン酸アンモニウムマグネシウム（ＭｇＮＨ４ＰＯ４）、硫酸アンモニウム（（ＮＨ４）２ＳＯ４）及びリン酸水素アンモニウムカリウム（ＮＨ４ＫＨＰＯ４）からなる群より選択される少なくとも１以上を選択して用いることができる。無機塩培地中のＮＨ４ ＋含有量は、好ましくは２０～１２０ｍｍｏｌ／Ｌ、より好ましくは４０～８０ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中においてＮＨ４ ＋含有量は、より一層効率よくＤ－乳酸を生産するという観点から、２０ｍｍｏｌ／Ｌ以上であることが好ましく、４０ｍｍｏｌ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中においてＮＨ４ ＋含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、１２０ｍｍｏｌ／Ｌ以下であることが好ましく、８０ｍｍｏｌ／Ｌ以下であるとさらに好ましい。 When NH 4 + is included as a constituent in the inorganic salt medium, the inorganic electrolyte to be used is not particularly limited. For example, ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ), diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), ammonium phosphate ((NH 4 ) 3 PO 4 ), ammonium magnesium phosphate (MgNH 4 PO 4 ), ammonium sulfate ((NH 4 ) 2 SO 4 ) and potassium ammonium hydrogen phosphate (NH 4 KHPO) It is possible to select and use at least one selected from the group consisting of 4 ). The NH 4 + content in the inorganic salt medium is preferably 20 to 120 mmol / L, more preferably 40 to 80 mmol / L. In addition, the NH 4 + content in the inorganic salt medium at the start of the culture is preferably 20 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and more preferably 40 mmol / L or more. preferable. On the other hand, the upper limit of the NH 4 + content in the inorganic salt medium at the start of the culture is preferably 120 mmol / L or less, more preferably 80 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid. More preferably.
　無機塩培地中にＳＯ４ ２－を構成成分として含ませる場合、用いる無機電解質は、特に限定されないが、たとえば、硫酸カリウム（Ｋ２ＳＯ４）、硫酸アンモニウム（（ＮＨ４）２ＳＯ４）及び硫酸マグネシウム（ＭｇＳＯ４）からなる群より選択される少なくとも１以上を選択して用いることができる。無機塩培地中のＳＯ４ ２－含有量は、好ましくは１０～５０ｍｍｏｌ／Ｌ、より好ましくは２０～４０ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中においてＳＯ４ ２－含有量は、より一層効率よくＤ－乳酸を生産するという観点から、１０ｍｍｏｌ／Ｌ以上であることが好ましく、２０ｍｍｏｌ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中においてＳＯ４ ２－含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、５０ｍｍｏｌ／Ｌ以下であることが好ましく、４０ｍｍｏｌ／Ｌ以下であるとさらに好ましい。 When SO 4 2− is included as a constituent in the inorganic salt medium, the inorganic electrolyte to be used is not particularly limited. For example, potassium sulfate (K 2 SO 4 ), ammonium sulfate ((NH 4 ) 2 SO 4 ) and sulfuric acid At least one selected from the group consisting of magnesium (MgSO 4 ) can be selected and used. The SO 4 2− content in the inorganic salt medium is preferably 10 to 50 mmol / L, more preferably 20 to 40 mmol / L. In addition, the SO 4 2− content in the inorganic salt medium at the start of the culture is preferably 10 mmol / L or more, more preferably 20 mmol / L or more, from the viewpoint of more efficiently producing D-lactic acid. Further preferred. On the other hand, the upper limit of the SO 4 2− content in the inorganic salt medium at the start of culture is preferably 50 mmol / L or less, more preferably 40 mmol / L or less from the viewpoint of producing D-lactic acid more efficiently. Is more preferable.
　無機塩培地中にＭｇ２＋を構成成分として含ませる場合、用いる無機電解質は、特に限定されないが、たとえば、リン酸水素マグネシウム（ＭｇＨＰＯ４）、リン酸二水素マグネシウム（Ｍｇ（Ｈ２ＰＯ４）２）、リン酸マグネシウム（Ｍｇ３（ＰＯ４）２）、リン酸アンモニウムマグネシウム（ＭｇＮＨ４ＰＯ４）及び硫酸マグネシウム（ＭｇＳＯ４）からなる群より選択される少なくとも１以上を選択して用いることができる。無機塩培地中のＭｇ２＋含有量は、好ましくは０．２～２ｍｍｏｌ／Ｌ、より好ましくは０．５～１.４ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中においてＭｇ２＋含有量は、より一層効率よくＤ－乳酸を生産するという観点から、０．２ｍｍｏｌ／Ｌ以上であることが好ましく、０．５ｍｍｏｌ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中においてＭｇ２＋含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、２ｍｍｏｌ／Ｌ以下であることが好ましく、１．４ｍｍｏｌ／Ｌ以下であるとさらに好ましい。 In the case where Mg 2+ is included as a constituent in the inorganic salt medium, the inorganic electrolyte used is not particularly limited. For example, magnesium hydrogen phosphate (MgHPO 4 ), magnesium dihydrogen phosphate (Mg (H 2 PO 4 ) 2 ), Magnesium phosphate (Mg 3 (PO 4 ) 2 ), ammonium magnesium phosphate (MgNH 4 PO 4 ) and magnesium sulfate (MgSO 4 ) can be selected and used. . The Mg 2+ content in the inorganic salt medium is preferably 0.2 to 2 mmol / L, more preferably 0.5 to 1.4 mmol / L. In addition, the Mg 2+ content in the inorganic salt medium at the start of the culture is preferably 0.2 mmol / L or more from the viewpoint of more efficiently producing D-lactic acid, and is 0.5 mmol / L or more. More preferably. On the other hand, the upper limit of the Mg 2+ content in the inorganic salt medium at the start of the culture is preferably 2 mmol / L or less, more preferably 1.4 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid. Is more preferable.
　また、本実施形態における「無機塩培地」は、チアミンや遷移金属イオンをさらに含んでいてもよい。無機塩培地中にチアミンや遷移金属イオンが含まれている場合、安価で、かつ、効率的にＤ－乳酸を生産する観点から、少なくとも上記（ｃ）又は上記（ｄ）のいずれか一方を満たすことが好ましく、精製をより簡便にする観点から上記（ｃ）及び上記（ｄ）の両方を満たすことがさらに好ましい。 In addition, the “inorganic salt medium” in the present embodiment may further contain thiamine or transition metal ions. When thiamine or transition metal ions are contained in the inorganic salt medium, at least one of the above (c) and (d) is satisfied from the viewpoint of inexpensive and efficient production of D-lactic acid It is preferable to satisfy both of the above (c) and (d) from the viewpoint of simplifying the purification.
　培養開始時の無機塩培地中においてチアミン含有量は、０．１ｍｇ／Ｌ以下であることが好ましく、０．０２ｍｇ／Ｌ以下であるとさらに好ましい。さらには、無機塩培地として、チアミンを含まないものを用いることがより一層好ましい。このため、培養開始時の無機塩培地中においてチアミン含有量は、０～０．１ｍｇ／Ｌであることが好ましく、より好ましくは０～０．０２ｍｇ／Ｌであるとより好ましい。また、下限値については、０ｍｇ／Ｌ以上であることが好ましい。 In the inorganic salt medium at the start of culture, the thiamine content is preferably 0.1 mg / L or less, and more preferably 0.02 mg / L or less. Further, it is more preferable to use an inorganic salt medium that does not contain thiamine. For this reason, the thiamine content in the inorganic salt medium at the start of culture is preferably 0 to 0.1 mg / L, more preferably 0 to 0.02 mg / L. Further, the lower limit is preferably 0 mg / L or more.
　また、遷移金属イオンは、特に限定されないが、たとえば、鉄イオン、コバルトイオン、銅イオン、亜鉛イオン及びモリブデン酸イオンからなる群より選択される少なくとも１以上のイオンを用いることができる。そして、培養開始時の無機塩培地中において遷移金属イオンの含有量は、１０ｍｇ／Ｌ以下であることが好ましく、０．８５ｍｇ／Ｌ以下であるとさらに好ましい。さらには、無機塩培地として、遷移金属イオンを実質的に含まないものを用いることが好ましい。このため、無機塩培地中の遷移金属イオンの含有量は、０～１０ｍｇ／Ｌであることが好ましく、０～０．８５ｍｇ／Ｌであるとより好ましい
　また、本実施形態に係るＤ－乳酸の生産方法において、培養開始時の無機塩培地は、さらに下記（ｅ）を満たすことが好ましい。こうすることで、より一層高品質のポリマーを得ることができ、より安価に、かつ、効率よくＤ－乳酸を生産することができる。
（ｅ）無機塩培地中の遷移金属イオンの含有量が０．８５ｍｇ／Ｌ以下である。 The transition metal ion is not particularly limited, and for example, at least one ion selected from the group consisting of iron ions, cobalt ions, copper ions, zinc ions, and molybdate ions can be used. The transition metal ion content in the inorganic salt medium at the start of the culture is preferably 10 mg / L or less, and more preferably 0.85 mg / L or less. Furthermore, it is preferable to use an inorganic salt medium that does not substantially contain transition metal ions. Therefore, the content of transition metal ions in the inorganic salt medium is preferably 0 to 10 mg / L, more preferably 0 to 0.85 mg / L. Also, the D-lactic acid according to this embodiment In the production method, it is preferable that the inorganic salt medium at the start of the culture further satisfies the following (e). In this way, a higher quality polymer can be obtained, and D-lactic acid can be produced at a lower cost and more efficiently.
(E) The content of transition metal ions in the inorganic salt medium is 0.85 mg / L or less.
　また、本実施形態における「無機塩培地」は、無機電解質を水に溶解又は懸濁して調製された液体であることが好ましい。この無機電解質を水に溶解又は懸濁して調製された液体に、炭素源を加えることにより、炭素源を原料として発酵によりＤ－乳酸を生産することができる。また、炭素源として用いる発酵性の糖は、特に限定されないが、たとえば、グルコース、フルクトース、キシロース、スクロース、グリセリン、アラビノース、メリビオース、トレハロース、マルトース、メリビオン酸、ラクトース、マルトトリオース、リボース、ガラクトース、ガラクツロン酸、グルコン酸、グルコサミン、グルクロン酸、マンニトール、マンノース、サッカリン酸、ソルビトール、フコース、ラムノース、アロース、及びＮ-アセチルグルコサミンからなる群より選択される１以上の化合物が挙げられる。また、デンプン加水分解物、草木質分解産物やセルロース加水分解物などを発酵性の糖（炭素源）として用いてもよい。培養開始時の、無機塩培地中の糖含有量は、高濃度の糖が微生物の生育を阻害することを考慮し、グルコース換算で、無機塩培地の全質量に対して２００ｇ／Ｌ以下とすることが好ましく、１５０ｇ／Ｌ以下であるとさらに好ましい。培養開始時の、無機塩培地中の糖含有量の下限は、２０ｇ／Ｌ以上であることが好ましく、５０ｇ／Ｌ以上であるとさらに好ましい。以上を踏まえ、培養開始時において無機塩培地中の糖含有量は、２０～２００ｇ／Ｌであることが好ましく、５０～１５０ｇ／Ｌのであるとより好ましい。 Further, the “inorganic salt medium” in the present embodiment is preferably a liquid prepared by dissolving or suspending an inorganic electrolyte in water. By adding a carbon source to a liquid prepared by dissolving or suspending the inorganic electrolyte in water, D-lactic acid can be produced by fermentation using the carbon source as a raw material. Further, fermentable sugar used as a carbon source is not particularly limited, for example, glucose, fructose, xylose, sucrose, glycerin, arabinose, melibiose, trehalose, maltose, melbionic acid, lactose, maltotriose, ribose, galactose, Examples thereof include one or more compounds selected from the group consisting of galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol, mannose, saccharic acid, sorbitol, fucose, rhamnose, allose, and N-acetylglucosamine. In addition, starch hydrolyzate, vegetative degradation product, cellulose hydrolyzate and the like may be used as fermentable sugar (carbon source). The sugar content in the inorganic salt medium at the start of the culture is 200 g / L or less in terms of glucose with respect to the total mass of the inorganic salt medium in consideration of the fact that a high concentration of sugar inhibits the growth of microorganisms. It is preferable, and it is further more preferable in it being 150 g / L or less. The lower limit of the sugar content in the inorganic salt medium at the start of culture is preferably 20 g / L or more, and more preferably 50 g / L or more. Based on the above, the sugar content in the inorganic salt medium at the start of culture is preferably 20 to 200 g / L, more preferably 50 to 150 g / L.
　本実施形態における「無機塩培地」には、更にベタインを含んでいてもよい。ここで、本実施形態におけるベタインとは、ＣＡＳ登録番号１０７－４３－７の化合物であり、トリメチルグリシン、グリシンベタインの名でも知られている化合物である。このベタインをさらに構成成分として含む無機塩培地を用いることで、Ｄ－乳酸生産大腸菌が良好なＤ－乳酸の発酵生産を行うことができるため好ましい。このように無機塩培地がベタインを含む場合、その含有量は、無機塩培地中に好ましくは０．０１～５ｍｍｏｌ／Ｌ、より好ましくは０．３～２ｍｍｏｌ／Ｌである。また、培養開始時の無機塩培地中において、ベタイン含有量は、より一層効率よくＤ－乳酸を生産するという観点から、０．０１ｍｍｏｌ／Ｌ以上であることが好ましく、０．３ｍｍｏｌ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中においてベタイン含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、５ｍｍｏｌ／Ｌ以下であることが好ましく、２ｍｍｏｌ／Ｌ以下であるとさらに好ましい。 The “inorganic salt medium” in the present embodiment may further contain betaine. Here, betaine in the present embodiment is a compound having CAS registration number 107-43-7, which is also known as trimethylglycine or glycine betaine. It is preferable to use an inorganic salt medium further containing betaine as a constituent component because D-lactic acid-producing Escherichia coli can perform excellent fermentation of D-lactic acid. Thus, when the inorganic salt medium contains betaine, the content thereof is preferably 0.01 to 5 mmol / L, more preferably 0.3 to 2 mmol / L in the inorganic salt medium. In addition, in the inorganic salt medium at the start of culture, the betaine content is preferably 0.01 mmol / L or more, more preferably 0.3 mmol / L or more, from the viewpoint of more efficiently producing D-lactic acid. More preferably. On the other hand, the upper limit of the betaine content in the inorganic salt medium at the start of the culture is preferably 5 mmol / L or less from the viewpoint of more efficiently producing D-lactic acid, and is 2 mmol / L or less. Further preferred.
　また、本実施形態における「無機塩培地」は、消泡剤を含んでいてもよい。消泡剤としては、公知のものがいずれも好適に使用できる。例えば、アデカノールＬＧ１２６（商品名、ＡＤＥＫＡ社製）等が挙げられる。消泡剤の含有量は、無機塩培地の全質量に対して０．０１～１ｇ／Ｌとすることが好ましく、０．０５～０．５ｇ／Ｌがより好ましい。また、培養開始時の無機塩培地中において消泡剤の含有量は、より一層効率よくＤ－乳酸を生産するという観点から、０．０１ｇ／Ｌ以上であることが好ましく、０．０５ｇ／Ｌ以上であるとさらに好ましい。一方、培養開始時の無機塩培地中において消泡剤の含有量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、１ｇ／Ｌ以下であることが好ましく、０．５ｇ／Ｌ以下であるとさらに好ましい。 In addition, the “inorganic salt medium” in the present embodiment may include an antifoaming agent. Any known antifoaming agent can be suitably used. Examples thereof include Adecanol LG126 (trade name, manufactured by ADEKA). The content of the antifoaming agent is preferably 0.01 to 1 g / L, more preferably 0.05 to 0.5 g / L with respect to the total mass of the inorganic salt medium. In addition, the content of the antifoaming agent in the inorganic salt medium at the start of the culture is preferably 0.01 g / L or more from the viewpoint of producing D-lactic acid more efficiently, and 0.05 g / L More preferably, the above is true. On the other hand, the upper limit of the content of the antifoaming agent in the inorganic salt medium at the start of the culture is preferably 1 g / L or less from the viewpoint of more efficiently producing D-lactic acid, and preferably 0.5 g / L. More preferably, it is L or less.
　本実施形態においてＤ－乳酸を発酵生産するにあたって、無機塩培地で培養する菌体は、炭素源、好ましくは糖からＤ－乳酸を発酵生産する能力があるＤ－乳酸生産大腸菌であれば特段の制限はないが、遺伝子組み換えにより、対象となる大腸菌の乳酸生産活性に関与する酵素活性の増強、若しくは不活性化、低減化又はこれらの組み合わせることによりＤ－乳酸生産活性及び／またはＤ乳酸光学純度を向上させた組換え大腸菌を、Ｄ－乳酸生産大腸菌として用いることが好ましい。 In fermentative production of D-lactic acid in this embodiment, the cells cultured in an inorganic salt medium are D-lactic acid-producing Escherichia coli capable of fermentatively producing D-lactic acid from a carbon source, preferably sugar. Although there is no limitation, D-lactic acid production activity and / or D-lactic acid optical purity by enhancing, inactivating, reducing or a combination of enzyme activities involved in lactic acid production activity of the target Escherichia coli by genetic recombination It is preferable to use recombinant Escherichia coli having improved D as a D-lactic acid producing Escherichia coli.
　本実施形態における「遺伝子組み換えにより」との文言は、生来の遺伝子の塩基配列に対する別のＤＮＡの挿入、あるいは遺伝子のある部分の置換、欠失又はこれらの組み合わせによって塩基配列上の変更が生じているものであれば全て包含し、例えば、突然変異が生じた結果得られたものであってもよい。Ｄ－乳酸生産活性及び／またはＤ－乳酸光学純度を向上させた組換え大腸菌としては、例えば、ＷＯ２００５／０３３３２４、ＷＯ２０１０／０３２６９７、ＷＯ２０１０／０３２６９８記載のもの等が挙げられる。 The term “by genetic recombination” in the present embodiment means that a change in the base sequence is caused by the insertion of another DNA into the base sequence of the native gene, or the substitution, deletion or combination of a part of the gene. It may be included, for example, as long as it is obtained as a result of mutation. Examples of recombinant Escherichia coli having improved D-lactic acid production activity and / or D-lactic acid optical purity include those described in WO2005 / 033324, WO2010 / 032697, and WO2010 / 032698.
　具体的には、Ｄ－乳酸生産大腸菌は、下記（ｉ）～（ｖ）からなる群より選択される１つ以上の遺伝子組み換えを含む組換え大腸菌であることが好ましく、（ｉ）～（ｉｉｉ）のすべての遺伝子組み換えを含む組換え大腸菌であるとさらに好ましい。このように遺伝子組み換えをして得られた組換え大腸菌は、通気条件下でＤ－乳酸を生産させる時にｌｄｈＡの発現が増強されていないものと比較してＤ－乳酸の蓄積量が向上し、不純物のピルビン酸濃度が減少すると共にＤ－乳酸の光学純度を向上させることが可能となる。
（ｉ）大腸菌が本来有しているＦＡＤ依存性Ｄ－乳酸デヒドロゲナーゼ（Ｄｌｄ）活性を不活化あるいは低減化する遺伝子組み換え
（ｉｉ）大腸菌が本来有しているピルベートホルメートリアーゼ（Ｐｆｌ）活性を不活化あるいは低減化する遺伝子組み換え
（ｉｉｉ）大腸菌由来ＮＡＤＨ依存性Ｄ－乳酸デヒドロゲナーゼ（ＬｄｈＡ）活性を増強する遺伝子組み換え
（ｉｖ）大腸菌が本来有しているリンゴ酸デヒドロゲナーゼ（Ｍｄｈ）活性を不活化あるいは低減化する遺伝子組み換え
（ｖ）大腸菌が本来有しているアスパラギン酸アンモニアリアーゼ（ＡｓｐＡ）活性を不活化あるいは低減化する遺伝子組み換え Specifically, the D-lactic acid-producing E. coli is preferably a recombinant E. coli containing one or more genetic recombination selected from the group consisting of the following (i) to (v): (i) to (iii) It is further preferred that the recombinant E. coli contains all of the genetic recombination. The recombinant Escherichia coli obtained by genetic recombination in this way has an increased amount of accumulated D-lactic acid compared to the one in which the expression of ldhA is not enhanced when producing D-lactic acid under aeration conditions. It is possible to improve the optical purity of D-lactic acid while reducing the concentration of impurity pyruvic acid.
(I) Genetic recombination that inactivates or reduces the FAD-dependent D-lactate dehydrogenase (Dld) activity inherent in E. coli (ii) The pyruvate formate lyase (Pfl) activity inherent in E. coli Inactivated or reduced genetic recombination (iii) E. coli-derived NADH-dependent D-lactate dehydrogenase (LdhA) activity enhanced (iv) Inactivated or malate dehydrogenase (Mdh) activity inherent in E. coli Gene recombination to reduce (v) Gene recombination to inactivate or reduce aspartate ammonia lyase (AspA) activity inherent in Escherichia coli
　（ｉ）のＤｌｄは、Ｄ－乳酸から、補酵素である酸化型フラビンアデニンジヌクレオチドの存在下でピルビン酸を生成する反応を触媒する酵素の総称を意味する。 (1) Dld means a general term for enzymes that catalyze the reaction of producing pyruvic acid from D-lactic acid in the presence of oxidized flavin adenine dinucleotide as a coenzyme.
　（ｉｉ）のＰｆｌは、国際生化学連合（Ｉ．Ｕ．Ｂ．）酵素委員会報告に準拠した酵素番号２．３．１．５４に分類され、ホルメートアセチルトランスフェラーゼとも呼ばれる酵素を指す。この酵素はピルビン酸からギ酸を生成する反応を可逆的に触媒する酵素の総称を意味している。 (Ii) Pfl refers to an enzyme classified as enzyme number 2.3.54 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee and also called formate acetyltransferase. This enzyme is a general term for enzymes that reversibly catalyze the reaction of producing formic acid from pyruvic acid.
　（ｉｉｉ）のＬｄｈＡは、ピルビン酸とＮＡＤＨからＤ－乳酸とＮＡＤを生成する大腸菌由来の酵素を指す。 (Iii) LdhA refers to an enzyme derived from E. coli that produces D-lactic acid and NAD from pyruvic acid and NADH.
　（ｉｖ）のＭｄｈは、国際生化学連合（Ｉ．Ｕ．Ｂ．）酵素委員会報告に準拠した酵素番号１．１．１．３７に分類され、リンゴ酸から、補酵素である酸化型ニコチンアミドアデニンジヌクレオチドの存在下でオキサロ酢酸を生成する反応を可逆的に触媒する酵素の総称を指す。 Miv of (iv) is classified into enzyme number 1.1.1.17 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee, from malic acid to oxidized nicotine which is a coenzyme. A generic term for enzymes that reversibly catalyze the reaction of producing oxaloacetate in the presence of amidoadenine dinucleotide.
　（ｖ）のＡｓｐＡは、国際生化学連合（Ｉ．Ｕ．Ｂ．）酵素委員会報告に準拠した酵素番号４．３．１．１に分類され、アスパルターゼとも呼ばれる酵素を指す。この酵素はＬ－アスパラギン酸からフマル酸を生成する反応を可逆的に触媒する酵素の総称を意味している。 (V) AspA refers to an enzyme classified as enzyme number 4.3.1.1 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee and also called aspartase. This enzyme is a general term for enzymes that reversibly catalyze the reaction of producing fumaric acid from L-aspartic acid.
　上記（ｉｉｉ）の遺伝子組み換えは、ＬｄｈＡをコードする遺伝子を、解糖系、核酸生合成系又はアミノ酸生合成系に関わる蛋白質の発現を司る遺伝子のプロモーターと連結した状態で発現プラスミドに組込み、これを大腸菌へ導入する方法や、大腸菌のゲノム上に存在する、大腸菌由来のＬｄｈＡをコードする遺伝子から、解糖系、核酸生合成系又はアミノ酸生合成系に関わる蛋白質の発現を司る遺伝子のプロモーターを使用することでＬｄｈＡを発現させる方法が挙げられるが、大腸菌のゲノム上に存在するＬｄｈＡをコードする遺伝子から、解糖系、核酸生合成系又はアミノ酸生合成系に関わる蛋白質の発現を司る遺伝子のプロモーターを使用してＬｄｈＡを発現させる方法であることが好ましい。 In the genetic recombination of (iii) above, the gene encoding LdhA is incorporated into an expression plasmid in a state of being linked to the promoter of a gene that controls the expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis. From the gene encoding LdhA derived from Escherichia coli, which is present in the genome of Escherichia coli, and the promoter of the gene responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis The method of expressing LdhA can be mentioned by using the gene, which is responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis from genes encoding LdhA present in the genome of E. coli. A method of expressing LdhA using a promoter is preferred.
　ここで、解糖系、核酸生合成系又はアミノ酸生合成系に関わる蛋白質の発現を司る遺伝子のプロモーターとは、恒常的に細菌内、好ましくは大腸菌内で機能する強力なプロモーターであって、且つグルコース存在下でも発現の抑制を受けにくいプロモーターを指す。具体的には、解糖系、核酸生合成系又はアミノ酸生合成系に関わる蛋白質の発現を司る遺伝子のプロモーターとしては、グリセルアルデヒド３リン酸デヒドロゲナーゼのプロモーターやセリンヒドロキシメチルトランスフェラーゼ（ｇｌｙＡ）プロモーター等が挙げられる。 Here, the promoter of the gene responsible for the expression of the protein involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis is a strong promoter that constantly functions in bacteria, preferably in E. coli, and A promoter that is less susceptible to expression suppression even in the presence of glucose. Specifically, examples of promoters for genes that are responsible for the expression of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis include glyceraldehyde 3-phosphate dehydrogenase promoter and serine hydroxymethyltransferase (glyA) promoter. Is mentioned.
　また、Ｄ－乳酸生産大腸菌は、スクロース非ＰＴＳ遺伝子群及びＦｒｕＫからなる群より選択された少なくとも一つが強化されていてもよいが、スクロース非ＰＴＳ遺伝子群及びＦｒｕＫの双方が強化されていることが好ましく、スクロース非ＰＴＳ遺伝子群のうちｃｓｃＡのみとＦｒｕＫとが共に強化されていることがさらに好ましい。 Further, in D-lactic acid-producing Escherichia coli, at least one selected from the group consisting of the sucrose non-PTS gene group and FruK may be strengthened, but both the sucrose non-PTS gene group and FruK may be strengthened. Preferably, it is more preferable that both cscA and FruK are enhanced in the sucrose non-PTS gene group.
　スクロース加水分解酵素（インベルターゼ、ＣｓｃＡ）とは、国際生化学連合（Ｉ．Ｕ．Ｂ）酵素委員会報告に準拠した酵素番号３．２．１．２６に分類され、スクロースからＤ－グルコースとＤ－フルクトースを生成する反応を触媒する酵素の総称を指す。ＣｓｃＡの強化は、例えば、大腸菌由来のＣｓｃＡをコードする遺伝子の塩基配列を有するＤＮＡを大腸菌に導入することで実現することができる。 Sucrose hydrolase (invertase, CscA) is classified into enzyme number 3.2.1.26 according to the report of the International Biochemical Union (I.U.B) Enzyme Committee. From sucrose to D-glucose and D -A generic term for enzymes that catalyze reactions that produce fructose. The enhancement of CscA can be realized, for example, by introducing a DNA having a base sequence of a gene encoding CscA derived from E. coli into E. coli.
　フルクトース－１－リン酸キナーゼ（ＦｒｕＫ）は、国際生化学連合（Ｉ．Ｕ．Ｂ）酵素委員会報告に準拠した酵素番号２．７．１．５６に分類され、ホスホフルクトキナーゼ１とも呼ばれる酵素を指す。ＦｒｕＫの強化は、例えば、大腸菌由来のＦｒｕＫをコードする遺伝子の塩基配列を有するＤＮＡを大腸菌に導入することで実現することができる。 Fructose-1-phosphate kinase (FruK) is classified as enzyme number 2.7.1.56 according to the report of the International Biochemical Union (I.B.) Enzyme Committee and is also called phosphofructokinase 1. Refers to an enzyme. Enhancement of FruK can be realized, for example, by introducing DNA having the base sequence of a gene encoding FruK derived from E. coli into E. coli.
　本実施形態のＤ－乳酸の生産方法は、Ｄ－乳酸生産大腸菌を用いて、炭素源、好ましくは糖からＤ－乳酸を生産させることを含むものである。即ち、Ｄ－乳酸生産大腸菌と炭素源とを接触させる工程と、接触により得られたＤ－乳酸を回収する工程とを含むものである。 The production method of D-lactic acid according to this embodiment includes producing D-lactic acid from a carbon source, preferably sugar, using D-lactic acid-producing Escherichia coli. That is, it includes a step of contacting D-lactic acid-producing Escherichia coli with a carbon source and a step of recovering D-lactic acid obtained by the contact.
　具体的には、本実施形態に係るＤ－乳酸の生産方法は、無機塩培地においてＤ－乳酸生産大腸菌と炭素源とを接触させてＤ－乳酸塩を得る工程と、Ｄ－乳酸塩を含む無機塩培地からＤ－乳酸生産大腸菌を除去した後にＤ－乳酸塩を脱塩してＤ－乳酸を得る工程と、を含む。 Specifically, the method for producing D-lactic acid according to the present embodiment includes a step of contacting D-lactic acid-producing Escherichia coli with a carbon source in an inorganic salt medium to obtain D-lactate, and D-lactate. Removing D-lactic acid-producing Escherichia coli from the inorganic salt medium and then desalting D-lactate to obtain D-lactic acid.
　また、本実施形態に係るＤ－乳酸の生産方法は、上記Ｄ－乳酸塩を得る工程において、Ｄ－乳酸のアルカリ土類金属塩を得て、その後、Ｄ－乳酸を得る工程において、Ｄ－乳酸のアルカリ土類金属塩を脱塩することでアルカリ土類金属を含む無機塩を析出させてもよく、Ｄ－乳酸塩を得る工程において、Ｄ－乳酸のアルカリ金属塩を得て、その後、Ｄ－乳酸を得る工程において、電解透析処理によりＤ－乳酸のアルカリ金属塩を脱塩してもよい。 In addition, in the method for producing D-lactic acid according to the present embodiment, in the step of obtaining D-lactate, the alkaline earth metal salt of D-lactic acid is obtained, and then in the step of obtaining D-lactic acid, D-lactic acid is obtained. An inorganic salt containing an alkaline earth metal may be precipitated by desalting the alkaline earth metal salt of lactic acid. In the step of obtaining D-lactate, an alkali metal salt of D-lactic acid is obtained, and then In the step of obtaining D-lactic acid, the alkali metal salt of D-lactic acid may be desalted by electrodialysis.
　また、本実施形態に係るＤ－乳酸の生産方法は、Ｄ－乳酸塩を脱塩してＤ－乳酸を得る工程の後に、Ｄ－乳酸の精製を行う精製工程をさらに含み、精製工程において、加水分解しながら蒸留を行うことが好ましい。こうすることで、蒸留工程のＤ－乳酸回収率をより一層上げることができ、より安価に、かつ、効率よくＤ－乳酸を生産することができる。 The method for producing D-lactic acid according to the present embodiment further includes a purification step of purifying D-lactic acid after the step of desalting D-lactate to obtain D-lactic acid, It is preferred to carry out the distillation while hydrolysis. By doing so, it is possible to further increase the D-lactic acid recovery rate in the distillation step, and to produce D-lactic acid more efficiently at a lower cost.
　Ｄ－乳酸生産大腸菌と炭素源との接触は、上述した無機塩培地中でＤ－乳酸生産大腸菌を培養することにより行われ、これによりＤ－乳酸を含む発酵液を得ることができる。 The contact between the D-lactic acid-producing Escherichia coli and the carbon source is carried out by culturing the D-lactic acid-producing Escherichia coli in the above-described inorganic salt medium, whereby a fermentation broth containing D-lactic acid can be obtained.
　Ｄ－乳酸生産大腸菌の培養条件としては使用する大腸菌、培養装置により変動するものの、培養温度は２０℃以上４０℃以下であることが好ましく、より好ましくは２５℃以上３５℃以下であるとさらに好ましい。 Although the culture conditions for D-lactic acid-producing E. coli vary depending on the E. coli used and the culture apparatus, the culture temperature is preferably 20 ° C. or higher and 40 ° C. or lower, more preferably 25 ° C. or higher and 35 ° C. or lower. .
　また、培養槽の圧力は、培養時に、常圧（０．１ＭＰａ）以上０．５ＭＰａ以下であることが好ましく、常圧（０．１ＭＰａ）以上０．２ＭＰａ以下であるとさらに好ましい。 Further, the pressure in the culture tank is preferably normal pressure (0.1 MPa) or more and 0.5 MPa or less, and more preferably normal pressure (0.1 MPa) or more and 0.2 MPa or less at the time of culture.
　また、培養のｐＨは、大腸菌の発酵生産力を高める観点から好ましくは４．０以上９．０以下とすればよく、ｐＨ６．０以上８．０以下であると好ましく、ｐＨ７．０以上７．８以下であるとさらに好ましい。なお、ｐＨの調製は、Ｄ－乳酸を生産しながら無機塩基を培養槽に添加しながら行うことが好ましい。これにより、生産するＤ－乳酸を無機塩基で中和し、乳酸塩とすることができる。 The pH of the culture is preferably 4.0 or more and 9.0 or less from the viewpoint of enhancing the fermentation productivity of Escherichia coli, preferably 6.0 or more and 8.0 or less, and more preferably 7.0 or more and 7. More preferably, it is 8 or less. The pH is preferably adjusted while adding an inorganic base to the culture tank while producing D-lactic acid. Thus, the produced D-lactic acid can be neutralized with an inorganic base to obtain a lactate.
　無機塩基としては、たとえば、水酸化リチウム、水酸化ナトリウム、水酸化カリウム及び水酸化ルビジウム等のアルカリ金属塩、水酸化マグネシウム、水酸化カルシウム及び水酸化バリウム等のアルカリ土類金属塩、並びにアンモニアからなる群より選択される少なくとも１種を用いればよい。また、無機塩基の種類を選択することで生産したＤ－乳酸に対応する乳酸塩の対イオンを、リチウムイオン、ナトリウムイオン、カリウムイオン、ルビジウムイオン、マグネシウムイオン、カルシウムイオン、バリウムイオン、アンモニウムイオンなど任意のカチオンにすることができる。 Examples of inorganic bases include alkali metal salts such as lithium hydroxide, sodium hydroxide, potassium hydroxide and rubidium hydroxide, alkaline earth metal salts such as magnesium hydroxide, calcium hydroxide and barium hydroxide, and ammonia. What is necessary is just to use at least 1 sort (s) selected from the group which consists of. In addition, counter ions of lactate corresponding to D-lactic acid produced by selecting the kind of inorganic base include lithium ion, sodium ion, potassium ion, rubidium ion, magnesium ion, calcium ion, barium ion, ammonium ion, etc. It can be any cation.
　培養時間は特に限定されず、菌体が十分に増殖し、且つＤ－乳酸が生成するに必要な時間であればよい。 The culture time is not particularly limited as long as it is necessary for the bacterial cells to grow sufficiently and to produce D-lactic acid.
　培養に際しては通常は温度、ｐＨ、通気条件、攪拌速度、圧力を制御し得る培養槽を用いるのが一般的であるが、本実施形態に係る培養に際しては培養槽を使用することに限定されない。なお、培養槽を用いて培養する場合には、培養槽内の培地に大腸菌を接種する必要があるが、接種する量に特段の限定はなく、用いる大腸菌について寒天培地にコロニーを形成させ、そのコロニーを白金耳などにより培養槽に直接接種してもよい。
   また、フラスコや別の培養槽などの容器で予め培養した培養液を調製し、これを必要量、培養槽内の培地に接種してもよい。このときの培養液の必要量は、特に制限されるものではないが、培養槽内の培地に大腸菌が１細胞でも接種される量であればよい。具体的には、培養液の量は、培養槽内の培地液量の０．０１％～４０％相当量が好ましい。より好ましくは０．１％～１０％相当量である。また、培養液の必要量は、より一層効率よくＤ－乳酸を生産するという観点から、０．０１％相当量以上であることが好ましく、０．１％相当量以上であるとさらに好ましい。一方、培養液の必要量の上限値は、より一層効率よくＤ－乳酸を生産するという観点から、４０％相当量以下であることが好ましく、１０％相当量以下であるとさらに好ましい。 In culturing, it is common to use a culture tank capable of controlling temperature, pH, aeration conditions, stirring speed, and pressure. However, the culturing according to the present embodiment is not limited to using a culture tank. In addition, when culturing using a culture tank, it is necessary to inoculate E. coli in the culture medium in the culture tank, but there is no particular limitation on the amount to be inoculated, and colonies are formed on the agar medium for the E. coli used. Colonies may be directly inoculated into the culture tank using platinum ears or the like.
Moreover, the culture solution previously cultured with containers, such as a flask and another culture tank, may be prepared, and this may be seed | inoculated to the culture medium in a culture tank. The required amount of the culture solution at this time is not particularly limited, but may be an amount that allows even one cell of E. coli to be inoculated into the medium in the culture tank. Specifically, the amount of the culture solution is preferably 0.01% to 40% equivalent to the amount of the medium solution in the culture tank. More preferably, the amount is equivalent to 0.1% to 10%. In addition, the necessary amount of the culture solution is preferably 0.01% or more, more preferably 0.1% or more, from the viewpoint of producing D-lactic acid more efficiently. On the other hand, the upper limit of the required amount of the culture broth is preferably 40% or less, more preferably 10% or less, from the viewpoint of more efficiently producing D-lactic acid.
　また、発酵工程における通気量は、乳酸生産効率の観点から、０ｖｖｍ以上であればよく、０．００１ｖｖｍ以上であることが好ましく、０．０１ｖｖｍ以上であるとさらに好ましい。一方、発酵工程における通気量は、乳酸生産効率の観点から、５ｖｖｍ以下であればよく、２ｖｖｍ以下であることが好ましく、１ｖｖｍ以下であるとさらに好ましい。
　なお、本実施形態では通気量としてｖｖｍとの表記を使用する場合がある。本明細書において「ｖｖｍ」とは、１分間で、液容量の何倍の通気をするかということを示すものであり、例えば、１０Ｌの発酵液に対して、５ｖｖｍの通気を行ったとは、毎分５０Ｌの通気を行ったということを意味するものである。 In addition, the aeration amount in the fermentation process may be 0 vvm or more, preferably 0.001 vvm or more, and more preferably 0.01 vvm or more from the viewpoint of lactic acid production efficiency. On the other hand, the amount of aeration in the fermentation process may be 5 vvm or less from the viewpoint of lactic acid production efficiency, preferably 2 vvm or less, and more preferably 1 vvm or less.
In this embodiment, the notation “vvm” may be used as the air flow rate. In the present specification, “vvm” indicates how many times the liquid volume is aerated in 1 minute. For example, 5 vvm aeration is performed on 10 L of fermentation broth. This means that 50 L was ventilated per minute.
　液中に通気する場合は内圧、撹拌羽根位置、撹拌羽根形状、撹拌速度の組み合わせにより液中への酸素の溶け込み速度が変化するために、Ｄ－乳酸の生産性、及び、Ｄ－乳酸以外の有機酸量などを指標に種々の条件が設定可能である。なお、設定された通気条件は、発酵初期から終了まで一貫して行う必要はなく、発酵工程の一部で行うことでも好ましい結果を得ることができる。上記のように通気を行うことでＤ－乳酸の生産性の向上、Ｄ－乳酸以外の有機酸量の削減を達成することができる。 In the case of aeration in the liquid, the rate of oxygen dissolution into the liquid varies depending on the combination of the internal pressure, the position of the stirring blade, the shape of the stirring blade, and the stirring speed, so the productivity of D-lactic acid and other than D-lactic acid Various conditions can be set by using the amount of organic acid as an index. In addition, it is not necessary to carry out the set aeration conditions consistently from the beginning of fermentation to completion | finish, and a favorable result can be obtained even if it carries out in a part of fermentation process. By performing aeration as described above, it is possible to improve the productivity of D-lactic acid and reduce the amount of organic acids other than D-lactic acid.
　また、酸素摂取速度（ＯＵＲ）は、乳酸生産性の観点から、０．０ｍｍｏｌ／Ｌ／ｈｒ以上であることが好ましく、１．０ｍｍｏｌ／Ｌ／ｈｒ以上であるとさらに好ましい。一方、酸素摂取速度（ＯＵＲ）の上限値は、乳酸生産性の観点から、１００．０ｍｍｏｌ／Ｌ／ｈｒ以下であることが好ましく、５０．０ｍｍｏｌ／Ｌ／ｈｒ以下であるとさらに好ましく、さらには１０．０ｍｍｏｌ／Ｌ／ｈｒ以下であることが好ましく、５．０ｍｍｏｌ／Ｌ／ｈｒ以下であるとより一層好ましい。
　なお、ＯＵＲは、Ｄ－乳酸の生産期に測定されたものであればよい。ここでＤ－乳酸の生産期とは、発酵開始後、大腸菌培養の適応期を経て大腸菌が増殖を開始しＤ－乳酸を生産する時期を指す。また大腸菌の培養と乳酸生産を分けて実施する場合は、乳酸生産工程を指す。 The oxygen uptake rate (OUR) is preferably 0.0 mmol / L / hr or more, and more preferably 1.0 mmol / L / hr or more, from the viewpoint of lactic acid productivity. On the other hand, the upper limit of the oxygen uptake rate (OUR) is preferably 100.0 mmol / L / hr or less, more preferably 50.0 mmol / L / hr or less from the viewpoint of lactic acid productivity, It is preferably 10.0 mmol / L / hr or less, and more preferably 5.0 mmol / L / hr or less.
The OUR may be any one measured during the production period of D-lactic acid. Here, the production phase of D-lactic acid refers to the time when E. coli starts to grow and produce D-lactic acid through the adaptation phase of E. coli culture after the start of fermentation. Moreover, when carrying out culture | cultivation of E. coli and lactic acid production separately, it refers to a lactic acid production process.
　本実施形態において酸素摂取速度（ＯＵＲ）とは、発酵液の単位容積あたりの酸素移動速度であり、すなわち大腸菌の酸素摂取速度とも言える。ＯＵＲは排ガス分析法によって以下の式１から求めたものを用いる。 In this embodiment, the oxygen uptake rate (OUR) is the oxygen transfer rate per unit volume of the fermentation broth, that is, it can be said that the oxygen uptake rate of E. coli. OUR obtained from the following formula 1 by exhaust gas analysis is used.
　（式１）
　　ＯＵＲ＝７．２２×１０６／ＶＬ×（ＱｉＰｉｙｉ／Ｔｉ－ＱｏＰｏｙｏ／Ｔｏ）
　　　　ＶＬ：発酵槽中の液量（Ｌ）
　　　　Ｑｉ及びＱｏ：空気入り口及び出口における空気流量（Ｌ／ｍｉｎ）
　　　　Ｐｉ及びＰｏ：空気入り口及び出口における空気圧（ＭＰａ）
　　　　Ｔｉ及びＴｏ：空気入り口及び出口における絶対温度（Ｋ）
　　　　ｙｉ及びｙｏ：空気入り口及び出口における酸素のモル分率 (Formula 1)
OUR = 7.22 × 10 6 / VL × (QiPiii / Ti-QoPoyo / To)
VL: Liquid volume in the fermenter (L)
Qi and Qo: air flow rate at the air inlet and outlet (L / min)
Pi and Po: Air pressure (MPa) at the air inlet and outlet
Ti and To: absolute temperature at the air inlet and outlet (K)
yi and yo: mole fraction of oxygen at the air inlet and outlet
　なお、上記式１に基づいてＯＵＲを求める際に、空気流量、空気圧、絶対温度の値が空気入り口及び出口で無視できる程度の差しかないときには１カ所での測定値を適用してもよい。また、本実施形態でいう圧力及び空気圧は、絶対圧力を指す。 When obtaining the OUR based on the above equation 1, the measured values at one place may be applied if the air flow rate, air pressure, and absolute temperature are not negligible at the air inlet and outlet. The pressure and air pressure referred to in the present embodiment refer to absolute pressure.
　ＯＵＲは、通気量、攪拌回転速度、温度、圧力、ｐＨなどによって変動する。従って、ＯＵＲを上述した範囲内に調整するには、通気流量、攪拌回転速度等を適宜調整すればよい。 OUR varies depending on aeration amount, stirring rotation speed, temperature, pressure, pH, and the like. Therefore, in order to adjust the OUR within the above-described range, the aeration flow rate, the stirring rotation speed, and the like may be adjusted as appropriate.
　また、上記ＯＵＲは他の指標に換算することもできる。このような他の指標としては、液境膜容量係数（ｋＬａ）を挙げることができる。液境膜容量係数（ｋＬａ）は通気量・攪拌回転速度の関数であり、以下の関係が知られている（式２、Ｒｉｃｈａｒｄｓ，　Ｊ．　Ｗ．　１９６１，　Ｐｒｏｇ．Ｉｎｄ．Ｍｉｃｒｏ．３，　１４３－１７２） The OUR can be converted into another index. Examples of such other indicators include a liquid film capacity coefficient (k L a). Ekisakaimaku capacity coefficient (k L a) is a function of the aeration rate and stirring speed, the following relationship is known (Equation 2, Richards, J. W. 1961, Prog.Ind.Micro.3, 143-172)
　（式２）
　　ｋＬａ∝（Ｐｇ/Ｖ）０．４Ｖｓ０．５Ｎ０．５
　　　　Ｐｇ：通気攪拌槽の消費動力
　　　　Ｖ：槽に張り込まれた液量
　　　　ＶＳ：見かけの空気線速度（通気量／槽断面積）
　　　　Ｎ：攪拌回転速度 (Formula 2)
k L aα (P g / V ) 0.4 Vs 0.5 N 0.5
P g : Power consumption of the aeration and stirring tank V: Liquid amount stuck in the tank V S : Apparent air linear velocity (aeration volume / tank cross-sectional area)
N: Stirring rotation speed
　図１は、Ｄ－乳酸生産大腸菌の培養に用いられる発酵装置の一例を示す。発酵装置１０には、発酵槽１２が設けられている。この発酵槽１２には、マスフローメータ１４を介して空気入り口から空気が供給されるようになっており（矢印Ａ）、一方、コンデンサ１６を介して槽内の空気が排気口から排出されるようになっている（矢印Ｂ）。コンデンサ１６と排気口との間には、槽内圧力計１８及び排ガス分析計２０が連結され、槽内の圧力及び出口の酸素モル分圧をそれぞれ測定可能になっている。発酵槽１２には、温度センサ２２、ＤＯセンサ２４及びｐＨセンサ２６がそれぞれ配置されており、発酵槽１２内の反応液中の、温度、ＤＯ（溶在酸素）及びｐＨが測定可能になっている。また、発酵槽１２には、攪拌機としてのディスクタービン翼２８が配置されており、ディスクタービン翼２８はモーター４４で攪拌・制御される。 FIG. 1 shows an example of a fermentation apparatus used for culturing D-lactic acid-producing E. coli. The fermentation apparatus 10 is provided with a fermentation tank 12. Air is supplied to the fermenter 12 from the air inlet via the mass flow meter 14 (arrow A), while the air in the tank is discharged from the exhaust port via the condenser 16. (Arrow B). An in-tank pressure gauge 18 and an exhaust gas analyzer 20 are connected between the condenser 16 and the exhaust port, and the pressure in the tank and the oxygen partial pressure at the outlet can be measured. In the fermenter 12, a temperature sensor 22, a DO sensor 24, and a pH sensor 26 are arranged, respectively, so that the temperature, DO (dissolved oxygen) and pH in the reaction solution in the fermenter 12 can be measured. Yes. The fermenter 12 is provided with a disc turbine blade 28 as a stirrer, and the disc turbine blade 28 is stirred and controlled by a motor 44.
　また、発酵槽１２の周囲は二重になっており温水にて加熱又は冷却することができる。発酵槽１２の外側には、ｐＨ調整剤を充填したｐＨ調整部３４が設けられている。ｐＨ調整部３４は、ポンプ３６を介して発酵槽１２へｐＨ調整剤を供給可能となっている。ｐＨ調整剤としては、前述の無機塩基を用いることができる。 Moreover, the periphery of the fermenter 12 is double and can be heated or cooled with warm water. A pH adjusting unit 34 filled with a pH adjusting agent is provided outside the fermenter 12. The pH adjuster 34 can supply a pH adjuster to the fermenter 12 via the pump 36. As the pH adjuster, the aforementioned inorganic base can be used.
　発酵槽１２には、全体を制御するコントローラ３８が備えられており、温度センサ２２、ＤＯセンサ２４及びｐＨセンサ２６に連結されて、それぞれのセンサからの情報が入力可能になっている。また、コントローラ３８は、各種センサからの情報に応じて、ジャケット３２の温度を調節して発酵槽内の溶液温度を制御すると共に、ポンプ３６の作動によりｐＨ制御するようになっている。 The fermenter 12 is provided with a controller 38 that controls the whole, and is connected to the temperature sensor 22, the DO sensor 24, and the pH sensor 26 so that information from each sensor can be input. Further, the controller 38 adjusts the temperature of the jacket 32 according to information from various sensors to control the solution temperature in the fermenter, and controls the pH by operating the pump 36.
　発酵槽１２中に蓄積したＤ－乳酸は、無機塩培地から分離・精製することで回収することができる。具体的には、無機塩培地からの菌体等の固形物の除去、脱塩、濃縮、精製などの工程であるが、その方法や順序は特に制限されないものの、発酵後の無機塩培地からＤ－乳酸生産大腸菌を除去した後にＤ－乳酸塩を脱塩してＤ－乳酸を得ることが得ることが好ましい。 D-lactic acid accumulated in the fermenter 12 can be recovered by separation and purification from the inorganic salt medium. Specifically, it is a process of removing solids such as cells from the inorganic salt medium, desalting, concentrating and purifying, but the method and order thereof are not particularly limited, but it is determined from the inorganic salt medium after fermentation. -It is preferable to obtain D-lactic acid by desalting D-lactate after removing lactic acid-producing Escherichia coli.
　例えば、菌体などの固形物の除去には、遠心分離やろ過などの方法が挙げられる。 For example, methods such as centrifugation and filtration can be used to remove solids such as bacterial cells.
　精製工程において、Ｄ－乳酸塩からＤ－乳酸へ変換する方法には、イオン交換カラムや電気透析などが挙げられる。無機酸の添加によりＤ－乳酸に脱塩してもよい。例えば、Ｄ－乳酸塩の対イオンがアルカリ土類金属イオンの場合には、Ｄ－乳酸塩を脱塩することでアルカリ土類金属を含む無機塩を析出させることができる。例えば、Ｄ－乳酸塩の対イオンがカルシウムイオンの場合には、硫酸を添加し、溶液中に硫酸カルシウムを析出させることで脱塩し、Ｄ－乳酸を得ることができる。 Examples of methods for converting D-lactate to D-lactic acid in the purification step include ion exchange columns and electrodialysis. It may be desalted to D-lactic acid by adding an inorganic acid. For example, when the counter ion of D-lactate is an alkaline earth metal ion, an inorganic salt containing an alkaline earth metal can be precipitated by desalting D-lactate. For example, when the counter ion of D-lactate is calcium ion, it can be desalted by adding sulfuric acid and precipitating calcium sulfate in the solution to obtain D-lactic acid.
　また、Ｄ－乳酸塩の対イオンがアルカリ金属イオン、好ましくはナトリウムイオン又はカリウムイオンの場合には、電解透析処理によりＤ－乳酸塩を脱塩することが好ましい。電解透析処理には、水分解電気透析装置を用いることができる。該装置は、バイポーラ膜と陽イオン交換膜とを交互に配列し、酸室と塩基室を形成させた電気透析装置のことである。その代表的なものとしては「二室式水分解電気透析装置」を挙げることができる。本実施形態においては、「水分解電気透析装置」を用いた電気透析操作のことを「水分解電気透析工程」と定義される。そして、更に具体的な処理として代表的には「二室式水分解電気透析工程」が例示される。 In addition, when the counter ion of D-lactate is an alkali metal ion, preferably sodium ion or potassium ion, it is preferable to desalinate D-lactate by electrodialysis. A water splitting electrodialyzer can be used for the electrodialysis treatment. The apparatus is an electrodialysis apparatus in which bipolar membranes and cation exchange membranes are alternately arranged to form an acid chamber and a base chamber. A typical example is a “two-chamber water-splitting electrodialysis apparatus”. In the present embodiment, the electrodialysis operation using the “water splitting electrodialysis apparatus” is defined as “water splitting electrodialysis step”. A more specific process is typically exemplified by a “two-chamber water-splitting electrodialysis process”.
　水分解電気透析工程においては、得られたＤ－乳酸塩を含む発酵液に対して、水分解電気透析装置を用いて、水分解電気透析工程を行い、Ｄ－乳酸とアルカリをそれぞれ回収する。すなわち、バイポーラ膜と陽イオン交換膜を使用した水分解電気透析装置に、Ｄ－乳酸塩溶液を供給して水分解電気透析工程を行い、酸室よりＤ－乳酸を、塩基室よりアルカリをそれぞれ得る。 In the water-splitting electrodialysis step, the obtained fermentation broth containing D-lactate is subjected to a water-splitting electrodialysis step using a water-splitting electrodialyzer, and D-lactic acid and alkali are recovered. That is, a water-splitting electrodialysis apparatus using a bipolar membrane and a cation exchange membrane is supplied with a D-lactate solution to carry out a water-splitting electrodialysis step, and D-lactic acid from the acid chamber and alkali from the base chamber, respectively. obtain.
　バイポーラ膜としては、従来公知のバイポーラ膜、すなわち陽イオン交換膜と陰イオン交換膜とを貼合わせた構造を有する公知のバイポーラ膜を使用することができる。また、水分解電気透析装置の陽イオン交換膜としては、公知の陽イオン交換膜を用いることができる。 As the bipolar membrane, a conventionally known bipolar membrane, that is, a known bipolar membrane having a structure in which a cation exchange membrane and an anion exchange membrane are bonded together can be used. Moreover, a well-known cation exchange membrane can be used as a cation exchange membrane of a water-splitting electrodialysis apparatus.
　図２は、本実施形態において使用される水分解電気透析装置の代表的な態様の概略図を示すものである。即ち、図２において、水分解電気透析装置は、陽電極３および陰電極４の間に、膜としてバイポーラ膜Ｂ、陽イオン交換膜Ｃの２種類が交互に配列され、酸室８、及び塩基室７の二室が形成されている。陽イオン交換膜Ｃと陽電極３との空隙（陽極室５）、及び、陽イオン交換膜Ｃと陰電極４との空隙（陰極室６）には電極液が満たされている。ここで、バイポーラ膜Ｂの陰イオン交換体側と陽イオン交換膜Ｃの間の室が塩基室７、バイポーラ膜Ｂの陽イオン交換体側と陽イオン交換膜Ｃの間の室が酸室８となる。 FIG. 2 shows a schematic diagram of a typical aspect of the water-splitting electrodialysis apparatus used in the present embodiment. That is, in FIG. 2, the water-splitting electrodialysis apparatus has two types of membranes, a bipolar membrane B and a cation exchange membrane C, arranged alternately between a positive electrode 3 and a negative electrode 4, and an acid chamber 8 and a base Two chambers of the chamber 7 are formed. The gap between the cation exchange membrane C and the positive electrode 3 (anode chamber 5) and the gap between the cation exchange membrane C and the negative electrode 4 (cathode chamber 6) are filled with an electrode solution. Here, the chamber between the anion exchanger side of the bipolar membrane B and the cation exchange membrane C is the base chamber 7, and the chamber between the cation exchanger side of the bipolar membrane B and the cation exchange membrane C is the acid chamber 8. .
　上記、水分解電気透析装置の構造は、公知の構造が採用される。
　本実施形態に係るＤ－乳酸の生産方法において、上記水分解電気透析装置を使用した水分解電気透析工程は、酸室８、塩基室７のそれぞれの室に供給する液の外部タンク（不図示）を設けて、それぞれの室と外部タンクとの間で液を循環しながら電気透析を行う方法が好適に採用される。 A known structure is adopted as the structure of the water-splitting electrodialysis apparatus.
In the method for producing D-lactic acid according to the present embodiment, the water-splitting electrodialysis step using the water-splitting electrodialysis apparatus includes an external tank for liquid to be supplied to each of the acid chamber 8 and the base chamber 7 (not shown). ), And a method of performing electrodialysis while circulating the liquid between the respective chambers and the external tank is preferably employed.
　上記、Ｄ－乳酸塩溶液を酸室８に供給して電気透析を行うと、酸室８のＤ－乳酸塩は通電と共にＤ－乳酸に変換していく。即ち、酸室８中に導入されたＤ－乳酸塩の陽イオンは、陽イオン交換膜Ｃを透過して塩基室７へ移動し、このとき、バイポーラ膜Ｂから生成したＯＨイオンと結合して塩基となる。また、酸室８ではバイポーラ膜Ｂから生成したプロトンとＤ－乳酸アニオンとが結合して非解離性のＤ－乳酸となり、そのまま酸室８にとどまる。 When the above-mentioned D-lactate solution is supplied to the acid chamber 8 and electrodialysis is performed, the D-lactate in the acid chamber 8 is converted to D-lactic acid with energization. That is, the cation of D-lactate introduced into the acid chamber 8 permeates through the cation exchange membrane C and moves to the base chamber 7. At this time, it binds to OH ions generated from the bipolar membrane B. Become a base. In the acid chamber 8, the proton generated from the bipolar membrane B and the D-lactic acid anion are combined to form non-dissociable D-lactic acid, and remains in the acid chamber 8 as it is.
　本実施形態において、水分解電気透析工程時の各種液の温度は、通常、５℃以上７０℃以下であり、２０℃以上５０℃以下とすることが好ましい。 In this embodiment, the temperature of various liquids during the water-splitting electrodialysis step is usually 5 ° C. or higher and 70 ° C. or lower, and preferably 20 ° C. or higher and 50 ° C. or lower.
　以上のように、水分解電気透析工程により、Ｄ－乳酸塩を分解しＤ－乳酸とアルカリを分離し、Ｄ－乳酸を回収することができる。なお、分離したアルカリは、Ｄ－乳酸等の有機酸発酵での中和剤として再利用することができる。 As described above, D-lactic acid can be decomposed and D-lactic acid and alkali can be separated by the water-splitting electrodialysis step to recover D-lactic acid. The separated alkali can be reused as a neutralizing agent in organic acid fermentation such as D-lactic acid.
　上記水分解電気透析工程によって、純度の高いＤ－乳酸を得ることができる。 D-lactic acid with high purity can be obtained by the water-splitting electrodialysis step.
　濃縮及び精製工程では、例えば、タンパク質や副生有機酸など不純物の除去には活性炭処理やＮＦ膜処理、イオン性物質の除去にはイオン交換カラムや陰イオン／陽イオン交換膜電気透析、蒸発による濃縮、精製及び乳酸の回収には、脱塩した後直接蒸留する方法、水蒸気蒸留する方法、ラクチドなどを形成させ蒸留する方法、アルコールと触媒を加えエステル化した後蒸留する方法、有機溶媒中に抽出する方法、クロマトカラムで分離する方法、イオン交換カラムで分離する方法、陰イオン／陽イオン交換膜電気透析により濃縮及び回収する方法、晶析により結晶化する方法が挙げられる。また、これらの方法を任意の順序で適宜組み合わせてもよい。 In the concentration and purification process, for example, activated carbon treatment or NF membrane treatment is used to remove impurities such as proteins and by-product organic acids, and ion exchange columns, anion / cation exchange membrane electrodialysis, and evaporation are used to remove ionic substances. Concentration, purification and recovery of lactic acid include desalting and direct distillation, steam distillation, lactide formation and distillation, alcohol and catalyst esterification followed by distillation, in organic solvents Examples of the method include extraction, chromatography column separation, ion exchange column separation, anion / cation exchange membrane electrodialysis concentration and recovery, and crystallization by crystallization. Moreover, you may combine these methods suitably in arbitrary orders.
　例えば、脱塩後に得られたＤ－乳酸溶液に対し、活性炭処理を行うことが好ましい。これにより、着色成分を除去し、ポリマー化の用途に適したＤ－乳酸を得ることができる。活性炭処理は、例えば、Ｄ－乳酸溶液の重量に対し、０．２重量％以上２重量％以下の活性炭を添加し、必要に応じて攪拌した後、濾過により活性炭を除去することで実行することができる。活性炭処理後のＤ－乳酸溶液の４００ｎｍの光透過率は、水の４００ｎｍの光透過率を１００％としたとき、８０％以上９９％以下とすることができる。 For example, it is preferable to perform activated carbon treatment on the D-lactic acid solution obtained after desalting. As a result, the coloring component can be removed, and D-lactic acid suitable for polymerizing use can be obtained. The activated carbon treatment is performed by adding, for example, 0.2% by weight or more and 2% by weight or less of activated carbon to the weight of the D-lactic acid solution, stirring the mixture as necessary, and then removing the activated carbon by filtration. Can do. The light transmittance at 400 nm of the D-lactic acid solution after the activated carbon treatment can be 80% or more and 99% or less when the light transmittance at 400 nm of water is 100%.
　また、例えば、脱塩後に得られたＤ－乳酸溶液を回分又は連続で蒸留する際、水を加え加水分解しながら蒸留することが好ましい。これにより、缶内液の重合を抑制でき、蒸留収率が向上する。重合度ｎは１以上４以下であることが好ましく、１以上２以下であるとさらに好ましく、１以上１．５以下であるとより一層好ましい。このとき蒸留収率は７０重量％以上９９．５重量％以下にすることができる。ここでいう蒸留収率とは、蒸留工程での乳酸成分の回収率であり、乳酸成分とは、乳酸と乳酸オリゴマーのことである。
　また、例えば、蒸発濃縮工程で重合が進行した場合は、蒸留の前に加水分解を行い、乳酸の重合度ｎを下げておくことも蒸留収率の向上には有効である。 For example, when the D-lactic acid solution obtained after desalting is distilled batchwise or continuously, it is preferable to distill while adding water to perform hydrolysis. Thereby, superposition | polymerization of a can internal liquid can be suppressed and a distillation yield improves. The polymerization degree n is preferably 1 or more and 4 or less, more preferably 1 or more and 2 or less, and even more preferably 1 or more and 1.5 or less. At this time, the distillation yield can be 70 wt% or more and 99.5 wt% or less. The distillation yield here is the recovery rate of the lactic acid component in the distillation step, and the lactic acid component is lactic acid and a lactic acid oligomer.
For example, when the polymerization proceeds in the evaporation and concentration step, it is also effective to improve the distillation yield by performing hydrolysis before the distillation to lower the polymerization degree n of lactic acid.
　ここで、本実施形態に係るポリマーの生産方法は、本実施形態に係るＤ－乳酸の生産方法により得られたＤ－乳酸を用いて重合反応を行うものである。以下、詳説する。 Here, the polymer production method according to the present embodiment is a polymerization reaction using D-lactic acid obtained by the D-lactic acid production method according to the present embodiment. The details will be described below.
　本実施形態に係るＤ－乳酸の生産方法において得られたＤ－乳酸を原料としてポリマー重合することにより、着色が少なく、高分子量のポリマー（ポリ乳酸や乳酸共重合体）を得ることができる。ここで言うポリ乳酸は、Ｄ－乳酸とＬ－乳酸とのステレオコンプレックス型重合体とＤ－乳酸の単独重合体を含む。Ｄ－乳酸とＬ－乳酸とのステレオコンプレックス型重合体の原料の一つであるＬ－乳酸については、公知のものが好適に使用できる。Ｌ－乳酸の製法は特に限定されることはなく、例えば発酵法や有機合成法などが挙げられる。これらポリ乳酸や乳酸共重合体は、様々な成形加工方法、例えば、射出成形、フィルム成形、シート成形、押出し成形、ブロー成形、発砲成形などにより、加工され各種用途に用いられる。用途としては、例えば、生分解性プラスチックの特性や耐熱性などを活かし、包装容器、緩衝材、各種電子・電気機器、ＯＡ機器、車両部品、機械部品、その他農業資材、漁業資材、搬送容器、遊戯具および雑貨などが挙げられる。 Polymer polymerization using D-lactic acid obtained in the method for producing D-lactic acid according to the present embodiment as a raw material makes it possible to obtain a high molecular weight polymer (polylactic acid or lactic acid copolymer) with little coloration. The polylactic acid referred to here includes a stereocomplex polymer of D-lactic acid and L-lactic acid and a homopolymer of D-lactic acid. As L-lactic acid, which is one of the raw materials for the stereocomplex polymer of D-lactic acid and L-lactic acid, known ones can be preferably used. The method for producing L-lactic acid is not particularly limited, and examples thereof include fermentation methods and organic synthesis methods. These polylactic acid and lactic acid copolymer are processed by various molding methods, for example, injection molding, film molding, sheet molding, extrusion molding, blow molding, foam molding, etc., and are used for various applications. Applications include, for example, the properties and heat resistance of biodegradable plastics, packaging containers, cushioning materials, various electronic and electrical equipment, OA equipment, vehicle parts, machine parts, other agricultural materials, fishery materials, transport containers, Examples include play equipment and miscellaneous goods.
　本実施形態に係るポリ乳酸の生産方法において、重合の方法は特に限定されるものではなく、公知の方法を採用することができる。例えば、乳酸ラクチドを経由する開環重合、乳酸や乳酸エステルを直接重合する縮重合、乳酸や乳酸エステルを直接重合したオリゴマーをポリイソシアネート等によりアミド結合やウレタン結合などを介するなどして鎖延長される重合方法などが挙げられる。また、重合の形式としては、固相重合や溶融重合などが挙げられる。
　本実施形態に係る乳酸共重合体の生産方法において、配列順序や重合の方法は特に限定されるものではなく、公知の方法を採用することができる。例えば、配列順序では、ランダム共重合、交互共重合、ブロック共重合、グラフト共重合などが挙げられ、また、重合の方法では、ラジカル共重合、イオン共重合、共縮合などが挙げられる。 In the method for producing polylactic acid according to this embodiment, the polymerization method is not particularly limited, and a known method can be adopted. For example, ring-opening polymerization via lactic acid lactide, condensation polymerization in which lactic acid or lactic acid ester is directly polymerized, and oligomer in which lactic acid or lactic acid ester is directly polymerized are chain-extended by amide bond or urethane bond with polyisocyanate. Polymerization methods and the like. Examples of the polymerization method include solid phase polymerization and melt polymerization.
In the method for producing a lactic acid copolymer according to the present embodiment, the arrangement order and the polymerization method are not particularly limited, and a known method can be employed. For example, the sequence order includes random copolymerization, alternating copolymerization, block copolymerization, graft copolymerization, and the like, and the polymerization method includes radical copolymerization, ionic copolymerization, cocondensation, and the like.
　このように本実施形態に係る方法で得られるＤ－乳酸は、ポリ乳酸の製造に用いることができる。得られるポリ乳酸の重量平均分子量（Ｍｗ）は、５万以上４０万以下であることが好ましく、８万以上３０万以下であると更に好ましい。 Thus, D-lactic acid obtained by the method according to this embodiment can be used for the production of polylactic acid. The weight average molecular weight (Mw) of the obtained polylactic acid is preferably 50,000 or more and 400,000 or less, and more preferably 80,000 or more and 300,000 or less.
　ここで、ポリマーの分子量分布は、通常、Ｍｗ／Ｍｎで表されるが、ポリ乳酸などの重縮合反応では、一般的に、反応が完結に近づくと分子量分布は２に近くなる（岡村誠三著　高分子化学序論　第２版 ｐ．２１０より）。分子量分布は、ポリマーの流動性、機械的性質に大きく影響し、分子量分布が広ければ、すなわち、Ｍｗ／Ｍｎの値が大きくなれば、非ニュートン特性が大きくなり流動性が向上するため、成形加工の工程において樹脂を成形しやすくなることが知られている。本実施形態に係る方法で得られたＤ－乳酸から製造されたポリ乳酸や乳酸共重合体は、一般的な重縮合系ポリマーより分子量分布が広く、その範囲は、好ましくは３以上２０以下、より好ましくは５以上１５以下、更に好ましくは８以上１２以下とすることができ、成形しやすいとこが特性である。更に、本実施形態に係る方法で得られるＤ－乳酸は、多量の活性炭処理や煩雑な精製工程を経なくても分子量分布の高いポリ乳酸や乳酸共重合体を製造することができる。例えば、活性炭処理においては、０．２重量％以上２重量％以下の少量の活性炭で処理しても分子量分布の広いポリ乳酸や乳酸共重合体を製造することができる。 Here, the molecular weight distribution of the polymer is usually expressed by Mw / Mn. However, in the polycondensation reaction such as polylactic acid, generally, the molecular weight distribution approaches 2 when the reaction approaches completion (Seizo Okamura) Introduction to Polymer Chemistry (2nd edition, p.210). The molecular weight distribution greatly affects the fluidity and mechanical properties of the polymer. If the molecular weight distribution is wide, that is, if the value of Mw / Mn increases, the non-Newtonian characteristics increase and the fluidity improves. It is known that the resin can be easily molded in this process. The polylactic acid or lactic acid copolymer produced from D-lactic acid obtained by the method according to this embodiment has a wider molecular weight distribution than a general polycondensation polymer, and the range is preferably 3 or more and 20 or less, More preferably, it is 5 or more and 15 or less, and further preferably 8 or more and 12 or less. Furthermore, D-lactic acid obtained by the method according to the present embodiment can produce polylactic acid and lactic acid copolymer having a high molecular weight distribution without going through a large amount of activated carbon treatment and complicated purification steps. For example, in the activated carbon treatment, a polylactic acid or a lactic acid copolymer having a wide molecular weight distribution can be produced even by treating with a small amount of activated carbon of 0.2 wt% or more and 2 wt% or less.
　また、本実施形態に係る方法で得られるＤ－乳酸から製造されたポリ乳酸や乳酸共重合体の着色度（ＹＩ値）は、好ましくはＹＩ値１０以下、より好ましくはＹＩ値５以下とすることができる。
　なお、ここでいうＹＩ値とは、理想上の白色（完全拡散反射面）を０として、この白色からの距離を示すものであり、白色から黄色方向の色差はプラス、青方向の色差はマイナスで表される。従って、マイナスの値が大きくなると青みが増し、プラスの値が大きくなると黄みが増し、何れも色相悪化の原因となる。
　色の三原色である赤、緑、青のうち、Ｘは主に赤を感じる値、Ｙは緑を感じる値、Ｚは青を感じる値とすると、ＹＩ値は、色差計（例えば、島津製作所紫外－可視分光計ＵＶ－２４００ＰＣ）を用いて三刺激値Ｘ，Ｙ，Ｚを測定し、式３により算出される。
ＹＩ値＝１００×（１．２８Ｘ－１．０６Ｚ）／Ｙ　　（式３） Further, the coloration degree (YI value) of polylactic acid or lactic acid copolymer produced from D-lactic acid obtained by the method according to this embodiment is preferably YI value of 10 or less, more preferably YI value of 5 or less. be able to.
Note that the YI value here indicates the distance from this white, assuming that the ideal white color (complete diffuse reflection surface) is 0, and the color difference from white to yellow is plus, and the color difference in blue is minus. It is represented by Therefore, when the negative value is increased, the bluish color is increased, and when the positive value is increased, the yellow color is increased, which causes deterioration of the hue.
Of the three primary colors, red, green, and blue, X is a value that feels red, Y is a value that feels green, and Z is a value that feels blue. -The tristimulus values X, Y, Z are measured using a visible spectrometer UV-2400PC) and calculated according to equation 3.
YI value = 100 × (1.28X−1.06Z) / Y (Formula 3)
　本実施形態に係る方法によれば、色相や分子量などＤ－乳酸ポリマーに求められる品質に、見合うようなＤ－乳酸ポリマーへと重合可能なＤ－乳酸モノマーを、より安価に、効率よく生産する方法を提供することができる。また、工業的な大量生産において、しばしば問題となるような廃棄物、廃塩をより削減したＤ－乳酸モノマー製造法を提供することができる。 According to the method according to the present embodiment, a D-lactic acid monomer that can be polymerized into a D-lactic acid polymer suitable for the quality required for the D-lactic acid polymer such as hue and molecular weight is produced at low cost and efficiently. A method can be provided. In addition, it is possible to provide a method for producing D-lactic acid monomer in which waste and waste salts, which are often problematic in industrial mass production, are further reduced.
　以上、本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。 As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above can also be employ | adopted.
　以下、本発明を実施例にて詳細に説明する。しかしながら、本発明はそれらに何ら限定されるものではない。特に断らない限り、得られたＤ－乳酸の光学純度はいずれも１００％ｅｅだった。光学純度はＨＰＬＣで定法に従って測定した（カラム：ＳＵＭＩＣＨＩＲＡＬ ＯＡ－５０００（住化分析センター社製）、溶離液：イソプロピルアルコール １０ｇ/Ｌおよび硫酸銅（ＩＩ）無水　３１９．２ｍｇ/Ｌよりなる水溶液、検出波長：２５４ｎｍ）。 Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them. Unless otherwise specified, the optical purity of the obtained D-lactic acid was 100% ee. The optical purity was measured by HPLC according to a conventional method (column: SUMICHILARＩＲOA-5000 (manufactured by Sumika Chemical Analysis)), eluent: aqueous solution consisting of 10 g / L of isopropyl alcohol イ ソ プ ロ ピ ル and 319.2 mg / L of anhydrous copper (II) sulfate, detection Wavelength: 254 nm).
（製造例１）ＷＯ２００５／０３３３２４の実施例２３に記載の方法と同様に大腸菌株、ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株／ＧＡＰｌｄｈＡゲノム挿入株を作製した。
　具体的には、以下の方法により、大腸菌株、ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株／ＧＡＰｌｄｈＡゲノム挿入株を作製した。 (Production Example 1) An E. coli strain, MG1655ΔpflΔdldΔmdhΔasp strain / GAPldhA genome insertion strain was prepared in the same manner as described in Example 23 of WO2005 / 033324.
Specifically, an E. coli strain, MG1655ΔpflΔdldΔmdhΔasp strain / GAPldhA genome insertion strain was prepared by the following method.
＜製造１：エシェリヒア・コリｄｌｄ遺伝子の近傍領域のクローニング＞
　エシェリヒア・コリのゲノムＤＮＡの全塩基配列は公知であり（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ０００９６）、エシェリヒア・コリのＦＡＤ依存性Ｄ－乳酸デヒドロゲナーゼをコードする遺伝子（以下ｄｌｄと略すことがある）の塩基配列も報告されている（Ｇｅｎｂａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｍ１００３８）。
　エシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡを、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（ＪｏｈｎＷｉｌｅｙ　&　Ｓｏｎｓ）記載の方法により調製し、得られたゲノムＤＮＡと、ＭＧ１６５５株由来ゲノムＤＮＡのｄｌｄ遺伝子近傍領域の遺伝子情報に基づいて作製された、ＣＡＡＣＡＣＣＡＡＧＣＴＴＴＣＧＣＧ（配列番号１）とＴＴＣＣＡＣＴＣＣＴＴＧＴＧＧＴＧＧＣ（配列番号２）、ＡＡＣＴＧＣＡＧＡＡＡＴＴＡＣＧＧＡＴＧＧＣＡＧＡＧ（配列番号３）とＴＧＴＴＣＴＡＧＡＡＡＧＴＴＣＴＴＴＧＡＣ（配列番号４）を用いてＰＣＲを行った。得られたフラグメントをそれぞれ、制限酵素ＨｉｎｄＩＩＩとＰｓｔＩ、ＰｓｔＩとＸｂａＩで消化することにより、それぞれ約１１４０ｂｐのフラグメントを得た。このフラグメントを温度感受性プラスミドｐＴＨ１８ｃｓ１（Ｈａｓｈｉｍｏｔｏ－Ｇｏｔｏｈ，　Ｔ．，　ｅｔ．ａｌ．，　Ｇｅｎｅ，　Ｖｏｌ．２４１（１），　ｐｐ１８５－１９１　（２０００））をＨｉｎｄＩＩＩ、ＸｂａＩで消化して得られるフラグメントと混合し、リガーゼを用いて結合した後、ＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で培養し、生育する形質転換体を得た。得られたコロニーをクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地で３０℃で一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的のｄｌｄ遺伝子近傍領域の配列が挿入されていることを確認した。本プラスミドをｐＴＨΔｄｌｄと命名した。 <Production 1: Cloning of the vicinity region of the Escherichia coli dld gene>
The entire base sequence of the genomic DNA of Escherichia coli is known (GenBank accession number U00096), and the base sequence of a gene encoding the FAD-dependent D-lactate dehydrogenase of Escherichia coli (hereinafter sometimes abbreviated as dld) is also reported. (Genbank accession number M10038).
The genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), and the obtained genomic DNA and the gene information of the region near the dld gene of the genomic DNA derived from MG1655 strain were used. PCR was performed using the prepared CAACACCAAGCTTTCGCG (SEQ ID NO: 1) and TTCCACTCCCTTGTGGTGCC (SEQ ID NO: 2), AACTGCAGAAATTACGGATGGCAGAG (SEQ ID NO: 3) and TGTTCTAGAAAGTTCTTTGAC (SEQ ID NO: 4). The obtained fragments were digested with restriction enzymes HindIII and PstI, and PstI and XbaI, respectively, to obtain fragments of about 1140 bp. This fragment was mixed with a fragment obtained by digesting the temperature sensitive plasmid pTH18cs1 (Hashimoto-Gotoh, T., et.al., Gene, Vol. 241 (1), pp185-191 (2000)) with HindIII and XbaI. Ligation using ligase, transformation into DH5α competent cell (DNA-903, Toyobo Co., Ltd.), growth on LB agar plate containing 10 μg / ml chloramphenicol, and transformation to grow Got the body. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of chloramphenicol, and the plasmid was recovered from the obtained cells. The inserted sequence into the plasmid was confirmed by sequencing, and it was confirmed that the sequence in the region near the target dld gene was inserted. This plasmid was designated as pTHΔdld.
＜製造２：エシェリヒア・コリＭＧ１６５５株ｄｌｄ遺伝子欠失株の作製＞
　上記製造１で得たプラスミドｐＴＨΔｄｌｄをＭＧ１６５５株に形質転換し、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で、生育する形質転換体を得た。得られた形質転換体をクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地に接種し、３０℃で一晩培養した。次にこれらの培養菌体をクロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに塗布し、４２℃で生育するコロニーを得た。
　さらにもう一度、４２℃で生育するシングルコロニーを得る操作を繰り返した。
　次に上記クローンを、ＬＢ液体培地（３ｍｌ／試験管）に接種し、３０℃で３～４時間、振とう培養した。これをシングルコロニーが得られるように適当に希釈（１０－２～１０－６程度）し、ＬＢ寒天プレートに塗布し、４２℃で一晩培養し、コロニーを得た。出現したコロニーの中から無作為に１００コロニーをピックアップしてそれぞれをＬＢ寒天プレートとクロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに生育させ、ＬＢ寒天プレートにのみ生育するクロラムフェニコール感受性のクローンを選んだ。さらにはこれらの目的クローンの染色体ＤＮＡからＰＣＲにより、ｄｌｄ遺伝子領域が欠失している株を選抜し、以上を満足するクローンをｄｌｄ欠失株とし、得られた株をＭＧ１６５５Δｄｌｄ株と命名した。
　なおエシェリヒア・コリＭＧ１６５５株は細胞・微生物・遺伝子バンクであるアメリカンタイプカルチャーコレクションより入手することができる。 <Production 2: Production of Escherichia coli MG1655 strain dld gene deletion strain>
The plasmid pTHΔdld obtained in Production 1 was transformed into the MG1655 strain, and a transformant that grew on an LB agar plate containing 10 μg / ml of chloramphenicol at 30 ° C. was obtained. The obtained transformant was inoculated into an LB liquid medium containing 10 μg / ml of chloramphenicol and cultured at 30 ° C. overnight. Next, these cultured cells were applied to an LB agar plate containing 10 μg / ml of chloramphenicol to obtain colonies that grew at 42 ° C.
Further, the operation of obtaining a single colony growing at 42 ° C. was repeated once more.
Next, the clones were inoculated into LB liquid medium (3 ml / tube) and cultured with shaking at 30 ° C. for 3 to 4 hours. This was appropriately diluted (about 10 −2 to 10 −6 ) so that a single colony was obtained, applied to an LB agar plate, and cultured overnight at 42 ° C. to obtain a colony. 100 colonies were picked up randomly from the colonies that appeared, and each was grown on an LB agar plate and an LB agar plate containing 10 μg / ml chloramphenicol. I picked a clone. Furthermore, a strain lacking the dld gene region was selected from the chromosomal DNA of these target clones by PCR, a clone satisfying the above was designated as a dld-deficient strain, and the resulting strain was designated as MG1655Δdld strain.
Escherichia coli MG1655 strain can be obtained from the American Type Culture Collection, which is a cell / microorganism / gene bank.
＜製造３：エシェリヒア・コリｐｆｌ遺伝子の近傍領域のクローニング＞
　エシェリヒア・コリのゲノムＤＮＡの全塩基配列は公知であり（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ０００９６），エシェリヒア・コリのピルベートホルメートリアーゼ（以下ｐｆｌと呼ぶことがある）をコードする遺伝子の塩基配列も報告されている（Ｇｅｎｂａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＥ０００１９２）。ｐｆｌをコードする遺伝子の塩基配列近傍領域をクローニングするため、配列番号５、６、７及び８に示すオリゴヌクレオチドプライマーを４種合成した。配列番号６、７のプライマーは５´末端側にＳｐｈＩ認識部位を有している。
　エシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡを、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（ＪｏｈｎＷｉｌｅｙ　&　Ｓｏｎｓ）記載の方法により調製し、得られたゲノムＤＮＡと、配列番号５の塩基配列を有するプライマーと配列番号６の塩基配列を有するプライマー、配列番号７の塩基配列を有するプライマーと配列番号８の塩基配列を有するプライマーの組み合わせで、通常の条件でＰＣＲを行うことにより約１．８ｋｂｐ（以下ｐｆｌＢ－Ｌ断片と呼ぶことがある）及び、約１．３ｋｂｐ（以下ｐｆｌＢ－Ｒ断片と呼ぶことがある）のＤＮＡ断片を増幅した。このＤＮＡ断片をアガロース電気泳動にて分離、回収し、ｐｆｌＢ－Ｌ断片をＨｉｎｄＩＩＩ及びＳｐｈＩで、ｐｆｌＢ－Ｒ断片をＳｐｈＩ及びＰｓｔＩでそれぞれ消化した。この消化断片２種と、温度感受性プラスミドｐＴＨ１８ｃｓ１（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＢ０１９６１０）（Ｈａｓｈｉｍｏｔｏ－Ｇｏｔｏｈ，　Ｔ．，　ｅｔ．ａｌ．，　Ｇｅｎｅ，　Ｖｏｌ．２４１（１），　ｐｐ１８５－１９１　（２０００））のＨｉｎｄＩＩＩ及びＰｓｔＩ消化物とをＴ４ＤＮＡリガーゼで反応した後、エシェリヒア・コリＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換して、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で培養し、生育する形質転換体を得た。得られたコロニーをクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地で３０℃で一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的とするｐｆｌＢをコードする遺伝子の５´上流近傍断片と３´下流近傍断片の２つの断片が挿入されていることを確認した。本プラスミドをｐＴＨΔｐｆｌと命名した。 <Production 3: Cloning of the vicinity region of the Escherichia coli pfl gene>
The entire nucleotide sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the nucleotide sequence of a gene encoding Escherichia coli pyruvate formate lyase (hereinafter sometimes referred to as pfl) has also been reported. (Genbank accession number AE000192). In order to clone the region near the base sequence of the gene encoding pfl, four oligonucleotide primers shown in SEQ ID NOs: 5, 6, 7 and 8 were synthesized. The primers of SEQ ID NOs: 6 and 7 have a SphI recognition site on the 5 ′ end side.
A genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), the obtained genomic DNA, a primer having the base sequence of SEQ ID NO: 5, and the base sequence of SEQ ID NO: 6 And a primer having the nucleotide sequence of SEQ ID NO: 7 and a primer having the nucleotide sequence of SEQ ID NO: 8, and performing PCR under normal conditions, it is about 1.8 kbp (hereinafter referred to as pflB-L fragment) And a DNA fragment of about 1.3 kbp (hereinafter sometimes referred to as a pflB-R fragment) was amplified. This DNA fragment was separated and collected by agarose electrophoresis, and the pflB-L fragment was digested with HindIII and SphI, and the pflB-R fragment was digested with SphI and PstI, respectively. Two kinds of the digested fragments and HindIII and PstI of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610) (Hashimoto-Gotoh, T., et., Gene, Vol. 241 (1), pp185-191 (2000)) The digested product was reacted with T4 DNA ligase, transformed into Escherichia coli DH5α competent cell (Toyobo Co., Ltd., DNA-903), and cultured at 30 ° C. on an LB agar plate containing 10 μg / ml of chloramphenicol. Thus, transformants that grow were obtained. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of chloramphenicol, and the plasmid was recovered from the obtained cells. The insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target pflB were inserted. This plasmid was named pTHΔpfl.
＜製造４：エシェリヒア・コリＭＧ１６５５ｐｆｌ、ｄｌｄ遺伝子欠失株作製＞
　上記製造３で得たプラスミドｐＴＨΔｐｆｌを、ＭＧ１６５５Δｄｌｄ株に形質転換し、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で生育する形質転換体を得た。得られた形質転換体を上記製造２と同様に培養、選抜を行い、最終的に、ｐｆｌ遺伝子が欠失している株の選抜を行うことにより、ｐｆｌ遺伝子が破壊されたＭＧ１６５５Δｄｌｄ株を取得し、本株をＭＧ１６５５ΔｐｆｌΔｄｌｄ株と命名した。 <Production 4: Escherichia coli MG1655 pfl, dld gene deletion strain preparation>
The plasmid pTHΔpfl obtained in Production 3 was transformed into the MG1655Δdld strain to obtain a transformant that grew at 30 ° C. on an LB agar plate containing 10 μg / ml of chloramphenicol. The obtained transformant was cultured and selected in the same manner as in Production 2 above, and finally a strain lacking the pfl gene was selected to obtain the MG1655Δdld strain in which the pfl gene was disrupted. This strain was designated as MG1655ΔpflΔdld strain.
＜製造５：エシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈ株の作製＞
　エシェリヒア・コリのゲノムＤＮＡの全塩基配列は公知であり（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ０００９６）、エシェリヒア・コリのｍｄｈ遺伝子の塩基配列も報告されている（Ｇｅｎｂａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＥ０００４０３）。ｍｄｈをコードする遺伝子の塩基配列近傍領域をクローニングするため、配列番号９、１０、１１及び１２に示すオリゴヌクレオチドプライマーを４種合成した。配列番号９の塩基配列を有するプライマーは５´末端側にＫｐｎＩ認識部位を、配列番号１０、１１の塩基配列を有するプライマーは５´末端側にＢａｍＨＩ認識部位を、配列番号１２の塩基配列を有するプライマーは５´末端側にＸｂａＩ認識部位をそれぞれ有している。
　エシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡを、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（ＪｏｈｎＷｉｌｅｙ　&　Ｓｏｎｓ）記載の方法により調製し、得られたゲノムＤＮＡ、配列番号９と配列番号１０、配列番号１１と配列番号１２の組み合わせで、通常の条件でＰＣＲを行うことにより約８００ｂｐ（以下ｍｄｈ－Ｌ断片と呼ぶことがある）及び、約１，０００ｂｐ（以下ｍｄｈ－Ｒ断片と呼ぶことがある）のＤＮＡ断片を増幅した。このＤＮＡ断片をアガロース電気泳動にて分離、回収し、ｍｄｈ－Ｌ断片をＫｐｎＩ及びＢａｍＨＩで、ｍｄｈ－Ｒ断片をＢａｍＨＩ及びＸｂａＩでそれぞれ消化した。この消化断片２種と、温度感受性プラスミドｐＴＨ１８ｃｓ１（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＢ０１９６１０）（Ｈａｓｈｉｍｏｔｏ－Ｇｏｔｏｈ，　Ｔ．，　ｅｔ．ａｌ．，　Ｇｅｎｅ，　Ｖｏｌ．２４１（１），　ｐｐ１８５－１９１　（２０００））のＫｐｎＩ及びＸｂａＩ消化物とをＴ４ＤＮＡリガーゼで反応した後、エシェリヒア・コリＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換して、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で培養し、生育する形質転換体を得た。得られたコロニーをクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地で３０℃で一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的とするｍｄｈをコードする遺伝子の５´上流近傍断片と３´下流近傍断片の２つの断片が挿入されていることを確認した。本プラスミドをｐＴＨΔｍｄｈと命名した。
　プラスミドｐＴＨΔｍｄｈをエシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄ株に形質転換し、上記製造２と同様に培養、選抜を行い、最終的に、ｍｄｈ遺伝子が欠失している株の選抜を行うことにより、ｍｄｈ遺伝子が破壊されたＭＧ１６５５ΔｐｆｌΔｄｌｄ株を取得した。本株をＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈ株と命名した。 <Production 5: Production of Escherichia coli MG1655ΔpflΔdldΔmdh strain>
The entire base sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the base sequence of the Escherichia coli mdh gene has also been reported (Genbank accession number AE000403). In order to clone the region near the base sequence of the gene encoding mdh, four types of oligonucleotide primers shown in SEQ ID NOs: 9, 10, 11, and 12 were synthesized. The primer having the base sequence of SEQ ID NO: 9 has a KpnI recognition site on the 5 ′ end side, the primer having the base sequences of SEQ ID NOS: 10 and 11 has a BamHI recognition site on the 5 ′ end side, and the base sequence of SEQ ID NO: 12. Each primer has an XbaI recognition site on the 5 ′ end side.
Genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons), and the resulting genomic DNA, SEQ ID NO: 9 and SEQ ID NO: 10, and combinations of SEQ ID NO: 11 and SEQ ID NO: 12 Thus, by performing PCR under normal conditions, DNA fragments of about 800 bp (hereinafter sometimes referred to as mdh-L fragment) and about 1,000 bp (hereinafter also referred to as mdh-R fragment) were amplified. This DNA fragment was separated and collected by agarose electrophoresis, and the mdh-L fragment was digested with KpnI and BamHI, and the mdh-R fragment was digested with BamHI and XbaI, respectively. Two of these digested fragments and KpnI and XbaI of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610) (Hashimoto-Gotoh, T., et., Gene, Vol. 241 (1), pp185-191 (2000)). The digested product was reacted with T4 DNA ligase, transformed into Escherichia coli DH5α competent cell (Toyobo Co., Ltd., DNA-903), and cultured at 30 ° C. on an LB agar plate containing 10 μg / ml of chloramphenicol. Thus, transformants that grow were obtained. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of chloramphenicol, and the plasmid was recovered from the obtained cells. The insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target mdh were inserted. This plasmid was named pTHΔmdh.
The plasmid pTHΔmdh is transformed into the Escherichia coli MG1655ΔpflΔdld strain, cultured and selected in the same manner as in Production 2 above, and finally the mdh gene is disrupted by selecting a strain lacking the mdh gene. MG1655ΔpflΔdld strain was obtained. This strain was designated as MG1655ΔpflΔdldΔmdh strain.
＜製造６：エシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株の作製＞
　エシェリヒア・コリのゲノムＤＮＡの全塩基配列は公知であり（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ０００９６）、エシェリヒア・コリのａｓｐＡ遺伝子の塩基配列も報告されている（Ｇｅｎｂａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＥ０００４８６）。ａｓｐＡをコードする遺伝子の塩基配列近傍領域をクローニングするため、配列番号１３、１４、１５及び１６に示すオリゴヌクレオチドプライマーを４種合成した。
　エシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡと、配列番号１３と配列番号１４、配列番号１５と配列番号１６の組み合わせで、通常の条件でＰＣＲを行うことにより約９１０ｂｐ（以下ａｓｐＡＬ断片と呼ぶことがある）及び、約１，１００ｂｐ（以下ａｓｐＡ－Ｒ断片と呼ぶことがある）のＤＮＡ断片を増幅した。このＤＮＡ断片をアガロース電気泳動にて分離、回収した。配列番号１４と配列番号１５は相補的な領域を含むことから、ａｓｐＡ－Ｌ断片とａｓｐＡ－Ｒ断片を混合し、プライマーを用いず、ＰＣＲ反応を行うことで、ａｓｐＡ－Ｌ断片とａｓｐＡ－Ｒ断片を連結することが可能である。これに基づき、ａｓｐＡ－Ｌ断片とａｓｐＡ－Ｒ断片を連結し、アガロース電気泳動にて分離、回収した。得られたＤＮＡ断片をＤＮＡ　Ｂｌｕｎｔｉｎｇ　Ｋｉｔ（宝バイオ）で末端を平滑化した後、Ｔ４ポリヌクレオチドキナーゼを用いて定法にて５´末端をリン酸化した。一方温度感受性プラスミドｐＴＨ１８ｃｓ１は、ＳｍａＩ消化後、アルカリフォスファターゼにて脱リン酸化処理を行った。上記のリン酸化した２種類の断片と、脱リン酸化したプラスミドをＴ４ＤＮＡリガーゼで反応した後、エシェリヒア・コリＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換して、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートで３０℃で培養し、生育する形質転換体を得た。得られたコロニーをクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地で３０℃で一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的とするａｓｐＡをコードする遺伝子の５´上流近傍断片と３´下流近傍断片の２つの断片が挿入されていることを確認した。本プラスミドをｐＴＨΔａｓｐと命名した。
プラスミドｐＴＨΔａｓｐをエシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈ株に形質転換し、上記製造２と同様に培養、選抜を行い、最終的に、ａｓｐＡ遺伝子が欠失している株の選抜を行うことにより、ａｓｐＡ遺伝子が破壊されたＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈ株を取得した。本株をＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株と命名した。 <Production 6: Production of Escherichia coli MG1655ΔpflΔdldΔmdhΔasp strain>
The entire base sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the base sequence of the aspA gene of Escherichia coli has also been reported (Genbank accession number AE000486). In order to clone the region near the base sequence of the gene encoding aspA, four oligonucleotide primers shown in SEQ ID NOs: 13, 14, 15 and 16 were synthesized.
About 910 bp (hereinafter sometimes referred to as an aspAL fragment) by performing PCR under the normal conditions with a combination of the genomic DNA of Escherichia coli MG1655 strain, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 15 and SEQ ID NO: 16. A DNA fragment of about 1,100 bp (hereinafter sometimes referred to as an aspA-R fragment) was amplified. This DNA fragment was separated and collected by agarose electrophoresis. Since SEQ ID NO: 14 and SEQ ID NO: 15 contain complementary regions, the aspA-L fragment and the aspA-R fragment are mixed with the aspA-L fragment and subjected to a PCR reaction without using a primer. It is possible to link the fragments. Based on this, the aspA-L fragment and the aspA-R fragment were ligated and separated and recovered by agarose electrophoresis. The resulting DNA fragment was blunted with DNA Blunting Kit (Takara Bio), and then phosphorylated at the 5 'end using T4 polynucleotide kinase by a conventional method. On the other hand, the temperature sensitive plasmid pTH18cs1 was digested with SmaI and then dephosphorylated with alkaline phosphatase. The above two phosphorylated fragments and the dephosphorylated plasmid were reacted with T4 DNA ligase, and transformed into Escherichia coli DH5α competent cell (Toyobo Co., Ltd. DNA-903), and chloramphenicol. Culture was performed at 30 ° C. on an LB agar plate containing 10 μg / ml to obtain a transformant that grew. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of chloramphenicol, and the plasmid was recovered from the obtained cells. The insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that two fragments, a 5 ′ upstream vicinity fragment and a 3 ′ downstream vicinity fragment, of the gene encoding the target aspA were inserted. This plasmid was designated as pTHΔasp.
The plasmid pTHΔasp is transformed into the Escherichia coli MG1655ΔpflΔdldΔmdh strain, cultured and selected in the same manner as in Production 2 above, and finally, the strain lacking the aspA gene is selected to destroy the aspA gene. MG1655ΔpflΔdldΔmdh strain was obtained. This strain was designated as MG1655ΔpflΔdldΔmdhΔasp strain.
＜製造７：エシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株のゲノム上ｌｄｈＡプロモーターのＧＡＰＤＨプロモーターへの置換＞
　エシェリヒア・コリのｌｄｈＡ遺伝子の塩基配列はすでに報告されている（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ３６９２８）。グリセルデヒド３－リン酸デヒドロゲナーゼ（ＧＡＰＤＨ）プロモーターを取得するためエシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡをテンプレートに用いてＡＡＣＧＡＡＴＴＣＴＣＧＣＡＡＴＧＡＴＴＧＡＣＡＣＧＡＴＴＣ（配列番号１７）、及びＡＣＡＧＡＡＴＴＣＧＣＴＡＴＴＴＧＴＴＡＧＴＧＡＡＴＡＡＡＡＧＧ（配列番号１８）によりＰＣＲ法で増幅し、得られたＤＮＡフラグメントを制限酵素ＥｃｏＲＩで消化することで約１００ｂｐのＧＡＰＤＨプロモーターをコードするフラグメントを得た。
　また、エシェリヒア・コリのゲノムＤＮＡの全塩基配列は公知であり（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ０００９６）、エシェリヒア・コリのｌｄｈＡ遺伝子の塩基配列も報告されている（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｕ３６９２８）。Ｄ－乳酸デヒドロゲナーゼ遺伝子（ｌｄｈＡ）を取得するためにエシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡをテンプレートに用いてＧＧＡＡＴＴＣＣＧＧＡＧＡＡＡＧＴＣＴＴＡＴＧＡＡＡＣＴ（配列番号１９）、及びＣＣＣＡＡＧＣＴＴＴＴＡＡＡＣＣＡＧＴＴＣＧＴＴＣＧＧＧＣ（配列番号２０）によりＰＣＲ法で増幅し、得られたＤＮＡフラグメントを制限酵素ＥｃｏＲＩ及びＨｉｎｄＩＩＩで消化することで約１．０ｋｂｐのＤ－乳酸デヒドロゲナーゼ（ｌｄｈＡ）遺伝子フラグメントを得た。上記の２つのＤＮＡフラグメントとプラスミドｐＵＣ１８を制限酵素ＥｃｏＲＩ及びＨｉｎｄＩＩＩで消化することで得られるフラグメントを混合し、リガーゼを用いて結合した後、エシェリヒア・コリＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、アンピシリン５０μｇ／ｍＬを含むＬＢ寒天プレートに生育する形質転換体を得た。得られたコロニーをアンピシリン５０μｇ／ｍＬを含むＬＢ液体培地で３０℃で一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的とするＧＡＰＤＨプロモーターとｌｄｈＡをコードする遺伝子が挿入され、ＧＡＰＤＨプロモーターによりｌｄｈＡが発現すると予測されるプラスミドを得た。本プラスミドをｐＧＡＰ－ｌｄｈＡと命名した。
　エシェリヒア・コリＭＧ１６５５株由来ｌｄｈＡ遺伝子の５'近傍領域の遺伝子情報に基づいて作成された、ＡＡＧＧＴＡＣＣＡＣＣＡＧＡＧＣＧＴＴＣＴＣＡＡＧＣ（配列番号２１）とＧＣＴＣＴＡＧＡＴＴＣＴＣＣＡＧＴＧＡＴＧＴＴＧＡＡＴＣＡＣ（配列番号２２）を用いて、エシェリヒア・コリゲノムＤＮＡを鋳型としてＰＣＲを行うことにより約１０００ｂｐのＤＮＡ断片を増幅した。
　また、エシェリヒア・コリＭＧ１６５５株のグリセルデヒド３－リン酸デヒドロゲナーゼ（ＧＡＰＤＨ）プロモーターの配列情報に基づいて作製されたＧＧＴＣＴＡＧＡＧＣＡＡＴＧＡＴＴＣＡＣＡＣＧＡＴＴＣＧ（配列番号２３）とエシェリヒア・コリＭＧ１６５５株のｌｄｈＡ遺伝子の配列情報に基づいて作製されたＡＡＣＴＧＣＡＧＧＴＴＣＧＴＴＣＴＣＡＴＡＣＡＣＧＴＣＣ（配列番号２４）を用いて、先に作製したベクターｐＧＡＰ－ｌｄｈＡを鋳型としてＰＣＲを行い、ＧＡＰＤＨプロモーターとｌｄｈＡ遺伝子の開始コドン近傍領域からなる約８５０ｂｐのＤＮＡフラグメントを得た。
　上記により得られたフラグメントをそれぞれ、制限酵素ＫｐｎＩとＸｂａＩ、ＸｂａＩとＰｓｔＩで消化し、このフラグメントを温度感受性プラスミドｐＴＨ１８ｃｓ１をＫｐｎＩとＰｓｔＩで消化して得られるフラグメントと混合し、リガーゼを用いて結合した後、ＤＨ５αコンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに３０℃で生育する形質転換体を得た。得られたコロニーをクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地で３０℃一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的とする、ｌｄｈＡをコードする遺伝子の５´上流近傍断片とＧＡＰＤＨプロモーターおよびｌｄｈＡ遺伝子の開始コドン近傍領域が挿入されていることを確認した。本プラスミドをｐＴＨＧＡＰｌｄｈＡと命名した。
　プラスミドｐＴＨＧＡＰｌｄｈＡをＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株に形質転換し、クロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに３０℃で一晩培養し、形質転換体を得た。得られた形質転換体をクロラムフェニコール１０μｇ／ｍｌを含むＬＢ液体培地に接種し、３０℃で一晩培養した。次にこれらの菌体をクロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに塗布し、４２℃で生育するコロニーを得た。得られたコロニーをＬＢ液体培地で３０℃一晩培養し、さらにＬＢ寒天プレートに塗布して４２℃で生育するコロニーを得た。
　出現したコロニーの中から無作為に１００コロニーをピックアップしてそれぞれをＬＢ寒天プレートとクロラムフェニコール１０μｇ／ｍｌを含むＬＢ寒天プレートに生育させ、クロラムフェニコール感受性のクローンを選んだ。さらにはこれらの目的クローンの染色体ＤＮＡからＰＣＲによりＧＡＰＤＨプロモーターとｌｄｈＡ遺伝子を含む約８００ｂｐ断片を増幅させ、ｌｄｈＡプロモーター領域がＧＡＰＤＨプロモーターに置換されている株を選抜し、以上を満足するクローンをＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株と命名した。 <Production 7: Replacement of ldhA promoter on the genome of Escherichia coli MG1655ΔpflΔdldΔmdhΔasp strain with GAPDH promoter>
The nucleotide sequence of the Escherichia coli ldhA gene has already been reported (GenBank accession number U36928). In order to obtain the glyceride 3-phosphate dehydrogenase (GAPDH) promoter, the genomic DNA of Escherichia coli MG1655 strain was used as a template, and AACGAATTCTCGCAATGTATTACACGAATTC (SEQ ID NO: 17), and AGAGAATTCGCTATTTGTTTAGGTGAATAAAAGG (SEQ ID NO: 18) The DNA fragment was digested with the restriction enzyme EcoRI to obtain a fragment encoding the GAPDH promoter of about 100 bp.
In addition, the entire base sequence of Escherichia coli genomic DNA is known (GenBank accession number U00096), and the base sequence of the Escherichia coli ldhA gene has also been reported (GenBank accession number U36928). In order to obtain the D-lactate dehydrogenase gene (ldhA), GGAATTCCCGGAGAAAGTCTTATGAAACT (SEQ ID NO: 19) and CCCAAGCTTTTAAACCAGTTCGGTCGGC (SEQ ID NO: 20) were obtained by PCR using the genomic DNA of Escherichia coli MG1655 strain as a template. The DNA fragment was digested with restriction enzymes EcoRI and HindIII to obtain an about 1.0 kbp D-lactate dehydrogenase (ldhA) gene fragment. The above two DNA fragments and the fragment obtained by digesting plasmid pUC18 with restriction enzymes EcoRI and HindIII were mixed and ligated using ligase, and then Escherichia coli DH5α competent cell (Toyobo Co., Ltd. DNA-903). And a transformant that grows on an LB agar plate containing 50 μg / mL of ampicillin was obtained. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin, and the plasmid was recovered from the obtained cells. The inserted sequence into the plasmid was confirmed by sequencing, and the target GAPDH promoter and the gene encoding ldhA were inserted, and a plasmid predicted to express ldhA by the GAPDH promoter was obtained. This plasmid was named pGAP-ldhA.
Using AAGGTACCACCAGAGCGTTCTCAAGC (SEQ ID NO: 21) and GCTTCTAGATTCTCCAGTGATGTTTGAATCAC (SEQ ID NO: 22), which was prepared based on the gene information of the 5 'vicinity region of the ldhA gene derived from Escherichia coli MG1655 strain, PCR using Escherichia coli genomic DNA as a template As a result, a DNA fragment of about 1000 bp was amplified.
Further, GTCTAGAGCAATGATCTACACGATTCG (SEQ ID NO: 23) prepared based on the sequence information of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter of Escherichia coli MG1655 strain and the sequence of the ldhA gene of Escherichia coli MG1655 strain. PCR was performed using the previously prepared vector pGAP-ldhA as a template using AACTGCAGGTTCGTTCTCATACACGTCC (SEQ ID NO: 24) to obtain a DNA fragment of about 850 bp consisting of the GAPDH promoter and the region near the start codon of the ldhA gene.
The fragments obtained above were digested with restriction enzymes KpnI and XbaI, XbaI and PstI, this fragment was mixed with a fragment obtained by digesting temperature sensitive plasmid pTH18cs1 with KpnI and PstI, and ligated using ligase. Thereafter, the cells were transformed into DH5α competent cells (DNA-903, Toyobo Co., Ltd.) to obtain transformants that grew at 30 ° C. on LB agar plates containing 10 μg / ml of chloramphenicol. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 10 μg / ml of chloramphenicol, and the plasmid was recovered from the obtained cells. The inserted sequence into the plasmid was confirmed by sequencing, and it was confirmed that the target fragment near the 5 'upstream of the gene encoding ldhA, the GAPDH promoter and the region near the start codon of the ldhA gene were inserted. This plasmid was designated as pTHGAPldhA.
Plasmid pTHGAPldhA was transformed into MG1655ΔpflΔdldΔmdhΔasp strain, and cultured on an LB agar plate containing 10 μg / ml of chloramphenicol at 30 ° C. overnight to obtain a transformant. The obtained transformant was inoculated into an LB liquid medium containing 10 μg / ml of chloramphenicol and cultured at 30 ° C. overnight. Next, these cells were applied to an LB agar plate containing 10 μg / ml of chloramphenicol to obtain colonies that grew at 42 ° C. The obtained colonies were cultured overnight at 30 ° C. in an LB liquid medium, and further applied to an LB agar plate to obtain colonies that grew at 42 ° C.
100 colonies were picked up randomly from the appearing colonies and grown on LB agar plates and LB agar plates containing 10 μg / ml of chloramphenicol, and chloramphenicol sensitive clones were selected. Furthermore, an approximately 800 bp fragment containing the GAPDH promoter and the ldhA gene is amplified from the chromosomal DNA of these target clones by PCR, a strain in which the ldhA promoter region is replaced by the GAPDH promoter is selected, and a clone satisfying the above is selected as MG1655ΔpflΔdldΔmdhΔasp / It was named GAPldhA genome insertion strain.
［実施例１］　ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株／ＧＡＰｌｄｈＡ挿入株による無機塩培地におけるＤ－乳酸生産
　前培養として三角フラスコにいれたＬＢ　Ｂｒｏｔｈ，　Ｍｉｌｌｅｒ培養液（Ｄｉｆｃｏ２４４６２０）１００ｍＬに、製造例１で作製したＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株／ｐＧＡＰｌｄｈＡ株を植菌し、一晩、１２０ｒｐｍで攪拌培養を行った。表１の組成よりなる無機塩培地４７５ｇの入った１Ｌ容の培養槽（ＡＢＬＥ社製培養装置ＢＭＪ－０１）をオートクレーブ滅菌し、そこに前培養液２５ｍｌを移し、培養を行った。培養は大気圧下、通気量０．５ｖｖｍ、撹拌速度３００ｒｐｍ、培養温度３５℃、ｐＨ７．５（２０ｗｔ％水酸化カルシウム、８０ｗｔ％純水のスラリーでｐＨ調整）でグルコースが枯渇するまで行った。培養終了後、得られた培養液中のＤ－乳酸蓄積量をＨＰＬＣで定法に従って測定した（カラム：ＵＬＴＲＯＮ ＰＳ－８０Ｈ（信和化工），溶離液：過塩素酸水溶液（ｐＨ＝２．１））。発酵終了時間は１８時間で、Ｄ－乳酸蓄積濃度は９２．７ｇ／Ｌであった。発酵終了時間とは、添加した糖が全て消費された時間のことであり、その長さによって生産性を評価できる。 [Example 1] Production of D-lactic acid in inorganic salt medium by MG1655ΔpflΔdldΔmdhΔasp strain / GAPldhA-inserted strain 100 mg of LB Broth, Miller culture solution (Difco244620d) in erlenmeyer flask as a pre-culture was prepared in MG1655ΔpflΔdhlΔdhlAdplAdhdAPdhdAPdhlAdpl The strain was inoculated and stirred and cultured overnight at 120 rpm. A 1 L culture tank (culture device BMJ-01 manufactured by ABLE) containing 475 g of the inorganic salt medium having the composition shown in Table 1 was sterilized by autoclave, and 25 ml of the preculture was transferred to the culture vessel. Cultivation was performed under atmospheric pressure until glucose was depleted at an aeration rate of 0.5 vvm, a stirring speed of 300 rpm, a culture temperature of 35 ° C., and a pH of 7.5 (pH adjustment with a slurry of 20 wt% calcium hydroxide and 80 wt% pure water). After completion of the culture, the amount of D-lactic acid accumulated in the obtained culture broth was measured by HPLC according to a conventional method (column: ULTRON PS-80H (Shinwa Kako), eluent: perchloric acid aqueous solution (pH = 2.1)) . The fermentation end time was 18 hours, and the D-lactic acid accumulation concentration was 92.7 g / L. The fermentation end time is the time when all the added sugar is consumed, and the productivity can be evaluated by the length.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
［実施例２］　無機塩培地で生産したＤ－乳酸の粗精製
　実施例１で得られたＤ－乳酸発酵液から菌体を除くために、８０００×ｇで遠心分離した。遠心分離上清中の乳酸カルシウムを脱塩して乳酸にするために、上清のｐＨが２．５になるまで９５重量％硫酸を添加した。析出した硫酸カルシウムを除くために、ろ紙（Ｗａｔｍａｎ４２）を使って吸引ろ過をした。脱色と粗精製をするために、ろ液重量の０．５重量％分の活性炭を添加して攪拌した。活性炭を除くために、ろ紙と０．４５μｍのメンブレンフィルターを使って吸引ろ過した。分光光度計における純水の４００ｎｍの波長の透過率を１００％とした場合、活性炭除去後のろ液の４００ｎｍの波長の透過率は９７．７％であった。 [Example 2] Crude purification of D-lactic acid produced in an inorganic salt medium In order to remove cells from the D-lactic acid fermentation broth obtained in Example 1, it was centrifuged at 8000 xg. In order to desalinate the calcium lactate in the centrifuged supernatant into lactic acid, 95% by weight sulfuric acid was added until the pH of the supernatant reached 2.5. In order to remove the precipitated calcium sulfate, suction filtration was performed using a filter paper (Watman 42). In order to perform decolorization and crude purification, activated carbon corresponding to 0.5% by weight of the filtrate was added and stirred. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 μm membrane filter. When the transmittance at a wavelength of 400 nm of pure water in the spectrophotometer was 100%, the transmittance at a wavelength of 400 nm of the filtrate after removal of activated carbon was 97.7%.
［比較例１］　ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株によるコーンスティープリカー培地におけるＤ－乳酸生産
　前培養として三角フラスコにいれたＬＢ　Ｂｒｏｔｈ，　Ｍｉｌｌｅｒ培養液（Ｄｉｆｃｏ２４４６２０）１００ｍＬに、製造例１で作製したＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株を植菌し、一晩、１２０ｒｐｍで攪拌培養を行った。前培養液２５ｍｌを、表２の組成よりなるコーンスティープリカー培地４７５ｇの入った１Ｌ容の培養槽（ＡＢＬＥ社製培養装置ＢＭＪ－０１）に移し、培養を行った。培養は大気圧下、通気量０．５ｖｖｍ、撹拌速度３００ｒｐｍ、培養温度３５℃、ｐＨ７．５（水酸化カルシウム水溶液で調整）でグルコースが枯渇するまで行った。培養終了後、得られた培養液中のＤ－乳酸蓄積量をＨＰＬＣで定法に従って測定した。Ｄ－乳酸蓄積濃度は、２０時間で９０．７ｇ／Ｌであった。 Comparative Example 1 Production of D-lactic acid in corn steep liquor medium by MG1655ΔpflΔdldΔmdhΔasp / GAPldhA genome-inserted strain MG1655ΔspflAPdhdhdphDhAPddhAdphAhdphΔdphAhdphΔphdApdΔhdhdApDhDhAdPhΔdhAdPhΔdhAhd The genome-inserted strain was inoculated and agitated and cultured at 120 rpm overnight. 25 ml of the preculture was transferred to a 1 L culture tank (culture device BMJ-01 manufactured by ABLE) containing 475 g of corn steep liquor medium having the composition shown in Table 2 and cultured. Cultivation was carried out under atmospheric pressure at an aeration rate of 0.5 vvm, a stirring speed of 300 rpm, a culture temperature of 35 ° C., pH 7.5 (adjusted with an aqueous calcium hydroxide solution) until glucose was depleted. After completion of the culture, the amount of D-lactic acid accumulated in the obtained culture broth was measured by HPLC according to a conventional method. The D-lactic acid accumulation concentration was 90.7 g / L in 20 hours.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
［比較例２］　コーンスティープリカー培地で生産したＤ－乳酸の粗精製
　比較例１で得られたＤ－乳酸発酵液から菌体を除くために、８０００×ｇで遠心分離した。遠心分離上清中の乳酸カルシウムを脱塩して乳酸にするために、上清のｐＨが２．５になるまで９５重量％硫酸を添加した。析出した硫酸カルシウムを除くために、ろ紙（Ｗａｔｍａｎ４２）を使って吸引ろ過をした。脱色と粗精製をするために、ろ液重量の０．５重量％分、または３．０重量％分の活性炭を添加して攪拌した。活性炭を除くために、ろ紙と０．４５μｍのメンブレンフィルターを使って吸引ろ過して粗精製液を得た。分光光度計における純水の４００ｎｍの波長の透過率を１００％とした場合、活性炭除去後のろ液の４００ｎｍの波長の透過率は、０．５重量％活性炭では７８．７％、３．０重量％活性炭では９７．７％だった。コーンスティープリカー培地を用いた検討においては、清澄なＤ－乳酸溶液を得るために、無機塩培地よりも多量の活性炭が必要となることが示された。 [Comparative Example 2] Rough purification of D-lactic acid produced in corn steep liquor medium In order to remove the cells from the D-lactic acid fermentation broth obtained in Comparative Example 1, it was centrifuged at 8000 xg. In order to desalinate the calcium lactate in the centrifuged supernatant into lactic acid, 95% by weight sulfuric acid was added until the pH of the supernatant reached 2.5. In order to remove the precipitated calcium sulfate, suction filtration was performed using a filter paper (Watman 42). In order to perform decolorization and rough purification, 0.5% by weight or 3.0% by weight of activated carbon was added and stirred. In order to remove the activated carbon, a crude purified solution was obtained by suction filtration using a filter paper and a 0.45 μm membrane filter. When the transmittance at a wavelength of 400 nm of pure water in the spectrophotometer is 100%, the transmittance at a wavelength of 400 nm of the filtrate after removal of activated carbon is 78.7% and 3.0% for 0.5 wt% activated carbon. It was 97.7% for the weight% activated carbon. In the study using corn steep liquor medium, it was shown that a larger amount of activated carbon was required than the inorganic salt medium in order to obtain a clear D-lactic acid solution.
［実施例３］　遷移金属イオンを添加した無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、培地に表３に示す組成の遷移金属含有水溶液を０．１体積％添加した。発酵終了時間、Ｄ－乳酸蓄積濃度は、遷移金属含有水溶液を添加しない場合と同等であった。 [Example 3] Production of D-lactic acid in inorganic salt medium supplemented with transition metal ions Under the culture conditions of Example 1, 0.1% by volume of a transition metal-containing aqueous solution having the composition shown in Table 3 was added to the medium. The fermentation end time and D-lactic acid accumulation concentration were the same as when no transition metal-containing aqueous solution was added.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
［実施例４］　塩化カルシウムとチアミンを添加した無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、実施例１の培地にさらに塩化カルシウム及びチアミンを共に１０ｍｇ／Ｌになるよう添加した培地を用い、培養を実施した。発酵終了時間、Ｄ－乳酸蓄積濃度は、塩化カルシウム及びチアミンを添加しない場合と同等であった。 [Example 4] Production of D-lactic acid in an inorganic salt medium supplemented with calcium chloride and thiamine Under the culture conditions of Example 1, both calcium chloride and thiamine were added to the medium of Example 1 to a concentration of 10 mg / L. Culture was performed using the prepared medium. The fermentation end time and D-lactic acid accumulation concentration were the same as when calcium chloride and thiamine were not added.
［実施例５］　遷移金属イオン、塩化カルシウムとチアミンを添加した無機塩培地でのＤ－乳酸の生産とＤ－乳酸の粗精製
　実施例１の培養条件で、実施例１の培地にさらに表３に示す組成の遷移金属含有溶液を０．１体積％添加し、そこにさらに、塩化カルシウム及びチアミンを共に１０ｍｇ／Ｌになるよう添加した培地を用い、培養を実施した。発酵終了時間、Ｄ－乳酸蓄積濃度は、実施例１と同等であった。
   得られたＤ－乳酸発酵液から菌体を除くために、８０００×ｇで遠心分離した。遠心分離上清中の乳酸カルシウムを脱塩して乳酸にするために、上清のｐＨが２．５になるまで９５重量％硫酸を添加した。析出した硫酸カルシウムを除くために、ろ紙（Ｗａｔｍａｎ４２）を使って吸引ろ過をした。脱色と粗精製をするために、ろ液重量の０．５重量％分、または１重量％分の活性炭を添加して攪拌した。活性炭を除くために、ろ紙と０．４５μｍのメンブレンフィルターを使って吸引ろ過した。分光光度計における純水の４００ｎｍの波長の透過率を１００％とした場合、活性炭除去後のろ液の４００ｎｍの波長の透過率は０．５重量％活性炭では９３．７％、１．０重量％活性炭では９８．１％であった。 [Example 5] Production of D-lactic acid and crude purification of D-lactic acid in an inorganic salt medium supplemented with transition metal ions, calcium chloride and thiamine Table 3 was further added to the medium of Example 1 under the culture conditions of Example 1. Culturing was carried out using a medium containing 0.1% by volume of a transition metal-containing solution having the composition shown in FIG. 5 and further adding calcium chloride and thiamine to 10 mg / L. The fermentation end time and D-lactic acid accumulation concentration were the same as in Example 1.
In order to remove the cells from the obtained D-lactic acid fermentation broth, it was centrifuged at 8000 × g. In order to desalinate the calcium lactate in the centrifuged supernatant into lactic acid, 95% by weight sulfuric acid was added until the pH of the supernatant reached 2.5. In order to remove the precipitated calcium sulfate, suction filtration was performed using a filter paper (Watman 42). In order to perform decolorization and rough purification, 0.5% by weight or 1% by weight of activated carbon was added and stirred. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 μm membrane filter. When the transmittance of 400 nm wavelength of pure water in the spectrophotometer is 100%, the transmittance of 400 nm wavelength of the filtrate after removal of activated carbon is 93.7% and 1.0 wt. It was 98.1% for% activated carbon.
［実施例６］　ベタイン含有量を変動させた無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、培地に添加するベタインを０～６ｇ／Ｌの含有量で添加した。それぞれの条件での発酵終了時間は表４に示す。Ｄ－乳酸蓄積濃度は、いずれの試験区においても同等であった。脱色と粗精製をするために、ろ液重量の０．５重量％分の活性炭を添加して攪拌した。活性炭を除くために、ろ紙と０．４５μｍのメンブレンフィルターを使って吸引ろ過した。いずれのベタイン量の場合でも、分光光度計における純水の４００ｎｍの波長の透過率を１００％とした場合、活性炭除去後のろ液の４００ｎｍの波長の透過率はいずれの試験区においても９８．０％以上であった。 [Example 6] Production of D-lactic acid in inorganic salt medium with varying betaine content Under the culture conditions of Example 1, betaine added to the medium was added at a content of 0 to 6 g / L. Table 4 shows the fermentation end time under each condition. The D-lactic acid accumulation concentration was the same in all test groups. In order to perform decolorization and crude purification, activated carbon corresponding to 0.5% by weight of the filtrate was added and stirred. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 μm membrane filter. Regardless of the amount of betaine, when the transmittance of 400 nm wavelength of pure water in the spectrophotometer is 100%, the transmittance of 400 nm wavelength of the filtrate after removal of activated carbon is 98. It was 0% or more.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
［実施例７］　硫酸アンモニウム含有量を低下させた無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、無機塩培地中の硫酸アンモニウム含有量を２ｇ／Ｌとした。発酵終了時間は３４時間となった。Ｄ－乳酸蓄積濃度は同等であった。 [Example 7] Production of D-lactic acid in an inorganic salt medium having a reduced ammonium sulfate content Under the culture conditions of Example 1, the ammonium sulfate content in the inorganic salt medium was set to 2 g / L. The fermentation end time was 34 hours. The D-lactic acid accumulation concentration was equivalent.
［実施例８］　硫酸マグネシウム含有量を低下させた無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、無機塩培地中の硫酸マグネシウム・７水和物を０．１ｇ／Ｌとした。発酵終了時間は３４時間となった。Ｄ－乳酸蓄積濃度は同等であった。 [Example 8] Production of D-lactic acid in inorganic salt medium with reduced magnesium sulfate content Under the culture conditions of Example 1, magnesium sulfate heptahydrate in inorganic salt medium was 0.1 g / L. did. The fermentation end time was 34 hours. The D-lactic acid accumulation concentration was equivalent.
［実施例９］　リン酸水素二カリウムをリン酸二水素カリウムに置換した無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、無機塩培地中のリン酸水素二カリウムを同じ含有量（ｇ／Ｌ）でリン酸二水素カリウムに置換して培養した。発酵終了時間、Ｄ－乳酸蓄積濃度は同等であり、リン酸水素二カリウムをリン酸二水素カリウムに置換してもよいことが明らかになった。 [Example 9] Production of D-lactic acid in an inorganic salt medium in which dipotassium hydrogen phosphate is replaced with potassium dihydrogen phosphate. Under the same culture conditions as in Example 1, the same content of dipotassium hydrogen phosphate in the inorganic salt medium is contained. The culture was carried out by substituting with potassium dihydrogen phosphate in an amount (g / L). It was revealed that the fermentation end time and D-lactic acid accumulation concentration were the same, and dipotassium hydrogen phosphate may be replaced with potassium dihydrogen phosphate.
［実施例１０］　リン酸水素二カリウム含有量を低下させた無機塩培地でのＤ－乳酸の生産
　実施例１の培養条件で、無機塩培地中のリン酸水素二カリウムを０．５ｇ／Ｌとした。発酵終了時間は２７時間となった。Ｄ－乳酸蓄積濃度は同等であった。 [Example 10] Production of D-lactic acid in an inorganic salt medium with a reduced content of dipotassium hydrogen phosphate Under the culture conditions of Example 1, 0.5 g / L of dipotassium hydrogen phosphate in the inorganic salt medium was used. It was. The fermentation end time was 27 hours. The D-lactic acid accumulation concentration was equivalent.
（製造例２）ＷＯ２０１０／０３２６９８の実施例９に記載の方法と同様に大腸菌株、ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株／ｐＧＡＰ－ｃｓｃＡ－ｆｒｕＫ株を作製した。
　具体的には、以下の方法により、大腸菌株、ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株／ｐＧＡＰ－ｃｓｃＡ－ｆｒｕＫ株を作製した。 (Production Example 2) In the same manner as in Example 9 of WO2010 / 032698, an E. coli strain, MG1655ΔpflΔdldΔmdhΔasp / GAPldhA genome insertion strain / pGAP-cscA-fruK strain was prepared.
Specifically, an E. coli strain, MG1655ΔpflΔdldΔmdhΔasp / GAPldhA genome insertion strain / pGAP-cscA-fruK strain was prepared by the following method.
＜製造８：エシェリヒア・コリＯ１５７由来インベルターゼ遺伝子、エシェリヒア・コリＭＧ１６５５由来フルクトース－１－リン酸キナーゼ遺伝子発現ベクターおよび該発現ベクター形質転換体の構築＞
　エシェリヒア・コリＯ１５７のインベルターゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、インベルターゼをコードする遺伝子（ｃｓｃＡ）はＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ ＡＥ００５１７４に記載のエシェリヒア・コリＯ１５７株ゲノム配列の３２７４３８３～３２７５８１６に記載されている。
　上記の遺伝子を発現させるために必要なプロモーターの塩基配列として、ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｘ０２６６２の塩基配列情報において、３９７－４４０に記されているエシェリヒア・コリ由来のグリセルアルデヒド３－リン酸デヒドロゲナーゼ（以下ＧＡＰＤＨと呼ぶことがある）のプロモーター配列を使用することができる。
　ＧＡＰＤＨプロモーターを取得するためエシェリヒア・コリＭＧ１６５５株のゲノムＤＮＡをテンプレートに用いてＣＧＡＧＣＴＡＣＡＴＡＴＧＣＡＡＴＧＡＴＴＧＡＣＡＣＧＡＴＴＣＣＧ（配列番号２５）、及びＴＣＴＡＧＡＧＣＴＡＴＴＴＧＴＴＡＧＴＧＡＡＴＡＡＡＡＧＧ（配列番号２６）によりＰＣＲ法で増幅し、得られたＤＮＡフラグメントを制限酵素ＮｄｅＩで消化することで約１１０ｂｐのＧＡＰＤＨプロモーターにあたるＤＮＡフラグメントを得た。得られたＤＮＡフラグメントとプラスミドｐＢＲ３２２（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　Ｊ０１７４９）を制限酵素ＮｄｅＩ及びＰｖｕＩＩで消化することで得られるフラグメントを混合し、リガーゼを用いて結合した後、エシェリヒア・コリＤＨ５α株コンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、アンピシリン５０μｇ／ｍＬを含むＬＢ寒天プレートに生育する形質転換体を得た。得られたコロニーをアンピシリン５０μｇ／ｍＬを含むＬＢ液体培地で３７℃一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的のＧＡＰＤＨプロモーター配列が正しく挿入されていることを確認した。本プラスミドをｐＢＲｇａｐＰと命名した。
　インベルターゼ遺伝子を取得するために、エシェリヒア・コリＯ１５７のゲノムＤＮＡ（ＳＩＧＭＡ-ＡＬＤＲＩＣＨ：ＩＲＭＭ４４９）をテンプレートに用いて、ＧＡＴＣＴＡＧＡＣＧＧＡＧＡＡＡＧＴＣＴＴＡＴＧＡＣＧＣＡＡＴＣＴＣＧＡＴＴＧＣＡＴＧ（配列番号２７）、及びＡＴＧＧＴＡＣＣＴＴＡＡＣＣＣＡＧＴＴＧＣＣＡＧＡＧＴＧＣ（配列番号２８）によりＰＣＲ法で増幅し、得られたＤＮＡフラグメントを制限酵素ＸｂａＩで消化することで約１．４ｋｂｐのインベルターゼ遺伝子フラグメントを得た。得られたＤＮＡフラグメントと先に作成したプラスミドｐＢＲｇａｐＰを制限酵素ＸｂａＩ及びＰｓｈＡＩで消化することで得られるフラグメントを混合し、リガーゼを用いて結合した後、エシェリヒア・コリＤＨ５α株コンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、アンピシリン５０μｇ／ｍＬを含むＬＢ寒天プレートに生育する形質転換体を得た。得られたコロニーをアンピシリン５０μｇ／ｍＬを含むＬＢ液体培地で３７℃一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的のｃｓｃＡが正しく挿入されていることを確認した。本プラスミドをｐＧＡＰ－ｃｓｃＡと命名した。
　エシェリヒア・コリＭＧ１６５５のフルクトース－１－リン酸キナーゼのアミノ酸配列と遺伝子の塩基配列は既に報告されている。すなわち、フルクトース－１－リン酸キナーゼをコードする遺伝子（ｆｒｕＫ）はＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒＵ０００９６に記載のエシェリヒア・コリＭＧ１６５５株ゲノム配列の２２６０３８７～２２５９４４９に記載されている。
　フルクトース－１－リン酸キナーゼ遺伝子を取得するために、エシェリヒア・コリＭＧ１６５５のゲノムＤＮＡをテンプレートに用いて、ＡＴＧＧＴＡＣＣＧＧＡＧＡＡＡＧＴＣＴＴＡＴＧＡＧＣＡＧＡＣＧＴＧＴＴＧＣＴＡＣ（配列番号２９）、及びＴＣＧＧＡＴＣＣＴＴＡＴＧＣＣＴＣＴＣＣＴＧＣＴＧＴＣＡＧ（配列番号３０）によりＰＣＲ法で増幅し、得られたＤＮＡフラグメントを制限酵素ＫｐｎＩで消化することで約１.０ｋｂｐのフルクトース－１－リン酸キナーゼ遺伝子フラグメントを得た。得られたＤＮＡフラグメントと先に作成したプラスミドｐＧＡＰ－ｃｓｃＡを制限酵素ＫｐｎＩ及びＥｃｏＲＶで消化することで得られるフラグメントを混合し、リガーゼを用いて結合した後、エシェリヒア・コリＤＨ５α株コンピテントセル（東洋紡績株式会社 ＤＮＡ－９０３）に形質転換し、アンピシリン５０μｇ／ｍＬを含むＬＢ寒天プレートに生育する形質転換体を得た。得られたコロニーをアンピシリン５０μｇ／ｍＬを含むＬＢ液体培地で３７℃一晩培養し、得られた菌体からプラスミドを回収した。プラスミドへの挿入配列をシーケンシングにより確認し、目的のｆｒｕＫが正しく挿入されていることを確認した。本プラスミドをｐＧＡＰ－ｃｓｃＡ－ｆｒｕＫと命名した。
　このプラスミドｐＧＡＰ－ｃｓｃＡ－ｆｒｕＫを上記製造７で作成したＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株に形質転換し、アンピシリン５０μｇ／ｍＬを含むＬＢ　Ｂｒｏｔｈ，Ｍｉｌｌｅｒ寒天プレートで３７℃一晩培養することにより、ＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株／ｐＧＡＰ－ｃｓｃＡ－ｆｒｕＫ株を得た。 <Production 8: Escherichia coli O157-derived invertase gene, Escherichia coli MG1655-derived fructose-1-phosphate kinase gene expression vector and construction of the expression vector transformant>
The amino acid sequence of Escherichia coli O157 invertase and the nucleotide sequence of the gene have already been reported. That is, the gene (cscA) encoding invertase is described in 3274383 to 3275816 of the Escherichia coli O157 strain genomic sequence described in GenBank accession number AE005174.
As a base sequence of a promoter necessary for expressing the above gene, glyceraldehyde 3-phosphate dehydrogenase (hereinafter referred to as GAPDH) derived from Escherichia coli described in 397 to 440 in the base sequence information of GenBank accession number X02662 Promoter sequences) may be used.
In order to obtain the GAPDH promoter, the genomic DNA of Escherichia coli MG1655 strain was used as a template, and the DNA fragment obtained was amplified by PCR using CGAGCTCACATATGCAATGATTGACACGATTCCG (SEQ ID NO: 25) and TCTAGAGCTATTTTGTTAGTGAAATAAAGG (SEQ ID NO: 26). The DNA fragment corresponding to the GAPDH promoter of about 110 bp was obtained by digestion. The obtained DNA fragment was mixed with the plasmid pBR322 (GenBank accession number J01749) digested with restriction enzymes NdeI and PvuII, ligated with ligase, Escherichia coli DH5α strain competent cell (Toyo Spinning Co., Ltd. DNA-903) was transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 μg / mL. The obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin, and the plasmid was recovered from the obtained cells. The insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that the target GAPDH promoter sequence was correctly inserted. This plasmid was designated as pBRgapP.
In order to obtain the invertase gene, Escherichia coli O157 genomic DNA (SIGMA-ALDRICH: IRMM449) was used as a template, and GATCTAGACGGGAGAAGTCTTATGACGCAATCTCGATTTGCATTG (SEQ ID NO: 27), and ATGGTACCTTAACCCAGTTGCAGGTG was obtained by PCR method. The obtained DNA fragment was digested with the restriction enzyme XbaI to obtain an invertase gene fragment of about 1.4 kbp. The obtained DNA fragment and the previously prepared plasmid pBRgapP were digested with restriction enzymes XbaI and PshAI, mixed together using ligase, and then Escherichia coli DH5α strain competent cell (Toyobo Co., Ltd.) Company DNA-903) was transformed to obtain transformants that grew on LB agar plates containing 50 μg / mL of ampicillin. The obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin, and the plasmid was recovered from the obtained cells. The insertion sequence into the plasmid was confirmed by sequencing, and it was confirmed that the target cscA was correctly inserted. This plasmid was named pGAP-cscA.
The amino acid sequence of the fructose-1-phosphate kinase of Escherichia coli MG1655 and the nucleotide sequence of the gene have already been reported. That is, the gene (fruK) encoding fructose-1-phosphate kinase is described in 2260387 to 2259449 of the Escherichia coli MG1655 strain genome sequence described in GenBank accession number U00096.
In order to obtain the fructose-1-phosphate kinase gene, using the genomic DNA of Escherichia coli MG1655 as a template, ATGGTACCGGAGAAAGTCTTATGGAGCAGACGTGTTCCTAC (SEQ ID NO: 29) and TCGGATCCTTATGCCTCTCCCTGCTGTCAG (SEQ ID NO: 30) were obtained by PCR. The obtained DNA fragment was digested with the restriction enzyme KpnI to obtain a fructose-1-phosphate kinase gene fragment of about 1.0 kbp. The obtained DNA fragment was mixed with the previously prepared plasmid pGAP-cscA by digestion with restriction enzymes KpnI and EcoRV, ligated with ligase, and then Escherichia coli DH5α strain competent cells (Toyo Spinning Co., Ltd. DNA-903) was transformed to obtain transformants that grew on LB agar plates containing ampicillin 50 μg / mL. The obtained colony was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin, and the plasmid was recovered from the obtained cells. The inserted sequence into the plasmid was confirmed by sequencing, and it was confirmed that the desired fruK was correctly inserted. This plasmid was designated as pGAP-cscA-fruK.
This plasmid pGAP-cscA-fruK was transformed into the MG1655ΔpflΔdldΔmdhΔasp / GAPldhA genome insertion strain prepared in Preparation 7 above, and cultured overnight at 37 ° C. on LB Broth, Miller agar plates containing ampicillin 50 μg / mL. A genome inserted strain / pGAP-cscA-fruK strain was obtained.
［実施例１１］スクロースを原料としたＤ－乳酸の生産
　大腸菌は、製造例２で作製したＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ／ＧＡＰｌｄｈＡゲノム挿入株／ｐＧＡＰ１８－ｃｓｃＡ－ｆｒｕＫ株を用い、実施例１の培養条件で、糖をグルコースからスクロースに変更し、またリン酸水素二カリウムと硫酸マグネシウムの含有量も変更した。培地に添加したリン酸水素二カリウムと硫酸マグネシウムの含有量と発酵終了時間を表５に示す。Ｄ－乳酸蓄積濃度は８５～９０ｇ／Ｌであり、グルコースを用いての試験とほぼ同等であった。 [Example 11] Production of D-lactic acid using sucrose as a raw material Escherichia coli uses the MG1655ΔpflΔdldΔmdhΔasp / GAPldhA genome insertion strain / pGAP18-cscA-fruK strain prepared in Production Example 2 under the culture conditions of Example 1, The content of dipotassium hydrogen phosphate and magnesium sulfate was changed from glucose to sucrose. Table 5 shows the contents of dipotassium hydrogen phosphate and magnesium sulfate added to the medium and the fermentation end time. The D-lactic acid accumulation concentration was 85 to 90 g / L, which was almost the same as the test using glucose.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
［実施例１２］
＜種培養＞
　ＬＢ培地（ＤｉｆｃｏＴＭ　ＬＢ　Ｂｒｏｔｈ　Ｍｉｌｌｅｒ）を、三角フラスコにフラスコ容量の１／５量入れ、１２１℃、１５分間オートクレーブ殺菌を行った。オートクレーブ殺菌後の培地に、製造例１で作製したエシェリヒア・コリＭＧ１６５５ΔｐｆｌΔｄｌｄΔｍｄｈΔａｓｐ株／ＧＡＰｌｄｈＡゲノム挿入株を０．１体積％接種した。３５℃の恒温室にて３時間振盪培養を行い、種菌体を増殖させた。
＜前培養＞
　ＬＢ培地（ＤｉｆｃｏＴＭ　ＬＢ　Ｂｒｏｔｈ　Ｍｉｌｌｅｒ）を、３Ｌ三角フラスコに８２０ｇ入れ、１２１℃、１５分間オートクレーブ殺菌を行った。オートクレーブ殺菌後の培地に、上記の種培養液１００ｍＬを接種した。３５℃の恒温室にて５時間、振盪培養を行い、種菌体を増殖させた。
＜Ｄ－乳酸発酵生産＞
   次いで、リン酸水素二カリウム６３．２ｇ／Ｌ、硫酸アンモニウム１５８．０ｇ／Ｌからなる混合液０．５Ｌ、および表６に示す組成の培地１５．３Ｌを各々オートクレーブ殺菌した後、３０Ｌ発酵槽（エイブル社製ＢＭＬ－０１ＰＩ）に入れ、上記の前培養液８２０ｍＬを接種した。発酵は圧力０．１２ＭＰａ、攪拌回転速度１４０ｒｐｍ、空気通気量０．１ｖｖｍ、発酵温度３５℃、ｐＨ＝７．５（２０ｗｔ％水酸化カルシウム、８０ｗｔ％純水のスラリーでＰＨ調整）に制御し、２４時間実施した。 [Example 12]
<Seed culture>
LB medium (Difco ™ LB Broth Miller) was placed in an Erlenmeyer flask in an amount 1/5 of the flask volume, and autoclaved at 121 ° C. for 15 minutes. The autoclave-sterilized medium was inoculated with 0.1% by volume of the Escherichia coli MG1655ΔpflΔdldΔmdhΔasp strain / GAPldhA genome insertion strain prepared in Production Example 1. Shaking culture was performed in a thermostatic chamber at 35 ° C. for 3 hours to grow seed cells.
<Pre-culture>
820 g of LB medium (Difco ™ LB Broth Miller) was placed in a 3 L Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. The medium after autoclaving was inoculated with 100 mL of the above seed culture. Shaking culture was performed in a thermostatic chamber at 35 ° C. for 5 hours to grow seed cells.
<D-lactic acid fermentation production>
Next, 0.5 L of a mixed solution composed of 63.2 g / L of dipotassium hydrogen phosphate, 158.0 g / L of ammonium sulfate, and 15.3 L of a medium having the composition shown in Table 6 were sterilized by autoclave, respectively. BML-01PI) and inoculated with 820 mL of the above preculture solution. The fermentation is controlled to a pressure of 0.12 MPa, a stirring rotation speed of 140 rpm, an air aeration amount of 0.1 vvm, a fermentation temperature of 35 ° C., pH = 7.5 (pH adjustment with a slurry of 20 wt% calcium hydroxide and 80 wt% pure water) Conducted for 24 hours.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　本実施例では、図１で示す発酵装置１０を用いた。本実施例では、空気入り口及び出口の空気流量は１．６Ｌ／ｍｉｎ、空気入り口及び出口の空気圧は０．１２ＭＰａ、空気入り口及び出口の温度は３５℃とした。また、空気入り口の酸素モル分率は０．２１とし、出口の酸素モル分率は、測定された酸素モル分率の値（０．２１～０．１８）を採用した。ここでは１分ごとに記録した各値を用いて、上述した式１に従って算出した値の培養５時間以降の平均値をＯＵＲとした。このとき、槽内の溶存酸素濃度（ＤＯ）は、発酵開始から３時間以降はほぼ０ｐｐｍであった。なお、空気入り口の空気流量はマスフローメータ１４の値を、また出口の空気流量も酸素消費による減少分は無視できる範囲としてマスフローメータ１４の値を採用した。同様に空気入り口及び出口の空気圧は共に槽内圧力計１８の値を採用した。また空気入り口及び出口における絶対温度は共に槽内の温度センサ２２の値を採用した。空気入り口の酸素モル分率は０．２１とし、出口の酸素モル分率は排ガス分析計２０の値を採用した。 In this example, the fermentation apparatus 10 shown in FIG. 1 was used. In this example, the air flow rate at the air inlet and outlet was 1.6 L / min, the air pressure at the air inlet and outlet was 0.12 MPa, and the temperature of the air inlet and outlet was 35 ° C. The oxygen mole fraction at the air inlet was 0.21, and the measured oxygen mole fraction (0.21 to 0.18) was used as the oxygen mole fraction at the outlet. Here, using each value recorded every minute, the average value after 5 hours of culture of the value calculated according to the above-mentioned formula 1 was defined as OUR. At this time, the dissolved oxygen concentration (DO) in the tank was almost 0 ppm after 3 hours from the start of fermentation. In addition, the value of the mass flow meter 14 was adopted as the air flow rate at the air inlet, and the value of the mass flow meter 14 was adopted as the range in which the decrease due to oxygen consumption was negligible. Similarly, the values of the pressure gauge 18 in the tank were adopted for the air pressure at the air inlet and outlet. The absolute temperature at the air inlet and outlet is the value of the temperature sensor 22 in the tank. The oxygen mole fraction at the air inlet was 0.21, and the value of the exhaust gas analyzer 20 was adopted as the oxygen mole fraction at the outlet.
　得られた発酵液中のＤ－乳酸濃度はＨＰＬＣで定法に従い測定した。（カラム：ＵＬＴＲＯＮ　ＰＳ－８０Ｈ，溶離液：過塩素酸水溶液（ｐＨ＝２．１））。 The D-lactic acid concentration in the obtained fermentation broth was measured by HPLC according to a conventional method. (Column: ULTRON PS-80H, eluent: perchloric acid aqueous solution (pH = 2.1)).
　得られた発酵液中の析出Ｃａ分は以下のようにして測定した。発酵液２００ｇを２５０ｍＬ遠心チューブに計り、１００Ｇ、２分の遠心条件で遠心する。菌体の浮遊した上清を除去する。さらに２，４００Ｇ、５分の遠心条件で遠心する。上清を取り除き、秤量する。この固形分の水分量を測定し、発酵液１ｋｇ当りの固形分量をカルシウム分（析出Ｃａ分）とした。２４時間発酵後の結果を表７に示す。 The precipitated Ca content in the obtained fermentation broth was measured as follows. 200 g of the fermentation broth is weighed into a 250 mL centrifuge tube, and centrifuged at 100 G for 2 minutes. Remove the floating supernatant of the cells. Centrifuge at 2,400 G for 5 minutes. Remove the supernatant and weigh. The water content of this solid content was measured, and the solid content per 1 kg of the fermentation broth was defined as calcium content (precipitated Ca content). The results after 24 hours fermentation are shown in Table 7.
［実施例１３］
　攪拌回転速度２２０ｒｐｍ、空気通気量０．０１３ｖｖｍ、窒素通気量０．２８７ｖｖｍで行った以外は実施例１２と同じ方法で発酵を実施し、目的物を得た。結果を表７に示す。 [Example 13]
Fermentation was carried out in the same manner as in Example 12 except that stirring was performed at a rotational speed of 220 rpm, an air flow rate of 0.013 vvm, and a nitrogen flow rate of 0.287 vvm to obtain the target product. The results are shown in Table 7.
［実施例１４］
　攪拌回転速度３５０ｒｐｍで行った以外は実施例１３と同じ方法で発酵を実施し、目的物を得た。結果を表７に示す。 [Example 14]
Fermentation was carried out in the same manner as in Example 13 except that the stirring was performed at a rotational speed of 350 rpm to obtain the target product. The results are shown in Table 7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
［実施例１５］
　実施例１２で得られた培養液２１．６ｋｇを遠心分離機にて菌体を分離し、上清に残った菌をフィルターに通して完全に除菌した。この上清液に硫酸ｐＨが２．５以下になるまで硫酸を添加し、硫酸カルシウムを析出させた。析出した硫酸カルシウムを濾過し、得られた分離液にろ液重量の０．５重量％分の活性炭を加えた。このスラリー液を一晩攪拌した後、再度濾過して活性炭を除去した。さらに陰イオン交換カラム、陽イオン交換カラムに通してカルシウムイオン（Ｃａ２＋）、及び、硫酸イオン（ＳＯ４ ２－）をそれぞれ５０ｐｐｍ以下に除去し、Ｄ－乳酸８．８％の粗乳酸水溶液１８．４ｋｇを得た。この液を蒸留精製し、乳酸濃度９０重量％の精製Ｄ－乳酸１．１４ｋｇを得た。 [Example 15]
Bacteria were separated from 21.6 kg of the culture solution obtained in Example 12 using a centrifuge, and the bacteria remaining in the supernatant were passed through a filter to be completely sterilized. Sulfuric acid was added to the supernatant until the sulfuric acid pH was 2.5 or less to precipitate calcium sulfate. The precipitated calcium sulfate was filtered, and activated carbon corresponding to 0.5% by weight of the filtrate was added to the obtained separated liquid. The slurry was stirred overnight and then filtered again to remove the activated carbon. Further, calcium ions (Ca 2+ ) and sulfate ions (SO 4 2− ) are respectively removed to 50 ppm or less through an anion exchange column and a cation exchange column, and a crude lactic acid aqueous solution 18 containing 8.8% D-lactic acid is obtained. 4 kg was obtained. This solution was purified by distillation to obtain 1.14 kg of purified D-lactic acid having a lactic acid concentration of 90% by weight.
［実施例１６］
　実施例１５で得られたＤ－乳酸２０ｇ及び酸化スズ０．０８３ｇを２００ｍＬの反応フラスコに入れ、攪拌翼、温度計、ディーンスターク管付の還流管を取り付けた。還流管上部より、真空ポンプと窒素導入ラインに接続した。減圧・窒素放圧を繰り返し、酸素を除去した後、１ｋＰａに減圧して１４０℃に加熱した。１４０℃で３時間保持して水分を減圧除去した。水分除去後、常温・常圧に戻した。次に反応フラスコにｏ－ジクロロベンゼン（ＯＤＣＢ）１４．４ｇを入れた。ディーンスターク管にも反応フラスコに溢れない程度にＯＤＣＢを入れた。酸素除去後、３０ｋＰａに減圧し、１６０℃に加熱した。１６０℃で６ｈｒ共沸脱水した。この際、２ｈｒおきにディーンスターク管内のＯＤＣＢを交換した。共沸脱水後、常温・常圧に戻した。さらに反応フラスコにＯＤＣＢ２８．８ｇを追加し、空のディーンスターク管にモレキュラシーブス３Ａを１０ｇ加え、溢れない程度にＯＤＣＢを入れた。酸素除去後、３０ｋＰａに減圧し、１６０℃に加熱した。１６０℃で３０ｈｒ重合した。放冷後、吸引ろ過しポリ乳酸組成物を得た。 [Example 16]
20 g of D-lactic acid and 0.083 g of tin oxide obtained in Example 15 were placed in a 200 mL reaction flask, and a stirring blade, a thermometer, and a reflux tube with a Dean-Stark tube were attached. From the upper part of the reflux tube, it was connected to a vacuum pump and a nitrogen introduction line. Depressurization and nitrogen release were repeated to remove oxygen, and then the pressure was reduced to 1 kPa and heated to 140 ° C. The water was removed under reduced pressure by holding at 140 ° C. for 3 hours. After removing water, the temperature was returned to normal temperature and normal pressure. Next, 14.4 g of o-dichlorobenzene (ODCB) was placed in the reaction flask. ODCB was also added to the Dean Stark tube so that it did not overflow into the reaction flask. After removing oxygen, the pressure was reduced to 30 kPa and the mixture was heated to 160 ° C. Azeotropic dehydration was performed at 160 ° C. for 6 hours. At this time, the ODCB in the Dean-Stark tube was replaced every 2 hours. After azeotropic dehydration, the temperature was returned to room temperature and normal pressure. Further, 28.8 g of ODCB was added to the reaction flask, 10 g of Molecular Sieves 3A was added to the empty Dean-Stark tube, and ODCB was added so as not to overflow. After removing oxygen, the pressure was reduced to 30 kPa and the mixture was heated to 160 ° C. Polymerization was performed at 160 ° C. for 30 hours. After cooling, suction filtration was performed to obtain a polylactic acid composition.
　得られたポリ乳酸組成物をクロロホルムに溶解し分子量をＧＰＣで測定した（カラム：Ｓｈｏｄｅｘ　ＧＰＣ　ＬＦ－８０４、溶離液：クロロホルム）。重合後の分子量は、数平均分子量（Ｍｎ）２．１万であった。重量平均分子量（Ｍｗ）はポリスチレン換算値として算出したところ、２０万であった。これより、分子量分布（Ｍｗ／Ｍｎ）が９．５であり、成形性などにおいて好適な影響をもたらす分子量分布の広いポリマーが得られた。ＹＩ値を島津製作所紫外－可視分光計ＵＶ－２４００ＰＣにて測定した。ジクロロメタンにポリ乳酸組成物を１重量％となるよう溶解し、測定に用いた。上述した式３により算出したところＹＩ値は０．６であった。 The obtained polylactic acid composition was dissolved in chloroform and the molecular weight was measured by GPC (column: Shodex GPC LF-804, eluent: chloroform). The molecular weight after the polymerization was a number average molecular weight (Mn) of 21,000. The weight average molecular weight (Mw) was 200,000 when calculated as a polystyrene equivalent value. As a result, a polymer having a molecular weight distribution (Mw / Mn) of 9.5 and having a wide molecular weight distribution that has a favorable influence on moldability and the like was obtained. The YI value was measured with a Shimadzu UV-visible spectrometer UV-2400PC. The polylactic acid composition was dissolved in dichloromethane to 1% by weight and used for measurement. The YI value calculated by the above-described formula 3 was 0.6.
［実施例１７］発酵液の精製
　ｐＨ調整剤に水酸化ナトリウム水溶液を用いること以外は実施例１と同様に培養を行い、得られた乳酸発酵液から菌体を除くために、８０００×ｇで遠心分離した。遠心分離した上清に、ろ液重量０．５重量％分の活性炭を添加して、２５℃で１時間攪拌した。活性炭を除くために、ろ紙と０．４５μｍのメンブレンフィルターを使って吸引ろ過した。分光光度計における純水の４００ｎｍの波長の透過率を１００％とした場合、活性炭除去後のろ液の４００ｎｍの波長の透過率は９８．０％だった。得られたろ液を、ロータリーバキュームエバポレーター（ＥＹＥＬＡ社製）を使用し、１０～２０ｈｐａ、５０℃にて減圧濃縮を行い、乳酸ナトリウム濃度１９．７重量％の乳酸ナトリウム溶液を得た。 [Example 17] Purification of fermentation broth The culture was conducted in the same manner as in Example 1 except that a sodium hydroxide aqueous solution was used as a pH adjuster. Centrifuged. Activated charcoal having a filtrate weight of 0.5% by weight was added to the centrifuged supernatant, and the mixture was stirred at 25 ° C. for 1 hour. In order to remove the activated carbon, suction filtration was performed using a filter paper and a 0.45 μm membrane filter. When the transmittance at a wavelength of 400 nm of pure water in the spectrophotometer was 100%, the transmittance at a wavelength of 400 nm of the filtrate after removal of activated carbon was 98.0%. The obtained filtrate was concentrated under reduced pressure at 10 to 20 hpa and 50 ° C. using a rotary vacuum evaporator (manufactured by EYELA) to obtain a sodium lactate solution having a sodium lactate concentration of 19.7% by weight.
　図２に示す二室式水分解電気透析装置を用いて乳酸ナトリウムの脱塩を行った。本実施例では、陽イオン交換膜（アストム社製、商品名：ネオセプタＣＭＸ）とバイポーラ膜（同、ネオセプタＢＰ－１）とが順番にそれぞれ１０枚ずつ（総有効膜面積は５５０ｃｍ２）配置され、酸室、塩基室が形成されたフィルタープレス型電気透析装置を用いた。酸室には活性炭処理後の乳酸ナトリウム溶液１０００ｇを、塩基室には純水１０００ｇをそれぞれ対応するタンクを設け、供給、循環した。なお、電極液としては５％硫酸水溶液１０００ｇを使用した。これらの装置を用いて、３０℃、一定電圧３０Ｖにて電気透析を行った。その結果、酸室から乳酸濃度２０．３重量％の乳酸溶液が９２０ｇ得られた。得られた乳酸溶液を上記と同様にロータリーバキュームエバポレーターで、乳酸濃度を９０．０重量％まで濃縮した。 Sodium lactate was desalted using the two-chamber water splitting electrodialysis apparatus shown in FIG. In this example, 10 cation exchange membranes (trade name: Neocepta CMX, manufactured by Astom Co., Ltd.) and 10 bipolar membranes (Neoceptor BP-1) are arranged in order (total effective membrane area is 550 cm 2 ). A filter press type electrodialyzer in which an acid chamber and a base chamber were formed was used. A tank corresponding to 1000 g of the sodium lactate solution after the activated carbon treatment was provided in the acid chamber, and 1000 g of pure water was provided in the base chamber and supplied and circulated. In addition, 1000 g of 5% sulfuric acid aqueous solution was used as the electrode solution. Using these apparatuses, electrodialysis was performed at 30 ° C. and a constant voltage of 30V. As a result, 920 g of a lactic acid solution having a lactic acid concentration of 20.3% by weight was obtained from the acid chamber. The obtained lactic acid solution was concentrated to 90.0% by weight with a rotary vacuum evaporator in the same manner as described above.
　得られた濃縮液に水を加え、水分濃度を３０％に調整した後、乳酸溶液１００ｇを滴下ロートに入れ、反応フラスコに滴下ロート、温度計、冷却管を取り付けた。１８０℃に加熱し、１０ｔｏｒｒ（１３３３Ｐａ）に減圧した。滴下温度を１００℃に保持し、０．５ｍＬ／ｍｉｎの滴下速度で滴下蒸留することにより、加水分解しながら、蒸留を行った。蒸留により得られた乳酸溶液の重合度ｎは１．１であり、このとき蒸留収率は９５重量％であった。 After adding water to the obtained concentrated liquid and adjusting the water concentration to 30%, 100 g of the lactic acid solution was placed in the dropping funnel, and a dropping funnel, a thermometer, and a cooling tube were attached to the reaction flask. It heated to 180 degreeC and pressure-reduced to 10 torr (1333 Pa). Distillation was carried out while hydrolyzing by maintaining the dropping temperature at 100 ° C. and performing drop distillation at a dropping rate of 0.5 mL / min. The polymerization degree n of the lactic acid solution obtained by distillation was 1.1, and the distillation yield was 95% by weight.
［実施例１８］重合
　実施例１７で得られた乳酸溶液を用いて、実施例１６と同様に重合を実施した。得られた重合物の分子量は、数平均分子量（Ｍｎ）２．１万、重量平均分子量（Ｍｗ）２１万であった。これより、分子量分布（Ｍｗ／Ｍｎ）は１０である。ＹＩ値は０．５であった。　 [Example 18] Polymerization Polymerization was carried out in the same manner as in Example 16 using the lactic acid solution obtained in Example 17. The molecular weight of the obtained polymer was a number average molecular weight (Mn) of 21,000 and a weight average molecular weight (Mw) of 210,000. Thus, the molecular weight distribution (Mw / Mn) is 10. The YI value was 0.5.
［実施例１９］
　実施例３～実施例４、実施例６～実施例１１でＤ－乳酸生産に使用した大腸菌と培地を用い、それら以外の条件を実施例１２と同様に設定し、Ｄ－乳酸発酵生産を行った。得られたＤ－乳酸塩を実施例１５と同様に精製した。
   また、実施例５でＤ－乳酸生産に使用した大腸菌と培地を用い、それら以外の条件を実施例１２と同様に設定し、培養を行った。得られたＤ－乳酸塩を精製で使用する活性炭量をろ液重量の１重量％とする以外の条件を実施例１５と同様に設定し精製した。
   上記により得られたＤ－乳酸を実施例１６と同様にそれぞれ重合した結果、数平均分子量（Ｍｎ）１．９万～２．１万、重量平均分子量（Ｍｗ）１８万～２５万、分子量分布８．５～１２、ＹＩ値０．２～２の重合物が得られ、いずれの試験区においても良好な重合物がえられた。 [Example 19]
Using E. coli and the medium used for D-lactic acid production in Examples 3 to 4 and 6 to 11, the other conditions were set in the same manner as in Example 12 to perform D-lactic acid fermentation production. It was. The obtained D-lactate was purified in the same manner as in Example 15.
In addition, E. coli and medium used for D-lactic acid production in Example 5 were used, and other conditions were set in the same manner as in Example 12 and cultured. The obtained D-lactate was purified in the same manner as in Example 15 except that the amount of activated carbon used for purification was 1% by weight of the filtrate.
The resulting D-lactic acid was polymerized in the same manner as in Example 16. As a result, the number average molecular weight (Mn) was 190000-21,000, the weight average molecular weight (Mw) 180,000-250,000, and the molecular weight distribution. A polymer having 8.5 to 12 and a YI value of 0.2 to 2 was obtained, and a good polymer was obtained in any test section.
［比較例３］
　比較例２の０．５重量％活性炭で処理したＤ－乳酸溶液を一晩攪拌した後、再度濾過して活性炭を除去した。さらに陰イオン交換カラム、陽イオン交換カラムに通してカルシウムイオン（Ｃａ２＋）、及び、硫酸イオン（ＳＯ４ ２－）をそれぞれ５０ｐｐｍ以下に除去し、Ｄ－乳酸８．５％の粗乳酸水溶液を得た。この液を蒸留精製し、乳酸濃度９９重量％の精製Ｄ－乳酸を得た。得られたＤ－乳酸を実施例１６と同様に重合した結果、数平均分子量（Ｍｎ）４．４万、重量平均分子量（Ｍｗ）８．０万、分子量分布（Ｍｗ／Ｍｎ）１．８、ＹＩ値１５の重合物が得られた。 [Comparative Example 3]
The D-lactic acid solution treated with 0.5% by weight activated carbon of Comparative Example 2 was stirred overnight and then filtered again to remove the activated carbon. Further, calcium ions (Ca 2+ ) and sulfate ions (SO 4 2− ) are respectively removed to 50 ppm or less through an anion exchange column and a cation exchange column, and a crude lactic acid aqueous solution of 8.5% D-lactic acid is obtained. Obtained. This solution was purified by distillation to obtain purified D-lactic acid having a lactic acid concentration of 99% by weight. As a result of polymerizing the obtained D-lactic acid in the same manner as in Example 16, the number average molecular weight (Mn) was 44,000, the weight average molecular weight (Mw) was 80000, the molecular weight distribution (Mw / Mn) was 1.8, A polymer having a YI value of 15 was obtained.
[比較例４]
　比較例２の３．０重量％活性炭を加えたＤ－乳酸溶液を一晩攪拌した後、再度濾過して活性炭を除去した。さらにさらに陰イオン交換カラム、陽イオン交換カラムに通してカルシウムイオン（Ｃａ２＋）、及び、硫酸イオン（ＳＯ４ ２－）をそれぞれ５０ｐｐｍ以下に除去し、Ｄ－乳酸８．５％の粗乳酸水溶液を得た。この液を蒸留精製し、乳酸濃度９９重量％の精製Ｄ－乳酸を得た。得られたＤ－乳酸を実施例１６と同様に重合した結果、数平均分子量（Ｍｎ）２．０万、重量平均分子量（Ｍｗ）１８．０万、分子量分布（Ｍｗ／Ｍｎ）９．０、ＹＩ値２の重合物が得られた。
　比較例３、４より、コーンスティープリカー培地で生産したＤ－乳酸では、活性炭処理工程の活性炭量を増やし精製度を高めなければ、高い質量平均分子量や広い分子量分布のポリ乳酸を得ることができなかった。 [Comparative Example 4]
The D-lactic acid solution added with 3.0% by weight activated carbon of Comparative Example 2 was stirred overnight and then filtered again to remove the activated carbon. Further, calcium ions (Ca 2+ ) and sulfate ions (SO 4 2− ) are each removed to 50 ppm or less through an anion exchange column and a cation exchange column, and a crude lactic acid aqueous solution of 8.5% D-lactic acid is used. Got. This solution was purified by distillation to obtain purified D-lactic acid having a lactic acid concentration of 99% by weight. As a result of polymerizing the obtained D-lactic acid in the same manner as in Example 16, the number average molecular weight (Mn) was 20,000, the weight average molecular weight (Mw) was 180000, the molecular weight distribution (Mw / Mn) was 9.0, A polymer having a YI value of 2 was obtained.
From Comparative Examples 3 and 4, with D-lactic acid produced in corn steep liquor medium, polylactic acid with high mass average molecular weight and broad molecular weight distribution can be obtained unless the amount of activated carbon in the activated carbon treatment process is increased and the degree of purification is increased. There wasn't.
　この出願は、２０１２年７月２３日に出願された日本出願特願２０１２－１６３０２８号を基礎とする優先権を主張し、その開示の全てをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2012-163028 filed on July 23, 2012, the entire disclosure of which is incorporated herein.

Claims (17)

  1. 　無機電解質と炭素源とを含み、下記（ａ）、（ｂ）、（ｃ）及び（ｄ）からなる群より選択される少なくとも１つを満たす無機塩培地でＤ－乳酸生産大腸菌を培養することによりＤ－乳酸を生産する、Ｄ－乳酸の生産方法。
    （ａ）前記無機塩培地中の前記炭素源以外の有機物の含有量が、培養開始時に１０ｇ／Ｌ以下である。
    （ｂ）前記無機塩培地中の前記無機電解質の含有量が、培養開始時に１１ｇ／Ｌ以下である。
    （ｃ）前記無機塩培地中のチアミンの含有量が０．１ｍｇ／Ｌ以下である。
    （ｄ）前記無機塩培地中の遷移金属イオンの含有量が１０ｍｇ／Ｌ以下である。     Culturing D-lactic acid-producing Escherichia coli in an inorganic salt medium containing an inorganic electrolyte and a carbon source and satisfying at least one selected from the group consisting of (a), (b), (c) and (d) below: A method for producing D-lactic acid, wherein D-lactic acid is produced by the method described above.
    (A) Content of organic substances other than the carbon source in the inorganic salt medium is 10 g / L or less at the start of culture.
    (B) The content of the inorganic electrolyte in the inorganic salt medium is 11 g / L or less at the start of culture.
    (C) The content of thiamine in the inorganic salt medium is 0.1 mg / L or less.
    (D) The content of transition metal ions in the inorganic salt medium is 10 mg / L or less.
  2. 　前記無機塩培地は、少なくとも前記（ｃ）または前記（ｄ）のいずれかを満たす、請求項１に記載のＤ－乳酸の生産方法。 2. The method for producing D-lactic acid according to claim 1, wherein the inorganic salt medium satisfies at least one of (c) and (d).
  3. 　ポリマー原料となるＤ－乳酸を生産するための、請求項１または２に記載のＤ－乳酸の生産方法。 The method for producing D-lactic acid according to claim 1 or 2, for producing D-lactic acid as a polymer raw material.
  4. 　培養開始時の前記無機塩培地がさらに下記（ｅ）を満たす、請求項１乃至３のいずれか一項に記載のＤ－乳酸の生産方法。
    （ｅ）前記無機塩培地中の前記遷移金属イオンの含有量が０．８５ｍｇ／Ｌ以下である。     The method for producing D-lactic acid according to any one of claims 1 to 3, wherein the inorganic salt medium at the start of culture further satisfies the following (e).
    (E) The content of the transition metal ion in the inorganic salt medium is 0.85 mg / L or less.
  5. 　前記炭素源が糖である、請求項１乃至４のいずれか一項に記載のＤ－乳酸の生産方法。 The method for producing D-lactic acid according to any one of claims 1 to 4, wherein the carbon source is sugar.
  6. 　前記糖は、グルコース、フルクトース、キシロース、スクロース、グリセリン、アラビノース、メリビオース、トレハロース、マルトース、メリビオン酸、ラクトース、マルトトリオース、リボース、ガラクトース、ガラクツロン酸、グルコン酸、グルコサミン、グルクロン酸、マンニトール、マンノース、サッカリン酸、ソルビトール、フコース、ラムノース、アロース、及びＮ-アセチルグルコサミンからなる群より選択される１以上の化合物を含む、請求項５に記載のＤ－乳酸の生産方法。 The sugar is glucose, fructose, xylose, sucrose, glycerin, arabinose, melibiose, trehalose, maltose, melibionic acid, lactose, maltotriose, ribose, galactose, galacturonic acid, gluconic acid, glucosamine, glucuronic acid, mannitol, mannose, 6. The method for producing D-lactic acid according to claim 5, comprising one or more compounds selected from the group consisting of saccharic acid, sorbitol, fucose, rhamnose, allose, and N-acetylglucosamine.
  7. 　前記無機電解質が、カリウムイオン、リン酸イオン、アンモニウムイオン、硫酸イオン及びマグネシウムイオンからなる群より選択される１以上のイオンを構成成分として含む、請求項１乃至６のいずれか一項に記載のＤ－乳酸の生産方法。 7. The inorganic electrolyte according to claim 1, wherein the inorganic electrolyte includes one or more ions selected from the group consisting of potassium ions, phosphate ions, ammonium ions, sulfate ions, and magnesium ions as a constituent component. A method for producing D-lactic acid.
  8. 　前記無機電解質が、カリウムイオンを含み、
    　培養開始時の前記無機塩培地において、前記カリウムイオンの濃度が５．８ｍｍｏｌ／Ｌ以上７３ｍｍｏｌ／Ｌ以下である、請求項１乃至７のいずれか一項に記載のＤ－乳酸の生産方法。     The inorganic electrolyte contains potassium ions;
    The method for producing D-lactic acid according to any one of claims 1 to 7, wherein a concentration of the potassium ion is 5.8 mmol / L or more and 73 mmol / L or less in the inorganic salt medium at the start of culture.
  9. 　前記無機塩培地が、前記無機電解質を水に溶解又は懸濁して調製された液体である、請求項１乃至８のいずれか一項に記載のＤ－乳酸の生産方法。 The method for producing D-lactic acid according to any one of claims 1 to 8, wherein the inorganic salt medium is a liquid prepared by dissolving or suspending the inorganic electrolyte in water.
  10. 　前記無機塩培地が、さらにベタインを含む、請求項１乃至９のいずれか一項に記載のＤ－乳酸の生産方法。 The method for producing D-lactic acid according to any one of claims 1 to 9, wherein the inorganic salt medium further contains betaine.
  11. 　前記Ｄ－乳酸生産大腸菌が、組換え大腸菌である、請求項１乃至１０のいずれか一項に記載のＤ－乳酸の生産方法。 The method for producing D-lactic acid according to any one of claims 1 to 10, wherein the D-lactic acid-producing E. coli is a recombinant E. coli.
  12. 　前記無機塩培地において前記Ｄ－乳酸生産大腸菌と前記炭素源とを接触させてＤ－乳酸塩を得る工程と、
    　前記Ｄ－乳酸塩を含む前記無機塩培地から前記Ｄ－乳酸生産大腸菌を除去した後に前記Ｄ－乳酸塩を脱塩してＤ－乳酸を得る工程と、
    　を含む、請求項１乃至１１のいずれか一項に記載のＤ－乳酸の生産方法。     Contacting the D-lactic acid-producing Escherichia coli with the carbon source in the inorganic salt medium to obtain D-lactate;
    Removing D-lactic acid-producing Escherichia coli from the inorganic salt medium containing the D-lactate and then desalting the D-lactate to obtain D-lactic acid;
    The method for producing D-lactic acid according to any one of claims 1 to 11, comprising:
  13. 　前記Ｄ－乳酸塩を得る前記工程において、Ｄ－乳酸のアルカリ土類金属塩を得て、
    　前記Ｄ－乳酸を得る前記工程において、Ｄ－乳酸の前記アルカリ土類金属塩を脱塩することでアルカリ土類金属を含む無機塩を析出させる請求項１２に記載のＤ－乳酸の生産方法。     In the step of obtaining the D-lactate, an alkaline earth metal salt of D-lactic acid is obtained,
    13. The method for producing D-lactic acid according to claim 12, wherein, in the step of obtaining the D-lactic acid, an inorganic salt containing an alkaline earth metal is precipitated by desalting the alkaline earth metal salt of D-lactic acid.
  14. 　前記Ｄ－乳酸塩を得る前記工程において、Ｄ－乳酸のアルカリ金属塩を得て、
    　前記Ｄ－乳酸を得る前記工程において、電解透析処理によりＤ－乳酸の前記アルカリ金属塩を脱塩する、請求項１２に記載のＤ－乳酸の生産方法。     In the step of obtaining the D-lactate, an alkali metal salt of D-lactic acid is obtained,
    13. The method for producing D-lactic acid according to claim 12, wherein, in the step of obtaining the D-lactic acid, the alkali metal salt of D-lactic acid is desalted by electrodialysis.
  15. 　前記Ｄ－乳酸塩を脱塩してＤ－乳酸を得る前記工程の後に前記Ｄ－乳酸の精製を行う精製工程を更に含み、
    　前記精製工程において、加水分解しながら蒸留を行う、請求項１２乃至１４のいずれか１項に記載のＤ－乳酸の生産方法。     And further comprising a purification step of purifying the D-lactic acid after the step of desalting the D-lactate to obtain D-lactic acid,
    The method for producing D-lactic acid according to any one of claims 12 to 14, wherein in the purification step, distillation is carried out while hydrolysis.
  16. 　請求項１乃至１５のいずれか一項に記載のＤ－乳酸の生産方法により得られたＤ－乳酸を用いて重合反応を行う、ポリマーの生産方法。 A method for producing a polymer, wherein a polymerization reaction is carried out using D-lactic acid obtained by the method for producing D-lactic acid according to any one of claims 1 to 15.
  17. 　請求項１６に記載のポリマーの生産方法で得られるポリマー。 A polymer obtained by the polymer production method according to claim 16.
PCT/JP2013/069876 2012-07-23 2013-07-23 Method for producing d-lactic acid, method for producing polymer, and polymer WO2014017469A1 (en)

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