JPS63269998A - Production of optically active carboxylic acid and antipode ester thereof - Google Patents

Abstract

PURPOSE:To obtain an optically active carboxylic acid and its antipode ester from the corresponding ester, in high efficiency, by the asymmetric hydrolysis of the ester with a microorganism belonging to Geotrichum genus. CONSTITUTION:An ester of formula II (R1 is alkyl, aralkyl or aryl; R2 and R3 are alkyl; n is 1 or 2) is treated with a cultured liquid of microorganism belonging to Geotrichum genus and capable of asymmetrically hydrolyzing an ester bond [e.g. Geotrichum sp. MR-3022 (FERM P-9318)], cell of the microorganism or its treated product to obtain an optically active carboxylic acid of formula I (R1, R2 and n are same as defined above) and its antipode ester.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention is based on the general formula % ((wherein R1 is an alkyl group, an aralkyl group, or an aryl group, a horse is an alkyl group, and n is 1 or 2) ) and its enantiomer Esthe μ.

[Conventional technology]

Ponic acid of formula I and its enantiomer ester are used as raw materials for synthesizing various physiologically active substances having optical activity. Conventionally, as a method for producing the optically active carboxylic acid of formula I, the force μ of the racemate is determined in advance by organic synthesis.
A method is known in which a bonic acid is synthesized and then resolved using an optical resolving agent, that is, a method in which it is physicochemically separated into one optically active form and its enantiomer (Japanese Unexamined Patent Publication No. 55-11).
No. 8455, No. 56-81557, No. 57-1885
65 and European Patent Publication No. 79200477).

On the other hand, optically active carboxylic acid esters are obtained by splitting a ponic acid and then carrying out an esterification reaction to produce an optically active ester.

[Problem that the invention seeks to solve]

However, these methods have drawbacks such as the need for a large amount of expensive resolving agent, the ease with which this resolving agent is mixed into the product as an impurity, and the complexity of the resolving process, making industrial production methods difficult. This is not necessarily satisfactory.

As a way to improve these drawbacks, the present inventors have developed the formula I
We have previously proposed a method for producing optically active carboxylic acids represented by and their enantiomers by the action of microorganisms. (
(See JP-A-60-12993, JP-A-60-30692, and JP-A-60-141297) The present inventors further conducted extensive research on a method for asymmetric hydrolysis of racemic carboxylic acid esters using microorganisms. As a result, a new species called Geotrichu
It has been found that optically active acid and its opposite ester can be efficiently produced by using microorganisms of the genus M).

[Failure to solve the problem]

The present invention is based on the general formula % formula %[:] (in the formula, the crow represents the 7L' group, RI, R2 and n
has the above-mentioned meaning) is treated with a culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Geotrichum that has the ability to asymmetrically hydrolyze an ester bond. This is a method for producing an optically active carboxylic acid represented by the general formula % [:] (wherein R, , R, and n have the above-mentioned meanings) and its enantiomer ester.

Examples of the alkyl group for the substituent R1 of the compounds of formulas (1) and (2) include methiμ group, ethylμ group, etc.
The / group includes, for example, a benzy/I/ group, and the ary/l/ group includes, for example, a phenyl group.

For example, the esthetic/I/@ used in the present invention includes S
-methyl acetyl-β-mercaptoisobutyrate, S-acetyp-γ-meμcapto α-methyl-n-methyl butyrate,
S-benzoyl-β-mercaptoisobutyric acid methyl A/, S
-phenylacetyl-β-mercaptoisomethyl acetate and the like.

Geotrichu used in the present invention
Microorganisms of the genus m; are microorganisms that have the ability to asymmetrically hydrolyze the ester bond of the above-mentioned compound [■], such as Geotrichum sp. MR-5022 (Geotrichum sp.
ichum sp, MR-3022).

This strain was collected from soil in the Otake area of Hiroshima, and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as copy number 9518.

This bacterial strain was identified as belonging to the known genus Geotrichum mainly by morphological observation. The mycological properties are described below.

Growth status on each medium (1) MY agar medium; cultured on an agar plate at 25°C for 2 weeks. Growth was good, and the resulting colonies expanded in a concentric circle as days passed. The appearance of the colony is yeast-like, with a milky white surface and a flat, ridged surface with a wavy periphery.

(2) Slide culture on cornmeal agar medium and potato agar medium: Culture at 25°C. Conidium-forming hyphae with septa are produced, and cylindrical conidia are formed by cutting the hyphae.

(3) MY liquid medium and wort medium: Stationary culture was performed at 25°C. Hyphae with septa were formed. Proliferates by division and forms conidia.

Its size is (10-15) μm x (20-40), a
It was m. After one week of culture, a film was formed on the surface of the medium, and the precipitate was flocculent.

(B) Physiological test (1) Fermentability: None 2) Assimilation of carbon source: Cultured with shaking at 30°C for up to 3 weeks. The types of carbon sources that were assimilated during 3 days, 1 week, and 3 weeks of culture, and the carbon sources that were not assimilated even after 3 weeks of culture are listed.

1) L-afvinose, D-xylose, D-glucose, assimilated in 3 days,
D-mannose, D-gafuctose, ma-μdose, sucrose, lactose, melibiose, cellobiose, trehalose, raffinose, α-methyμmD-gμ
Coside, arbutin, dextrin, inodite, ethanol, lu' 11) Things that were assimilated in 1 week In addition to the above carbon sources, L-womenose, melezitose 11) Things that were assimilated in 3 weeks, the above carbon sources In addition, D-abbinose, atonite, D
-Mannitol, D-sorbitol, succinate IV) Non-assimilable carbon source L-sorbose, dulcitate, citrate (3) Assimilation of nitrate: Assimilable (4) Degradability of urea: Urease activity Cultivation of microorganisms can be carried out either in liquid culture or solid culture. The culture medium contains carbon sources, nitrogen sources, and
A mixture of vitamins, minerals, and other ingredients is used as appropriate. A small amount of ester may be added to the medium to improve the hydrolytic ability of the microorganism. Cultivation is carried out at a temperature and pH that allow microorganisms to grow, but is usually carried out at a temperature of 50° C. or lower and at a pH in the range of 2 to 11. Aeration and stirring may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, ester/I/ (n) may be added to the medium at the beginning or during the culture, or ester (n) may be added to the culture solution after culturing the microorganisms in advance. . Alternatively, the cells of the grown microorganism may be collected by centrifugation or the like and added to the reaction medium containing the ester.

In this case, for convenience of handling, the bacterial cells are dried bacterial cells such as freeze-dried bacterial cells, spray-dried bacterial cells, bacterial cells treated with an organic solvent such as acetone or toluene, or destroyed bacterial cells.
A processed product such as a bacterial cell extract can also be used. For example, ion-exchanged water or a buffer solution is used as the reaction medium.

The concentration of ester in the reaction medium or culture solution is [LO1-5
0% by weight is preferred. Esthep can also be added in a suspended state in water. The solubility of the ester can also be improved by adding an organic solvent such as methanol to acetone to the reaction solution. The pa of the reaction solution is 2 to 11, preferably 5 to 8.
is within the range of As the reaction progresses, the pH of the reaction solution decreases due to the generated acid, but in this case, it is preferable to maintain it at the optimum pI with an appropriate neutralizing agent.The reaction temperature is 5 to 50°C. is preferred.

Separation and purification of the product from the reaction solution or culture solution can be carried out by conventional methods such as extraction, recrystallization, cuff chromatography, etc.

In the following examples, ``chi'' means % by weight.

Example Geotrichum sp. MR-3022 (Feikoken Bibori No. 9318) was cultured in a liquid medium (p.
H7,0) 100+d was inoculated and cultured with shaking at 30°C for 1 day. After completion of the culture, the culture solution was centrifuged, and the entire amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in M/10 phosphate buffer (pH 7.0) 50-. This bacterial body s
2.5-methyl 8-acetyl-β-mercaptoisobutyrate was added to the cloudy solution, and the mixture was shaken at 30° C. for 48 hours to react.

At this time, S-acetyμ-β-meμcaptoisobutyric acid methyl
The decomposition rate was 49. The reaction solution was adjusted to pH 7.0, and methyl S-acetiμmβ-meμcaptoisobutyrate was extracted with ethyl acetate.

Next, the pa of the aqueous layer was lowered to 2.0 with sulfuric acid, and then S-acetyl-β-mecaptoisobutyric acid in the aqueous layer was extracted with ethyl acetate. After dehydrating the extract by adding anhydrous sodium sulfate, the solvent was removed by evaporation. Extracted 8-acetyumβ-mecaptoisobutyric acid and S-acetyl-β
- The specific optical rotation of methyl mercaptoisobutyrate was measured using an optical rotation needle (Model PM1mI) manufactured by Union Giken.

The results are shown in Table 1.

From this, it can be seen that optically active μ-bonic acid and its enantiomer ester are produced.

Table 1

Claims (1)

[Claims] 1. General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. Geotrichum (Geotrichum), which has the ability to asymmetrically hydrolyze the ester bond, is added to the expressed ester.
There are general formulas ▲ mathematical formulas, chemical formulas, tables, etc. ▼ (in the formulas, R_1, R_2 and n have the meanings above A method for producing an optically active carboxylic acid and its enantiomer ester having the following: