JPH0731486A - Method for producing optically active dicarboxylic acid - Google Patents

Abstract

(57) [Summary] [Structure] A method for producing an optically active dicarboxylic acid by allowing a microorganism belonging to the genus Arthrobacter or a treated product thereof to act on citraconic acid or halomaleic acid. [Effect] According to the method of the present invention, an optically active dicarboxylic acid useful as a synthetic raw material for medicines and agricultural chemicals can be efficiently produced with higher stereoselectivity as compared with conventional methods.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an optically active dicarboxylic acid from an unsaturated dicarboxylic acid by allowing a microorganism belonging to the genus Arthrobacter or a treated product thereof to act. Optically active dicarboxylic acids are
It is useful as a raw material or intermediate when synthesizing physiologically active substances such as pesticides.

[0002]

2. Description of the Related Art Optically active dicarboxylic acids such as malic acid derivatives and citramalic acid usually contain one or more asymmetric carbon atoms, so that they are not suitable for chemical synthesis. There were problems such as a low rate, a large number of reaction steps, and a low optical purity of the final product. On the other hand, biological reactions utilizing the action of microorganisms and enzymes have advantages such as high reaction specificity and the fact that the reaction proceeds at room temperature and atmospheric pressure, and are useful for fermentation of amino acids and production of antibiotics. It has been widely studied in the past. Also for optically active dicarboxylic acids, a production method utilizing a biological reaction has been reported.

For example, with respect to the production of R-(-)-citramalic acid, S. S. Subramanian et al. Used citraconic acid from a microorganism belonging to the genus Pseudomonas as R-
An enzyme activity of converting to (−)-citramalic acid has been obtained (J. Biol. Chem., 243 , 2367 (19).
68)). Katsuki et al. Reported that a microorganism belonging to the genus Bacillus converts citraconic acid into R-(-)-citramalic acid (J. Biochem., 70 , 44.
1 (1971)), Hasegawa, et al. R-(-)-citramalic acid is produced from citraconic acid using citracohinate hydratase (Japanese Patent Laid-Open No. 60-196192). Regarding the production of malic acid derivatives, a method for producing 2S, 3R-methylmalic acid by allowing a microorganism to act on citraconic acid has been reported (JP-A 61-19495). However, the production efficiency of the optically active dicarboxylic acid by these microbiological methods is not always sufficient, and development of a more excellent method is desired. Further, although the derivative of malic acid is highly useful, it is the current situation that production studies by microbiological methods have not been sufficiently conducted.

[0004]

Means for Solving the Problems The present inventors have conducted a study on a method for efficiently producing an optically active dicarboxylic acid by a microbiological method, and as a result, a microorganism belonging to the genus Arthrobacter or its The inventors have found that an optically active dicarboxylic acid can be efficiently produced by causing a treated product of cells to act on an unsaturated dicarboxylic acid, and have completed the present invention.

That is, the gist of the present invention is the following general formula (I)

[0006]

[Chemical 3]

[0007] A unsaturated dicarboxylic acid represented by the formula (R ¹ represents a methyl group or a halogen atom) is reacted with a microorganism belonging to the genus Arthrobacter or a treated product thereof. The following general formula (II)

[0008]

[Chemical 4]

(In the above formula, R ¹ is as defined in the above general formula (I), and R ² and R ³ each independently represent a hydrogen atom or a hydroxyl group, but at the same time represent the same substituent. However, when R ¹ represents a methyl group,
R ² represents a hydroxyl group. ) Exists in the manufacturing method of the optically active dicarboxylic acid.

The present invention will be described in detail below. The unsaturated dicarboxylic acid represented by the general formula (I) is a substance that serves as a substrate in the microbiological method of the present invention. In the general formula (I), when R ¹ is a methyl group, it represents citraconic acid, and when R ¹ is a halogen atom, it represents halomaleic acid. Examples of the halogen atom defined by R ¹ include a fluorine atom, a chlorine atom and a bromine atom. Therefore, in the present invention, citraconic acid produces R-(−)-citramalic acid, and halomaleic acid produces 2S, 3S-halomalic acid.

Microorganisms that catalyze the reaction are microorganisms belonging to the genus Arthrobacter, and include unsaturated dicarboxylic acids represented by the general formula (I) to optically active dicarboxylic acids represented by the general formula (II). There is no particular limitation as long as it has the ability to generate. In the present invention, the microorganism belonging to the genus Arthrobacter preferably has malate hydratase. Specifically, Arthrobacter sp that the present inventors have isolated from natural soil (A
rthrobacter sp. ) MCI2612 (F
ERM P-12562). The mycological properties of the microorganism are described in detail in JP-A-5-103680.

In the present invention, the above-mentioned bacterium can be easily grown by culturing it in a medium containing a nutrient which can be normally utilized by the microorganism. As a nutrient source,
Glucose, starch syrup, dextrin, sucrose, starch, sugar alcohol, molasses, organic acid, animal / vegetable oil and the like can be used. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed oil, meat extract, peptone,
Yeast extract, ammonium sulfate, sodium nitrate, ammonium nitrate, urea and the like can be used. Other as needed
It is useful to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, manganese, chlorine, phosphoric acid, sulfuric acid and other ions. Further, nutrients such as vitamins used for usual culture can be appropriately mixed in the medium.

As a culture method, the pH of the nutrient medium is 4.
The culture is performed at 0 to 9.5 and aerobically cultivated at 20 to 40 ° C for about 10 to 96 hours. The microorganism used in the present invention,
It means a microbial cell or a treated product of the microbial cell. The "treated microbial cell product" in the present invention is an extract of microbial cells,
It means a malate hydratase-containing material such as a ground product and an ultrasonically crushed product, and in the present invention, the microbial cell itself (viable cell or dried cell) may be used, or a microbial cell-treated product may be used. You may use.

The microbial cells or treated cells obtained by the above culture are adsorbed and bound to an immobilizing carrier such as polyacrylamide gel, photocrosslinkable resin, agar and carrageenan.
It is also possible to react by adding a substrate to a suspension suspended in an appropriate buffer solution or a suspension packed with such an immobilized product in a column. When the microbial cells themselves are allowed to act, a method of adding unsaturated dicarboxylic acid as a substrate to the nutrient medium to carry out the reaction at the same time with the culture, or a method of adding the substrate after the completion of the culture to carry out the reaction, etc. Can be used. The temperature during the reaction is preferably in the range of 20 to 50 ° C,
The pH is preferably in the range of 5.0 to 10.0. The concentration of the added substrate is preferably in the range of 0.1 to 10%. Further, it is possible to accelerate the reaction rate by adding an organic solvent such as toluene during the reaction.

When the treated product of the bacterial cells is allowed to act, the microorganism itself is allowed to act in an appropriate buffer using a malate hydratase-containing material such as an extract of the bacterial cells, a ground product, or an ultrasonically crushed product. The reaction can be carried out under the same conditions as in the case.
Specifically, after the completion of the culture, cells are separated from the culture solution by centrifugation or the like, the obtained cells are disrupted by an ultrasonic disruptor, etc., and the supernatant obtained by centrifugation or the like is ammonium sulfate ( (30-60%) treatment to obtain a precipitate, which is suspended in a small amount of water and then dialyzed to obtain a crude enzyme solution. This crude enzyme solution was adjusted to pH 7.5, for example, 0.01 to 0.
React with substrate in 1M Tris buffer. Such crude enzymes are further described in DEAE-toyopearl, Octyl.
-Purification using ion exchange chromatography such as sepharose, Ether-5PW,
Since a purified enzyme preparation can be obtained, such a purified enzyme preparation may be subjected to the reaction. The optimum p of the purified enzyme preparation
H is 8.0 and shows maximum activity at 45 ° C. Also pH
It is stable in the range of 7.0 to 9.0, and is stable up to 40 ° C. in the heat treatment for 30 minutes. It is composed of two subunits, and their molecular weights are 28,000 and 5 respectively by electrophoresis on SDS-polyacrylamide gel.
It is 8,000.

The optically active dicarboxylic acid of the present invention thus obtained can be extracted and purified by a conventional method. For example, the pH of the obtained optically active dicarboxylic acid-containing solution is lowered to around 1.0 using sulfuric acid or the like, and ammonium sulfate is added until saturation is reached. After that, extraction is performed several times with an equal amount of ethyl acetate or the like. The solvent is removed under reduced pressure to obtain an optically active dicarboxylic acid-containing material. Furthermore, dissolve this in a small amount of hexane, etc.
By performing silica gel chromatography eluting with a mixed solvent such as ethyl acetate, it can be easily separated from other impurities.

[0017]

EXAMPLES The method of the present invention will be further described with reference to the following examples.
The present invention will be specifically described, but the present invention does not exceed the gist thereof.
The present invention is not limited to the following examples. Example 1 Sorbitol 10 g, NH_FourNO₃ 6g, KH₂PO
_Four 3g, K₂HPO _Four 10.5 g, MgSO_Four・ 7
H₂O 250mg, yeast extract 5.5g, L-pu
Lorin 2 g, maleic acid 2 g, water 1 liter, pH 6.
In a liquid medium consisting of 0 composition,Arthrobacter
r sp. Inoculated with MCI2612 at 28 o'clock at 20:00
It was cultured for a while. The obtained culture solution is centrifuged to collect bacterial cells.
Therefore, the cells were washed with 25 mM phosphate buffer (pH 7.0).
This contains 40 g of citraconic acid and 10 ml of toluene.
Add 25 mM phosphate buffer (pH 7.0)
It was made 1 liter and reacted at 27 ° C. Reaction time
And the generated R-(-)-citramalic acid,
It shows in Table 1. In either case, the reaction proceeds rapidly and
45 hours after the start of the reaction, R-(-) with 100% optical purity was obtained.
-Citramalic acid was obtained in a yield of 43.5%. Still raw
The product can be confirmed by elemental analysis, NMR, and liquid chromatography.
It was carried out using

[0018]

[Table 1] Table 1 ────────────────────────────────── Reaction time Generated citramalic acid R-(- ) -Citramal Molar yield (hr) (g / l) Optical purity of acid (%) (%) ─────────────────────────── ──────── 3 2.5 100 5.5 5.5 8 6.5 100 14.3 11 8.6 100 18.9 25 15.0 100 32.9 45 19.8 100 43.5 ── ────────────────────────────────

Example 2 Cells were separated from the cell culture solution of Example 1 to obtain purified malate hydratase (80 units). 25mM Tris-HCl buffer containing 10g of chloromaleic acid (pH)
7.5) was added to bring the total volume to 100 ml, and the mixture was reacted at 28 ° C. After 10 hours, 2S, 3S- with an optical purity of 100%
6 g of chloromalic acid was obtained.

Example 3 To the purified malate hydratase obtained in Example 2, 25 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 10 g of bromomaleic acid was added to bring the total amount to 100 ml, and the temperature was 28 ° C.
It was made to react with. After 10 hours, 2S with an optical purity of 100%,
6 g of 3S-bromomalic acid was obtained.

[0021]

Industrial Applicability According to the method of the present invention, an optically active dicarboxylic acid useful as a synthetic raw material for medicines and agricultural chemicals can be produced with higher stereoselectivity and higher efficiency than conventional methods.

Claims (2)

[Claims] 1. The following general formula (I): (Wherein R ¹ represents a methyl group or a halogen atom) is reacted with a microorganism belonging to the genus Arthrobacter or a treated product thereof, which is represented by the following general formula: (II) [Chemical formula 2] (In the above formula, R ¹ is as defined in the above general formula (I), and R ² and R ³ each independently represent a hydrogen atom or a hydroxyl group, but do not represent the same substituent at the same time. , R ¹ represents a methyl group, and R ² represents a hydroxyl group.). 2. The method according to claim 1, wherein the microorganism belonging to the genus Arthrobacter or a treated product thereof has malate hydratase.