JPH06169795A - Production of optically active carboxylic acid and its antipode ester - Google Patents

Abstract

PURPOSE:To efficiently separate the subject compound from a reactional system and purify the compound because of a low elusion of proteins from a catalyst into the reactional system by asymmetrically hydrolyzing a carboxylate ester with a specific immobilized vital catalyst. CONSTITUTION:A carboxylate ester of formula I (R1 is alkyl, aryl, etc.; R2, R3 are alkyl; (n) is 1, 2) (e.g. beta-acetylthio-alpha-methylpropionic acid methyl ester) is treated with an immobilized vital catalyst to provide the objective compound of formula II, the immobilized vital catalyst being produced by treating bacterial cells with a multifunctional crosslinking agent, and the bacterial cells having an ability to asymmetrically hydrolyze the ester bond. The bacterial cells asymmetrically hydrolyzing the ester bond is preferably Pseudomonas putida MR-2068 strain (FERN P-3846), etc. The multifunctional agent includes glutar aldehyde. The hydrolysis reaction is preferably performed at >=45 deg.C for 4-15hrs.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the general formula [1]:

[0002]

[Chemical 3]

(Wherein 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 an immobilized biocatalyst is attached to the carboxylic acid ester. By acting, the general formula [2]:

[0004]

[Chemical 4]

The present invention relates to a method for producing an optically active carboxylic acid represented by the formula: wherein R ₁ , R ₂ and n have the same meanings as defined above, and an antipodal ester thereof.

[0006]

The optically active carboxylic acid represented by the above general formula [2] and its antipodal ester are useful as raw materials for synthesizing various physiologically active substances. For the first time, a method of asymmetrically hydrolyzing a racemic carboxylic acid ester represented by [1] using an enzyme or a microorganism has been proposed (for example, JP-A-60-12992 and JP-A-60-12992).
-See the 12993 publication, etc.).

The present inventors have isolated and obtained Pseudomonas putida MR-2068 from soil as this esterase-producing strain having a high asymmetric hydrolysis activity.
We are proposing a stock strain (Microtechnical Lab. No. 3846).
1-222798). The inventors have further shown that recombinant D
Applying NA technology, Pseudomo
nas putida ) MR-2068 strain (Microtechnology Research Institute No. 3846), a recombinant DNA microorganism that expresses a large amount of esterase gene Escherichia coli MR-2103 strain
We have succeeded in the development of (Microtechnology Research Institute No. 3835) (see Japanese Patent Application No. 3-249923).

[0008]

When asymmetrically hydrolyzing the above-mentioned carboxylic acid ester [1], it is preferable to omit complicated operations such as purification of the enzyme and use the bacterial cell itself as the enzyme source. Usually done. At this time, separation and purification of the product from the enzyme reaction-terminated liquid can be carried out by an ordinary method such as extraction, recrystallization, column chromatography and the like. In this operation, the presence of denatured protein causes a decrease in operability and a decrease in purification yield, and it has been desired to develop an immobilized biocatalyst with a small amount of protein eluted into the reaction system. Therefore, an object of the present invention is to efficiently separate and purify the product from the reaction system when asymmetrically hydrolyzing the carboxylic acid ester [1]. It is to provide a method using a catalyst.

[0009]

Means for Solving the Problems In the reaction for asymmetrically hydrolyzing the above-mentioned carboxylic acid ester [1] to obtain optically active carboxylic acid [2] and its enantiomer ester, in the reaction system, As a result of extensive research on biocatalysts that can efficiently separate and purify the product from bacterium, immobilization by treating cells having asymmetric hydrolysis of ester bond with polyfunctional crosslinking agent By using a biocatalyst, it was found that the product can be efficiently separated and purified from the reaction system, and the present invention has been completed.

That is, the present invention has the general formula [1]:

[0011]

[Chemical 5]

(Wherein 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 the ester bond is asymmetric. A general formula [2], characterized in that an immobilized biocatalyst obtained by treating a microbial cell having the ability to hydrolyze with a polyfunctional crosslinking agent is allowed to act.

[0013]

[Chemical 6]

The present invention relates to a method for producing an optically active carboxylic acid represented by the formula (wherein R ₁ , R ₂ and n have the same meanings as defined above) and its enantiomer ester. The general formula [1]
And [2], the alkyl group represented by R ₁ is preferably a lower alkyl group having 1 to 6 carbon atoms, for example, a methyl group or an ethyl group, the aralkyl group, for example, a benzyl group, or the aryl group. Examples thereof include a phenyl group, and the alkyl group represented by R ₂ or R ₃ is preferably a lower alkyl group having 1 to 6 carbon atoms, such as a methyl group or an ethyl group. And
Examples of the carboxylic acid ester [1] include methyl β-acetylthio-α-methylpropionate, methyl S-acetyl-β-mercaptoisobutyrate, and S-acetyl-γ-.
Examples thereof include methyl mercapto-α-methyl-n-butyrate, methyl S-benzoyl-β-mercaptoisobutyrate and methyl S-phenylacetyl-β-mercaptoisobutyrate.

The immobilized biocatalyst used in the present invention is preferably Pseudomonas.
putida ) MR-2068 strain or Escherichi coli ( Escherichi
a coli ) MR-2103 strain immobilized. The Pseudomonas putid
a ) MR-2068 strain and Escherichia col
i ) The MR-2103 strain has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, as Micro Engineering Research Article No. 3846 and Micro Engineering Research Article No. 3835, respectively.

The above Pseudomona
s putida ) MR-2068 strain and Eschericia coli ( Escheric
Cultivation of the hia coli ) MR-2103 strain can be carried out 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. The 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.

Generally, there are various methods for immobilizing a biocatalyst. For example, 1) a method in which an enzyme is bound to a water-insoluble carrier, 2) a cross-linking method in which an enzyme is cross-linked with a reagent having two or more functional groups (a polyfunctional cross-linking agent), 3) an enzyme is a polymer The encapsulation method includes encapsulation in a fine lattice of gel or coating with a semipermeable membrane of polymer. The immobilization of microorganisms is essentially the same as the enzyme immobilization method.

In the present invention, an immobilized biocatalyst obtained by treating a microbial cell having the ability to asymmetrically hydrolyze an ester bond with a polyfunctional crosslinking agent is used. The polyfunctional cross-linking agent that can be used in the present invention is not particularly limited in its type, and includes glutaraldehyde that forms a Schiff base, an isocyanic acid derivative that forms a peptide bond, N, N′-ethylene bismaleimide, and diazo coupling. It is possible to use bisdiazobenzidine which does the above, N, N′-polymethylenebisiodoacetamide which alkylates, and the like.

The form of the biocatalyst used in the production of the immobilized biocatalyst used in the present invention is a culture-finished solution, wet cells, dried cells (eg freeze-dried cells), spray-dried cells or an organic solvent (eg A bacterial cell treated with acetone, toluene, etc.) can be used. As a method for crosslinking the biocatalyst, any method can be applied, for example, cells are suspended in a pH buffer solution or physiological saline, a polyfunctional crosslinking agent is added thereto, and the cells are crosslinked to immobilize the living body. A method using a catalyst can be used. When using glutaraldehyde as a polyfunctional crosslinking agent, the amount added is usually 0.1-10.0% (wt%) based on the dry weight of the cells.
However, it is preferable to add it in the range of 0.5 to 5.0% (wt%). The cross-linking treatment temperature is usually within the range of 5 to 70 ° C., but it is possible to simultaneously sterilize the cells by treating at a temperature of 60 ° C. or higher, and it is also possible to deactivate the non-thermostable enzyme. . The cross-linking treatment time is usually in the range of 10 minutes to 5 hours, but it is desirable to perform the treatment for 1 hour or more.

In carrying out the hydrolysis reaction, a method of adding the immobilized biocatalyst to the carboxylic acid ester and a method of adding the immobilized biocatalyst to the reaction medium containing the carboxylic acid ester are possible. As the reaction medium, for example, ion-exchanged water or a buffer solution is used. The carboxylic acid ester concentration in the reaction medium is preferably 0.01 to 50% by weight. The amount of immobilized biocatalyst used varies depending on the catalytic activity, but is usually 0.001.
-10%, preferably 0.01-1%. The carboxylic acid ester may be added in a state of being suspended 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 usually 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 usually within the range of 5 to 80 ° C, but the immobilized biocatalyst used in the present invention has good thermal stability, and therefore the reaction is preferably performed at 45 ° C or higher. The reaction time varies depending on the reaction temperature and the like, but is usually 2 to
It is 48 hours, preferably 4 to 15 hours.

Separation and purification of the product from the reaction solution can be carried out by a conventional method such as extraction, recrystallization, column chromatography and the like. In the production method of the present invention, since the amount of protein eluted into the reaction system is small, separation and purification can be efficiently performed.

[0022]

EXAMPLES The present invention will be described in more detail below with reference to preparation examples and examples, but the scope of the present invention is not limited thereto. (Preparation Example 1) (1) Preparation of immobilized biocatalyst Pseudomonas putida MR-206
8 strains (Mikoken Kenjoyori No. 3846) were inoculated into 100 ml of liquid medium (pH 6.8) consisting of 0.5% meat extract, 0.75% peptone, 0.25% NaCl, 0.5% glucose and 0.15% malt extract, and 30 Shaking culture was carried out at 0 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized bacterial cell solution containing 16 mg of immobilized bacterial cells was added to 20 ml of M / 20 phosphate buffer solution, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 1. In each case, the untreated one was set to 100%.

[0023]

[Table 1]

(Preparation Example 2) (1) Preparation of immobilized biocatalyst Escherichia coli MR-2103 strain (Kikuko Kenjoyori No. 3835) was meat extract 0.5%, peptone 1.
100 ml of a liquid medium (pH 7.0) containing 0% and 0.5% of NaCl was inoculated and shake culture was carried out at 37 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized bacterial cell solution containing 16 mg of immobilized bacterial cells was added to 20 ml of M / 20 phosphate buffer solution, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 2. In each case, the untreated one was set to 100%.

[0025]

[Table 2]

[0026]

Example 1 Asymmetric hydrolysis of methyl (±) -β-acetylthio-α-methylpropionate In 90 ml of M / 20 phosphate buffer (pH 7.0) (±) -β-acetylthio-α-methylpropionic acid 10 g of methyl was added. 1 ml of an aqueous solution containing 500 mg of the immobilized biocatalyst prepared in Preparation Example 1 was added and reacted at 45 ° C. for 8 hours. During this time,
The pH of the reaction solution was adjusted to 7.0 with a 10% aqueous NaOH solution. The decomposition rate of methyl (-)-β-acetylthio-α-methylpropionate was 96%. After completion of the reaction, unreacted methyl β-acetylthio-α-methylpropionate was extracted with methyl isobutyl ketone (MIBK). Then, the pH of the aqueous layer of the extraction residual liquid was lowered to 2.0 or less with dilute sulfuric acid, and then (-)-β-acetylthio-α-methylpropionic acid was extracted with MIBK. Anhydrous sodium sulfate was added to the extract for dehydration, and the solvent was removed by evaporation to give an oil. The target (-)-β-acetylthio-α-methylpropionic acid was obtained in an amount of 4.2 g. Compared with the case of using the biocatalyst without immobilization treatment, the operability in the extraction operation and the extraction efficiency were improved. Table 3 shows a comparison of extraction efficiency of reaction products.

[0027]

[Table 3]

[0028]

Example 2 Non-proliferative hydrolysis of methyl (±) -β-acetylthio-α-methylpropionate In 90 ml of M / 20 phosphate buffer (pH 7.0), (±) -β-acetylthio-α-methylpropionic acid 10 g of methyl was added. 1 ml of an aqueous solution containing 3 mg of the immobilized biocatalyst prepared in Preparation Example 2 was added and reacted at 45 ° C. for 8 hours. During this time, 10
The pH of the reaction solution was adjusted to 7.0 with a% NaOH aqueous solution.
The decomposition rate of methyl (-)-β-acetylthio-α-methylpropionate was 95%. After reaction, unreacted β
Methyl -acetylthio-α-methylpropionate was extracted with methyl isobutyl ketone (MIBK). Then
After reducing the pH of the aqueous layer of the extraction residue to 2.0 or less with diluted sulfuric acid,
(−)-Β-Acetylthio-α-methylpropionic acid was extracted with MIBK. Anhydrous sodium sulfate was added to the extract for dehydration, and then the solvent was removed by evaporation to obtain a raffinate. The target (-)-β-acetylthio-α-methylpropionic acid was obtained in an amount of 4.3 g. Compared with the case of using the biocatalyst without immobilization treatment, the operability in the extraction operation and the extraction efficiency were improved. Table 4 shows a comparison of extraction efficiencies of reaction products.

[0029]

[Table 4]

[0030]

INDUSTRIAL APPLICABILITY In producing the optically active carboxylic acid represented by the general formula [2] or its enantiomer ester by the action of the biocatalyst, the present invention uses an immobilized biocatalyst as described in the specification. In comparison with the conventional production method, the operability and the yield in the separation and purification step of the reaction product can be significantly improved.

Claims (2)

[Claims] 1. A general formula [1]: (In the formula, R ₁ represents an alkyl group, an aralkyl group or an aryl group, R ₂ and R ₃ represent an alkyl group, and n represents 1 or 2.)
The carboxylic acid ester represented by the formula [2] is characterized by reacting an immobilized biocatalyst obtained by treating a microbial cell having an ability to asymmetrically hydrolyze an ester bond with a polyfunctional crosslinking agent. ]: [Chemical 2] (Wherein R ₁ , R ₂ and n have the same meanings as defined above), and a process for producing an optically active carboxylic acid and its enantiomer ester. Wherein the immobilized biocatalyst is Pseudomonas putida (Pseudomonas putida) MR-2068 strain or Eserikia
The method for producing an optically active carboxylic acid and its enantiomer ester according to claim 1, which is obtained by immobilizing Escherichia coli MR-2103 strain.