JPH0632635B2 - Process for producing optically active carboxylic acid and its enantiomer ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] (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 a process for producing an optically active carboxylic acid and an antipodal ester thereof.

[Conventional technology]

The carboxylic acid of formula I and its antipodal ester are used as raw materials for synthesizing various physiologically active substances having optical activity. Conventionally, as a method for producing an optically active carboxylic acid of the formula I, a method of synthesizing a racemic carboxylic acid by organic synthesis in advance and then resolving it using an optical resolving agent, that is, one optically active physicochemically There is known a method of classifying the substance into its enantiomer (JP-A-55-11).
No. 8455, No. 56-81557, No. 57-1885.
63, European Patent Publication No. 79200477). On the other hand, an optically active carboxylic acid ester has a method in which a carboxylic acid is cleaved and then an esterification reaction is performed to obtain an optically active ester.

However, these methods have drawbacks that a large amount of expensive resolving agent is required, this resolving agent is easily mixed in the product as an impurity, and the resolving process is complicated. As a result, I am not always satisfied.

As a way to remedy these drawbacks, we have described the formula I
Has previously proposed a method for producing an optically active carboxylic acid represented by the formula (1) or its enantiomer ester by the action of a microorganism (JP-A-60-12993 and JP-A-60-30692,
No. 60-94091), it is also described that microorganisms of the genus Cichudomonas can be used in the latter two, and in the examples, C. fluorescens (IFO3081) and C. varius (IAM1046) are described. Along with that, Cyudmonas putida (IFO12996) is described.

[Problems to be solved by the invention]

For these conventionally known microorganisms of the genus Cichudomonas, one of the isomers of the racemic carboxylic acid ester is selectively asymmetrically hydrolyzed to obtain a carboxylic acid with high optical purity and its enantiomer ester. Although it is excellent in terms of production when it is produced, it has a drawback that the productivity of the optically active carboxylic acid or ester per cell is low because the enzyme activity of the cell is low, and the creation of a microorganism that solves this point is strong. Was wanted.

As a result of intensive studies on the method of asymmetrically hydrolyzing racemic carboxylic acid ester using microorganisms, the present inventors have found that the strain Pseudomonas putida (Pseudomonas putida) newly isolated from soil in Otake district of Hiroshima prefecture is newly isolated. , Microtechnology Research Institute, No. 9677) has a very high enzyme activity,
It was found that the optically active carboxylic acid of formula I and its enantiomer ester can be efficiently produced by using the microorganism.

[Means for solving problems]

The present invention has the general formula (Wherein R ₃ represents an alkyl group, and R ₁ , R ₂ and n have the above-mentioned meanings), the deposit number having the ability to asymmetrically hydrolyze the ester is provided by 9
No. 677 microorganism Pseudomonas
Sputidb) culture solution, bacterial cells or treated product of bacterial cells (Wherein R ₁ , R ₂ and n have the above-mentioned meanings) and a method for producing an optically active carboxylic acid and its enantiomer ester.

Examples of the alkyl group for the substituent R ₁ of the compounds of the formulas I and II include a methyl group and an ethyl group, an aralkyl group such as a benzyl group, and an aryl group such as a phenyl group.

Examples of the ester (II) used in the present invention include S-
Acetyl-β-mercaptoisobutyrate methyl, S-acetyl-γ-mercapto-α-methyl-n-butyrate methyl, S
-Methyl benzoyl-β-mercaptoisobutyrate, methyl S-phenylacetyl-β-mercaptoisobutyrate and the like.

Microorganisms Microbiology Research Institute No. 9677 used in the present invention was collected from soil in the Otake district of Hiroshima Prefecture, and from the mycological properties shown in Table 1, it is possible to use C.
was identified as Eudomonas Putida), but this strain belongs to other microorganisms and IFO12 belonging to the same S.
The cell activity is higher than that of 996 or IFO03738.

Cultivation of the microorganism used in the present invention can be performed by liquid culture or solid culture. As the medium, a medium in which components such as a carbon source, a nitrogen source, vitamins and minerals that can normally be assimilated by microorganisms are appropriately mixed is used. A small amount of ester may be added to the medium in order to improve the ability of the microorganism to hydrolyze. Culturing is carried out at a temperature and pH at which the microorganism can grow, but it is usually carried out at a temperature of 50 ° C. or lower and a pH range of 2-11. Aeration and agitation may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, ester (II) may be added to the medium at the start or during the culture, or the ester (II) 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 ester-containing reaction medium.
In this case, the cells are, for convenience of handling, dried cells such as freeze-dried cells, spray-dried cells or cells treated with an organic solvent such as acetone or toluene, or a disrupted cell,
A microbial cell-treated product such as a microbial cell extract can also be used. As the reaction medium, for example, ion-exchanged water or a buffer solution is used. The concentration of the ester in the reaction medium or the culture solution is 0.01 to 5
0% by weight is preferred. The ester can also be added in suspension in water. An organic solvent such as methanol or acetone may be added to the reaction solution to improve the solubility of the ester. The pH of the reaction solution is in the range of 2 to 11, preferably 5 to 8. As the reaction proceeds, the pH of the reaction solution decreases due to the generated carboxylic acid. In this case, it is preferable to maintain the pH at an optimum level with a suitable neutralizing agent. The reaction temperature is preferably 5 to 50 ° C.

Separation and purification of the product from the reaction solution or the culture solution can be carried out by an ordinary method such as extraction, recrystallization, column chromatography and the like.

〔Example〕

 The present invention will be further described below with reference to examples.

In the following examples,% means% by weight. In addition, in the examples, the specific activity of the bacterial cells is S-acetyl-β-mercaptoisobutyric acid or S-per 1 g of the bacterial cells per 1 hour of enzyme reaction.
Production of methyl acetyl-β-mercaptoisobutyrate (mg)
Express with.

Example 1 0.5% of meat extract, 0.75% of peptone, and N of Pseudomonas putida
The cells were inoculated into 100 ml of a liquid medium (pH 6.8) consisting of aCl 0.25%, glucose 0.5%, malto extract 0.15%, and yeast extract 0.15%, and shake culture was carried out at 30 ° C for 1 day. After culturing,
The culture solution was centrifuged, and the whole amount of the obtained bacterial cells was washed with ion-exchanged water and then suspended in 50 ml of M / 10 phosphate buffer (pH 7.0). (±) -S-acetyl-β was added to the cell suspension.
-Add 2.5 ml of methyl mercaptoisobutyrate and add 2 at 30 ° C.
The reaction was carried out by shaking for 4 hours.

At this time, the specific activity of the cells used in the reaction was 3038 units, which was a very high value, and the decomposition rate of methyl S-acetyl-β-mercaptoisobutyrate was 49%. The reaction solution was adjusted to pH 7.0 and methyl S-acetyl-β-mercaptoisobutyrate was extracted with ethyl acetate. Then the pH of the aqueous layer was adjusted to 2.
After reducing to 0, S-acetyl-β-mercaptoisobutyric acid in the aqueous layer was extracted with acetic ester. After anhydrous sodium sulfate was added to the extract for dehydration, the solvent was removed by evaporation. The specific rotation of the extracted S-acetyl-β-mercaptoisobutyric acid and methyl S-acetyl-β-mercaptoisobutyrate was measured by Union Giken Optical Rotometer (PM101 type).
It was measured at. The results are shown in Table 2.

From this, it is understood that the optically active carboxylic acid and its antipodal ester are produced.

Comparative Examples 1 and 2 Instead of C. petitidae (Microtechnical Research Institute No. 9677), C. pupida IFO 03738 (Comparative Example 1) and C. petitda IFO 12996
An experiment was conducted under the same conditions as in Example 1 except that (Comparative Example 2) was used.

The results are shown in Table 3.

In Comparative Examples 1 and 2, the specific activity of the cells was lower than that of Example 1, and as a result, S-acetyl-β-after 24 hours of reaction was reacted.
It can be seen that the decomposition rate of methyl mercaptoisobutyrate is low.

〔The invention's effect〕

In producing the optically active carboxylic acid represented by the formula I or its enantiomer ester by the action of a microorganism, the present invention describes that the microorganism P. putida microbe containing an extremely highly active enzyme as described in the specification. Koken Bacteria No. 9
677), the productivity of the optically active carboxylic acid or its enantiomer ester per enzyme can be shortened by shortening the reaction time, charging the reaction solution, and lowering the bacterial cell concentration as compared with the conventional production method. It can be dramatically improved. Based on the effect, the present invention is industrially extremely significant in efficiently producing the compound.

Claims (1)

[Claims] 1. A general formula (In the formula, R ₁ is an alkyl group, an aralkyl group or an aryl group,
R ₂ and R ₃ are alkyl groups, and n is 1 or 2), and have the ability to asymmetrically hydrolyze the ester. Deposit number Microorganism Research Institute No. 9677 microorganism Pseudomonas putida ( Pseudomonas putida) culture solution, cells or a treated product of cells, the general formula A process for producing an optically active carboxylic acid represented by the formula (wherein R ₁ , R ₂ and n have the above-mentioned meanings) and an antipodal ester thereof.