JPH08245497A - Production of d(-)-tartaric acid - Google Patents

Abstract

PURPOSE: To provide a method for producing D(-)-tartaric acid by which the D(-)-tartaric acid important to a raw material for chemical industry is produced in high yield by stereospecifically hydrolyzing cisepoxysuccinic acid capable of being readily synthesized from maleic acid or maleic anhydride available in a large amount at a low cost according to a well-known method using a specific microorganism. CONSTITUTION: A cultured product of a microorganism, belonging to the genus Pseudomonas and having capability of hydrolyzing the epoxy ring of cisepoxysuccinic acid and producing D(-)-tartaric acid or its treated substance is mixed with the cisepoxysuccinic acid in a solution and reacted to enzymically convert the cisepoxysuccinic acid and produce the D(-)-tartaric acid. Thereby, the D(-)-tartaric acid is produced.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for producing D (-)-tartaric acid. Specifically, the biological reaction is used to stereospecifically hydrolyze cis-epoxysuccinic acid to give D (-)
The present invention relates to a method for industrially producing D (-)-tartaric acid, which is important as a raw material for the chemical industry, by converting it into tartaric acid.

[0002]

2. Description of the Related Art D (-)-tartaric acid is an optically active organic compound, and is an important compound used when synthesizing, for example, pharmaceutical and agrochemical products or their production intermediates.
However, D (-)-tartaric acid rarely exists in nature, and the present situation is that it is obtained mainly by optically resolving DL-tartaric acid.

By the way, DL-tartaric acid can be obtained by preparing it from natural tartar or by oxidizing fumaric acid with potassium permanganate or the like. However, it is simple because of problems such as stable supply of raw materials and cost. There was a problem of lacking.

On the other hand, utilizing the reaction specificity of living organisms,
There is a report on producing D (-)-tartaric acid from cis-epoxysuccinic acid using a microorganism belonging to the genus Alcaligenes (Japanese Patent Laid-Open No. 145586/50), but sufficient productivity is not obtained. It was the current situation.

[0005]

The object of the present invention is to
It is an object of the present invention to provide a method for producing (-)-tartaric acid from a raw material that can be inexpensively and easily supplied in high yield.

[0006]

The present inventors stereospecifically hydrolyze cis-epoxysuccinic acid, which can be easily supplied from maleic acid or maleic anhydride, which can be inexpensively supplied in large quantities, by a known method. , D (-)-tartaric acid in a high yield, the results of studies have revealed that such an object can be achieved by a biological reaction utilizing a specific microorganism, and the present invention is completed. I arrived.

That is, the gist of the present invention is to use a culture of a microorganism belonging to the genus Pseudomonas and having the ability to hydrolyze the epoxy ring of cis-epoxysuccinic acid to produce D (-)-tartaric acid, or a treated product thereof. Then, cis-epoxysuccinic acid is enzymatically converted to produce D (−)-tartaric acid, which is a method for producing D (−)-tartaric acid.

The present invention will be described in detail below. Cis-epoxysuccinic acid used as a raw material is described in, for example, J.
Org. Chem. , 24 , 54 (1959), Japanese Patent Publication Nos. 51-20490, 54-29486, and JP-A-6-271557, and the like.
It is obtained by epoxidizing maleic acid or maleic anhydride with hydrogen peroxide. Cis-epoxysuccinic acid is used in the reaction as a free acid or an appropriate salt.
Any salt may be used as long as it does not inhibit the reaction of the present invention, but in consideration of economical efficiency, etc., a monovalent or divalent ammonium salt, potassium salt, sodium salt, calcium salt or the like.
Valuable salts are preferred.

The microorganism used in the present invention belongs to the genus Pseudomonas, and hydrolyzes the epoxy ring of cisepoxysuccinic acid to produce D (-)-.
It is a microorganism capable of producing tartaric acid and is not particularly limited as long as it satisfies this condition. Specifically, Pseudomonas (Pseudomonas) is used.
putida), particularly Pseudomonas (Pseudomonas) separated from nature by the present inventors.
putida) MCI 3037 strain (hereinafter, "this strain")
Alternatively, it may be abbreviated as “MCI 3037 strain”. ) Is mentioned.

The mycological properties of the MCI 3037 strain are as follows. a. Morphological properties Cultivated on ordinary agar medium (Eiken) at 30 ° C. for 24 hours (1) Shape and size of cells; rod-shaped / 0.7-1.0 ×
2.0-4.0 μm (2) Cell polymorphism; None (3) Motility; Yes / Multiple polar flagella (4) Presence or absence of spores; None b. Cultivation properties (1) Meat broth agar culture: Half-lens-shaped circular colonies are formed by culturing at 30 ° C. for 24 hours. The color tone is yellowish white to light orange, opaque, and the surface of the colony is smooth. (2) Liquid culture of broth; good growth and strong turbidity are observed, but no film formation is observed on the liquid surface. (3) Meat broth gelatin puncture culture; at 22 ° C. culture, it grows linearly along the puncture line, but no liquefaction of gelatin is observed. (4) Litmus milk; no changes such as acid production, peptone formation and coagulation were observed. c. Physiological properties (1) Gram stain negative (2) Nitrate reduction negative (3) Vaginal reaction negative (4) MR test negative (5) VP test negative (6) Indole formation negative (7) Hydrogen sulfide formation negative (8) Starch hydrolysis Negative (9) Utilization of citric acid Positive (10) Pigment formation; Fluorescent pigment formation on King B medium (11) Urease negative (12) Oxidase positive (13) Catalase positive (14) Growth range: temperature = 9 to 37 ° C., pH = 5-1
0 (15) Attitude toward oxygen; did not grow under anaerobic conditions (16) OF test Oxidation (17) Acid formation from sugars L-arabinose + D-xylose + D-glucose + D-mannose + D- Fructose + D-galactose + maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol + inositol-glycerin + starch-(+: acid is generated,-: acid is not generated) (18) Hydrolysis of arginine Decomposition positive (19) Esculin hydrolysis negative (20) β-galactosidase negative (21) Assimilation of organic acids Potassium gluconate + n-capric acid + adipic acid-DL-malic acid + phenyl acetate + (+: substances (22) Cleavage of the benzene ring; ortho type d Chemotaxonomic properties (1) G + C content in DNA; 59.3% (2) isoprenoid quinone; ubiquinone Q9 (3) microbial cell fatty acid C12: 0 C16: 0 C16: 1 C18: 0 ΔC17: 0 2OH- C12: 0 3OH-C10: 0 e. Taxonomic consideration (1) Identification of family level This strain (MCI 3037 strain) is 1) an aerobic gram-negative bacillus, 2) oxidase- and catalase-positive, and 3) motility due to multiple polar flagella, 4) O
-F test uses glucose oxidatively 5) D
The (G + C) content in NA showed 59.3%, and the like.

[0012] Cowan and Steel's Guide to Medical Bacterial Identification, 3rd Edition (1993) and Bergey's Manual of Systematic Bacteriology, 1st.
Vol. 140-219 (1984), this strain is characterized by Pseudomonady
onacacea) family. (2) Identification of genus and species level Further, this strain produces 6) yellow-green fluorescent pigment on King B medium, 7) cleavage of benzene ring is ortho type, 8)
It does not have the liquefaction ability of gelatin. 9) It does not generate acid from sugars such as trehalose, sorbitol and inositol.
10) Isoprenoid quinone has ubiquinone Q9. 10) 2OH-C12: 0 and 3 as bacterial cell fatty acids.
It has characteristics such as having OH-C 10: 0. These characteristics are due to the fact that the strain is Pseudomonas.
as) Pseudomonas putida (Pseudo)
monas putida).

[0012] From the above, the strain MCI3037 of the present strain was obtained from Pseudomonas.
putida). In addition, MCI3037 strain was founded by the Institute of Biotechnology, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, Life Research Institute No. 1454.
Deposited as No. 8 (FERM P-14548).

In the present invention, the above-mentioned microorganism is used to enzymatically hydrolyze the epoxy ring of cis-epoxysuccinic acid to convert it into D (-)-tartaric acid. (Live cells or dried cells) may be used, or a treated product thereof may be used.

As used herein, the term "treated product" means an extract of microbial cells or a ground product of microbial cells, or a product obtained by separating and purifying it by a known method such as ammonium sulfate fractionation, ion exchange chromatography or gel filtration. The obtained crude product or purified product of the enzyme that catalyzes the hydrolysis reaction of the epoxy ring is obtained.

The culture of the microorganism may be either liquid culture or solid culture, but industrially, aeration-agitation culture using a liquid medium is convenient. Any medium can be used for the culture as long as the microorganism can grow and produce an enzyme that converts cisepoxysuccinic acid into D (−)-tartaric acid. For example, carbon sources include sugars such as glucose and fructose, alcohols, organic acids,
Corn stapler and molasses are used. As the nitrogen source, ammonia salt, nitrate, urea and the like are used. As the inorganic salt, various phosphates, sulfates, metal salts such as magnesium, potassium, manganese, iron and zinc are used.

If necessary, growth promoting factors such as yeast extract, vitamins and nucleotides are added.
Further, in order to induce the enzyme, it is advantageous to add cis-epoxysuccinic acid to the medium at the start of the culture or during the culture.

The pH of the medium is 5-10, preferably 6-
9. Culture temperature is 10 to 37 ° C, preferably 25 to 35 ° C
Aerobically for about 1 to 7 days. When the treated product of the microorganism is used in the present invention, for example, the culture of the microorganism is collected as it is or by centrifugation, filtration or the like,
A suspension of this in water or a buffer is used.

In the present invention, the culture of the above-mentioned microorganism or a treated product thereof and cis-epoxysuccinic acid are mixed in a solution to react. The concentration of cis-epoxysuccinic acid in the reaction solution is not particularly limited, but it is advantageous to make it as high as possible within a range that does not inhibit the activity of the culture or treated product. A preferable concentration is 30% by weight or less.

The reaction may be carried out by any of standing, stirring and shaking, but stirring or shaking is preferred for efficient reaction. Alternatively, a method in which the culture or treated product of the microorganism is immobilized on an appropriate support, packed in a column, and a cis-epoxysuccinic acid solution is flown can be used. Reaction temperature is 10 to 60 ° C, preferably 20 to 50 ° C, pH
Is 5-9, preferably 6-8.

The D (-)-tartaric acid produced in the medium or the reaction solution as described above is separated and purified by a known method. Specifically, after removing the bacterial cells, calcium salts such as calcium chloride and calcium carbonate are added to precipitate as D (-)-tartaric acid, which is filtered, washed with water, and then adjusted to pH 1.8 with sulfuric acid. The D (-)-tartaric acid solution obtained by removing the precipitate by
(-)-A method of precipitating crystals of tartaric acid, or the liquid after separating and removing the bacterial cells is adsorbed on a basic ion exchange resin, and the eluate obtained by elution with formic acid or the like after washing is concentrated under reduced pressure. There is a method of precipitating crystals of D (-)-tartaric acid.

The conversion of D (-)-tartaric acid thus obtained from cis-epoxysuccinic acid can be 99% or more if appropriate reaction conditions are selected.

[0022]

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to the methods described below as long as the gist thereof is not exceeded. <Example> Cis-epoxy succinic acid 1%, ammonium sulfate 0.2%, potassium phosphate 1 0.14%, disodium phosphate (12 hydrate) 0.31%, magnesium sulfate (heptahydrate) 0 0.05%, ferrous sulfate 0.001%, yeast extract 0.1%, 100 ml of liquid medium adjusted to pH 7.0 and sterilized at 120 ° C. for 20 minutes, M
The CI3037 strain was inoculated and shake-cultured at 30 ° C. for 24 hours. The bacterial cells were collected from the culture solution by centrifugation and suspended in 50 ml of water.

On the other hand, the substrate cis-epoxysuccinic acid 1.3
Dissolve 2 g in water, adjust the pH to 8 with sodium hydroxide, and then to 50 ml with water.
It was made to react at 50 ° C. for 5 hours.

Analysis of this reaction solution revealed that 52.6% of the cis-epoxysuccinic acid had been converted to tartaric acid. The reaction mixture after reacting at 30 ° C. for 10 hours was analyzed to find that cis-epoxysuccinic acid was 99.6.
It was found that% had been converted to tartaric acid.

The conversion rate to tartaric acid was determined by high performance liquid chromatography [analysis column: MCIGEL CK0
8E (manufactured by Mitsubishi Chemical Co., Ltd.)] was used to measure the tartaric acid concentration in the reaction solution using tartaric acid as a commercial reagent as a standard, and was calculated based on the following formula from the charged amount of cis-epoxysuccinic acid at the start of the reaction. It was done.

[0026]

[Equation 1] After removing bacterial cells from this reaction solution by centrifugation, 1M
11 ml of a calcium chloride solution was added, and the mixture was left overnight at 4 ° C., and the resulting precipitate was collected by centrifugation. The obtained precipitate was suspended in 50 ml of water and then centrifuged again to collect the precipitate. After suspending this precipitate in 50 ml of water, 1
The N sulfuric acid solution was added to adjust the pH to 1.8, and the supernatant obtained by centrifugation again was concentrated under reduced pressure to obtain 0.93 g of tartaric acid crystals.

The obtained tartaric acid crystals were made into an aqueous solution having a concentration of 10%, and the optical rotation of the aqueous solution was measured by JASCO DIP-3.
70 (manufactured by JASCO Corporation) was measured at a measurement temperature of 25 ° C., which showed an optical rotation of -15.3. As a standard, the optical rotation of commercially available D (-)-tartaric acid was measured by the same method to find that it was -15.6.

As described above, the optical rotation of the obtained tartaric acid is in good agreement with the optical rotation of the standard D (-)-tartaric acid, and the tartaric acid obtained in this Example is D (-)-tartaric acid. confirmed.

[0029]

INDUSTRIAL APPLICABILITY According to the method of the present invention, cis-epoxysuccinic acid which can be easily supplied from maleic anhydride or maleic anhydride, which can be supplied in large quantities at low cost, can be stereospecifically produced by using a specific microorganism. By hydrolysis, D (-)-tartaric acid, which is important as a raw material for the chemical industry, can be produced in high yield. Specifically, if appropriate reaction conditions are selected, a conversion rate to D (-)-tartaric acid of 99% or more can be achieved.

Further, compared with the case of using a conventional microorganism belonging to the genus Alcaligenes, the concentration rate of bacterial cells for the reaction is low, and higher productivity can be obtained.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshiya Kawaguchi 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi Chemical Corporation Yokohama Research Institute

Claims (2)

[Claims] 1. A culture of a microorganism which belongs to the genus Pseudomonas and has the ability to hydrolyze the epoxy ring of cis-epoxysuccinic acid to produce D (-)-tartaric acid, or a treated product thereof. To enzymatically convert D
(-)-D (-), characterized by producing tartaric acid
-A method for producing tartaric acid. 2. A microorganism belonging to the genus Pseudomonas and having the ability to hydrolyze the epoxy ring of cisepoxysuccinic acid to produce D (−)-tartaric acid is Pseudomonas putida. The manufacturing method described.