JPH0520074B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to the general formula The present invention relates to a method for producing an optically active carboxylic acid represented by the formula (wherein R ₁ is an alkyl group, an aralkyl group, or an aryl group, R ₂ is an alkyl group, and n is 1 or 2) and its enantiomer ester. [Prior Art] Carboxylic acids of the formula and their enantiomers are used as raw materials for synthesizing various physiologically active substances having optical activity. Conventionally, the method for producing the optically active carboxylic acid of the formula is to first synthesize a racemic carboxylic acid by organic synthesis and then resolve it using an optical resolving agent. A method of separating the enantiomers is known (Japanese Unexamined Patent Publication No. 118455-1989).
No. 56-81557, No. 57-188563, and European Patent Publication No. 79200477). on the other hand,
Optically active carboxylic acid esters are obtained by splitting the carboxylic acid and then performing an esterification reaction to produce optically active esters. [Problems to be solved by the invention] However, these methods require a large amount of expensive dividing agent, the dividing agent is likely to be mixed into the product as an impurity, and the dividing process is complicated. There are drawbacks such as, and it is not necessarily satisfactory as an industrial manufacturing method. As a method for improving these drawbacks, the present inventors previously proposed a method for producing an optically active carboxylic acid represented by the formula or its enantiomer ester by the action of microorganisms. (Unexamined Japanese Patent Publication No. 12993/1983, No. 60-
(See No. 30692 and No. 60-141297) The present inventors further conducted intensive research on a method for asymmetric hydrolysis of racemic carboxylic acid esters using microorganisms, and as a result, they newly discovered that Geotrichum spp. By using microorganisms,
It has been found that optically active carboxylic acids of the formula and their enantiomer esters can be efficiently produced. [Means for solving the problems] The present invention provides the general formula (In the formula, R ₃ represents an alkyl group, R ₁ , R ₂ 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. general formula (In the formula, R ₁ , R ₂ and n have the above meanings)
This is a method for producing an optically active carboxylic acid represented by the formula and its enantiomer ester. Examples of the alkyl group for the substituent R ₁ of the formula and the compound of the formula include methyl group, ethyl group, etc., examples of the aralkyl group include benzyl group, and examples of the aryl group include phenyl group. Examples of the ester () used in the present invention include methyl S-acetyl-β-mercaptoisobutyrate, methyl S-acetyl-γ-mercapto-α-methyl-n-butyrate, and methyl S-benzoyl-β-mercaptoisobutyrate. , S-phenylacetyl-β
-Methyl mercaptoisobutyrate and the like. The microorganism of the genus Geotrichum used in the present invention contains the above-mentioned compound []
Microorganisms that have the ability to asymmetrically hydrolyze the ester bonds of Geotrichum sp.
MR-3022 (Geotrichum sp. MR-3022) is mentioned. This bacterial strain was collected from soil in the Otake district of Hiroshima Prefecture, and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Science and Technology Research Institute No. 9318. This bacterial strain was identified as belonging to the known genus Geotrichum mainly by morphological observation. The mycological properties are described below. (A) 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 colonies that formed expanded almost concentrically as days passed. The colony is yeast-like in appearance;
The surface is milky white, slightly viscous, and has a flat, raised surface with wavy edges. (2) Slide culture on cornmeal agar medium and potato agar medium: Culture at 25℃. Conidium-forming hyphae with septa are produced, and cylindrical conidia are formed by cutting the hyphae. (3) MY liquid medium and wort medium: statically cultured at 25°C. Hyphae with septa were formed.
Proliferates by division and forms conidia. Its size was (10-15) μm×(20-40) μm. 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 on the 3rd day of culture, 1 week, and 3 weeks, and the carbon sources that were not assimilated even after 3 weeks of culture are listed. ) Assimilated on the 3rd day L-arabinose, D-xylose, D
-glucose, D-mannose, D-galactose, maltose, sucrose,
Lactose, melibiose, cellobiose, trehalose, raffinose, α-methyl-D-glucoside, alptin, dextrin, inosyte, ethanol. ) Assimilated in one week In addition to the above carbon sources, L-rhamnose, melezitose) Assimilated in 3 weeks In addition to the above carbon sources, D-arabinose, adonite, D-mannite, D-sorbit, succinate . ) Non-assimilable carbon sources L-sorbose, dulcitrate, citrate (3) Assimilation of nitrate: assimilation possible (4) Degradability of urea: with urease activity Microorganisms can be cultured in liquid culture or solid culture. But it can be done. As a medium, one containing appropriately blended components such as a carbon source, nitrogen source, vitamins, and minerals that can be normally assimilated by microorganisms is used. 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 at which microorganisms can grow, but it is usually carried out at a temperature of 50°C or less and a pH range of 2 to 11. Aeration and stirring may be performed to promote the growth of microorganisms. When carrying out the hydrolysis reaction, the ester (2) may be added to the medium at the beginning or during the culture, or the ester (2) may be added to the culture solution after culturing the microorganism 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, such as freeze-dried bacterial cells, spray-dried bacterial cells, bacterial cells treated with organic solvents such as acetone, toluene, etc., or bacterial cell destruction materials, bacterial cell extracts, etc. A processed product of bacterial cells 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 preferably 0.01 to 50% by weight.
The ester can also be added in suspension in water. The solubility of the ester can also be improved by adding an organic solvent such as methanol or acetone to the reaction solution. The pH 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 carboxylic acid, but in this case it is preferable to maintain the pH at an optimum level using a suitable neutralizing agent. The reaction temperature is preferably 5 to 50°C. Separation and purification of the product from the reaction solution or culture solution can be carried out by conventional methods such as extraction, recrystallization, column chromatography, etc. In the following examples, % means weight %. Example: Geotrichum sp. MR-3022 (Feikoken Bibori No. 9318) was mixed with 1% glucose, 0.5% peptone,
The cells were inoculated into 100 ml of a liquid medium (PH7.0) consisting of 0.5% meat extract and 0.1% yeast extract, and cultured with shaking at 30°C for 1 day. After the culture was completed, the culture solution was centrifuged, and the entire amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in 50 ml of M/10 phosphate buffer (PH7.0).
To this cell suspension, 25 ml of methyl (±)-S-acetyl-β-mercaptoisobutyrate was added and reacted by shaking at 30°C for 48 hours. The decomposition rate of methyl S-acetyl-β-mercaptoisobutyrate at this time was 49%. PH of reaction solution
7.0, and methyl S-acetyl-β-mercaptoisobutyrate was extracted with ethyl acetate. Next, after lowering the pH of the aqueous layer to 2.0 with sulfuric acid, the S in the aqueous layer was
-Acetyl-β-mercaptoisobutyric acid was extracted with ethyl acetate. After dehydrating the extract by adding anhydrous sodium sulfate, the solvent was removed by evaporation. The specific optical rotation of the extracted S-acetyl-β-mercaptoisobutyric acid and methyl S-acetyl-β-mercaptoisobutyrate was measured using a polarimeter (Model PM101, manufactured by Union Giken Co., Ltd.). The results are shown in Table 1. It is clear from this that an optically active carboxylic acid and its enantiomer ester are produced. 【table】

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ is an alkyl group, an aralkyl group, or an aryl group, R ₂ and R ₃ are an alkyl group, and n is 1 or 2). (Geotrichum), characterized by the action of a culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Geotrichum.
general formula (In the formula, R ₁ , R ₂ and n have the above meanings)
A method for producing an optically active carboxylic acid represented by and its enantiomer ester.