JP2003250595A - Method for optically resolving carboxylic acid ester by using microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active hydroxycarboxylic acid alkyl ester or an optically active hydroxycarboxylic acid, or an optically active tetrahydrofuran-2-carboxylic acid alkyl ester or an optically active tetrahydrofuran-2-carboxylic acid, capable of simply producing the esters or the acids at low costs in an industrially advantageous manner. <P>SOLUTION: This method for producing the optically active hydroxycarboxylic acid alkyl ester or the optically active hydroxycarboxylic acid, or the optically active tetrahydrofuran-2-carboxylic acid alkyl ester or the optically active tetrahydrofuran-2-carboxylic acid comprises making a microorganism belonging to Enterobacter or Citrobacter act on a racemic hydroxycarboxylic acid ester expressed by the formula: R<SP>1</SP>-(CH<SB>2</SB>)<SB>n</SB>-CHOH-(CH<SB>2</SB>)<SB>m</SB>- COOR<SP>2</SP>(R<SP>1</SP>is methyl or benzyl; R<SP>2</SP>is an alkyl; and (n) is 0 or 1 when (m) is 0, and (n) is 0 when (m) is 1) or a racemic tetrahydrofuran-2-carboxylic acid alkyl ester so as to optically resolving the ester, and collecting one of the optically active carboxylic acid alkyl esters which is remained or another optically active carboxylic acid alkyl ester which is formed. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optically active hydroxycarboxylic acid and an optically active tetrahydrofuran-2-carboxylic acid which can be useful as chiral building blocks or intermediates in the synthesis of optically active compounds such as pharmaceuticals, agricultural chemicals and physiologically active substances. And a method for producing such alkyl esters by microbial treatment.

[0002]

2. Description of the Related Art Halobacterium Halobacte
It has been reported that when Rium halobium) is allowed to act on racemic 3-hydroxybutanoic acid ester, R-form 3-hydroxybutanoic acid ester remains due to the carboxylic acid ester hydrolyzing reaction (see Non-Patent Document 1). However, a method for producing an S-form 3-hydroxybutanoic acid ester of high optical purity from a racemic 3-hydroxybutanoic acid ester using a microorganism having stereoselective esterolysis activity is not known.

Further, optically active tetrahydrofuran-2-
As a biochemical method of carboxylic acid alkyl ester (sometimes abbreviated as THFAE) or its acid (sometimes abbreviated as THFA), esterases derived from microorganisms or animals are allowed to act on THFAE, Patent Document 1 and Patent Document 2 disclose a method of dividing the optically active THFAE remaining by stereoselective hydrolysis and the optically active THFA produced. However, in both methods, the stereoselectivity is affected by the type of the alkyl side chain of THFAE, the stereoselectivity is low, and the yield of the optically active substance obtained is low. We have
For chloroalcohols such as racemic 4-chloro-3-hydroxybutanoate, enterobacter (Enter
bacterium), microbial cell of the genus Chitrobacter (Citrobacter) or its microbial extract is allowed to act to convert to S-form 3-hydroxy-γ-butyrolactone, and the remaining R-form 4-chloro-3-hydroxybutane Method for simultaneously recovering acid ester (Patent Document 3, Non-Patent Document 2)
Is found and reported.

As a result of further diligent research on the substrate specificity of the above-mentioned microbial cell reaction, the present inventors have found a property of stereoselectively hydrolyzing a carboxylic acid ester, and completed the present invention. The racemic hydroxycarboxylic acid alkyl ester or tetrahydrofuran-2-carboxylic acid alkyl ester used as a substrate in the present invention is different from a chloroalcohol compound such as 4-chloro-3-hydroxybutanoic acid ester, and therefore it is stereoselective. It is considered that the splitting reaction proceeds due to stereoselective carboxylic acid ester hydrolysis activity, rather than the reaction in which hydrochloric acid is generated as the dechlorination reaction proceeds. Further, in this reaction according to the present invention, it is a very interesting reaction to stereoselectively hydrolyze the carboxylic acid ester while stereoselectively recognizing the asymmetric carbons at the α-position and the β-position.

[0005]

[Patent Document 1] International Publication No. 01/92554 Pamphlet

[Patent Document 2] JP-A-14-171994

[Patent Document 3] Japanese Patent Laid-Open No. 9-47296

[Non-Patent Document 1] Ehrler and Seebach, Helv. Chim. Ac
ta, 72,793-799 (1989)

[Non-Patent Document 2] Suzuki et al., Enzyme and Microb.
Technol., 24, 13-20 (1999)

[0006]

SUMMARY OF THE INVENTION The object of the present invention is to provide a simple, inexpensive and industrially advantageous method for producing optically active hydroxycarboxylic acid and optically active tetrahydrofuran-2-carboxylic acid, and alkyl esters thereof. To provide.

[0007]

The present invention provides the following formula [1] R ^1- (CH ₂ ) _n -CHOH- (CH ₂ ) _m -COOR ² [1] (wherein R ¹ is a methyl group or A benzyl group, R ² is an alkyl group, and when m is 0, n is 0 or 1
And when m is 1, n is 0. ) Represented by racemic hydroxycarboxylic acid alkyl ester, or racemic tetrahydrofuran-2-carboxylic acid alkyl ester, a microorganism belonging to the genus Enterobacter or the genus Chitrobacter or having a carboxylic acid ester degrading activity or a product thereof is allowed to act, The ester is optically resolved, and one remaining optically active carboxylic acid alkyl ester or the other optically active carboxylic acid produced is collected to obtain an optically active hydroxycarboxylic acid alkyl ester or optically active hydroxycarboxylic acid, or Active tetrahydrofuran-2-carboxylic acid alkyl ester or optically active tetrahydrofuran-2
-Regarding a method for producing a carboxylic acid.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a racemic hydroxycarboxylic acid alkyl ester represented by the formula [1] or a racemic tetrahydrofuran-2-carboxylic acid alkyl ester (hereinafter simply referred to as racemic carboxylic acid alkyl ester). A microbial cell having a carboxylic acid ester-decomposing activity or a product thereof, which belongs to the genus Enterobacter or the genus Chitrobacter, is allowed to act, and one of the optically active carboxylic acids is converted into an optically active carboxylic acid by stereoselective ester hydrolysis reaction. The method is characterized in that the other optically active carboxylic acid alkyl ester is allowed to remain, and then one of the optically active substances is isolated and recovered.

In the present invention, preferred examples of the racemic hydroxycarboxylic acid alkyl ester used as the substrate are 3-hydroxybutanoic acid methyl or ethyl ester, 2-hydroxybutanoic acid methyl or ethyl ester, lactate methyl or ethyl ester, or 4-phenyl-2-hydroxybutanoic acid methyl or ethyl ester. Particularly preferred substrates are 3-hydroxybutanoic acid methyl or ethyl ester, 2-
Hydroxybutanoic acid methyl or ethyl ester. A preferred example of racemic tetrahydrofuran-2-carboxylic acid alkyl ester is its methyl or ethyl ester. The bacterial cell-producing ester-degrading enzyme used in the present invention has high specificity for its methyl ester among THFAEs, is efficient, and has a high yield.
For the first time, we have found to prepare FAE. The microorganisms used in the present invention are microorganisms belonging to the genus Enterobacter and microorganisms belonging to the genus Chitrobacter that produce an enzyme that catalyzes a stereoselective esterolysis reaction with respect to a racemic carboxylic acid alkyl ester. Specifically, Enterobacter sp DS isolated from soil by the present inventors
-S-75 strain, Citrobacter freundii DS-S-13 strain, Citrobact
er freundii DS-K-40 strain can be mentioned. These microorganisms have already been deposited at the National Institute of Advanced Industrial Science and Technology (formerly Institute of Industrial Science, Institute for Microbial Industry) with the following international deposit numbers. Enterobacter sp DS-S-75 strain: FERM BP-5494, Citrobact
er freundii DS-S-13 strain: FERM BP-5492, Citrobacter f
reundii DS-K-40 strain: FERM BP-5493.

The above microorganism Enterobacter sp DS-S-75 strain, C
itrobacter freundii DS-S-13 strain, and Citrobacter f
As a medium composition for culturing the reundii DS-K-40 strain, any medium can be used as long as it is a medium in which these microorganisms are normally grown. For example, as a carbon source, alcohols such as glycerol, D-sorbitol, D-mannitol, organic acids such as acetic acid, citric acid, malic acid, maleic acid, fumaric acid, gluconic acid and salts thereof, D-glucose, D-fructose. , A carbohydrate such as D-galactose or a mixture thereof with an inorganic nitrogen compound such as ammonium sulfate, ammonium nitrate and ammonium phosphate as a nitrogen source and an organic nitrogen compound such as urea, peptone, casein, yeast extract, meat extract and corn steep liquor. Mention may be made of mixtures thereof. In addition, phosphates, magnesium salts, potassium salts, manganese salts, iron salts, zinc salts, copper salts, and the like as inorganic salts, and vitamins may be added as necessary. Cultivation of the above microorganisms may be carried out by a conventional method, for example, a pH of 5 to 9, preferably 6.5 to 7.0, and a culture temperature of 20 to 40 ^° C., preferably 25 to 37 ^° C. It is preferable to carry out for 10 to 96 hours.

As a method for obtaining the optically active carboxylic acid alkyl ester remaining and the optically active carboxylic acid which is the product thereof by allowing the above-mentioned microorganism or its microbial product to act on the racemic carboxylic acid alkyl ester,
The microorganism according to the present invention is 1) a culture solution of the microorganism obtained by the above culture method, or 2) a bacterial cell obtained by centrifugation or membrane separation from the culture solution and a treated product of the bacterial cell (crushed bacterial cell or bacterial cell). (Extract) or 3) they are immobilized by a conventional method, and a substrate is added to a mixture of microbial cells mixed with a buffer. The reaction temperature is preferably 15 to 50 ^° C,
The reaction pH is preferably 6-9. The substrate concentration in the reaction solution is preferably 0.1 to 20% (v / v), and the substrates may be added all at once at the beginning or may be added in portions. In the case of divided addition, the amount of the added group may be calculated using the amount of alkali required for neutralization as an index, and the substrate may be added according to the decomposition activity. The alkali used is not particularly limited, and both strong bases and weak bases can be used, but when a weak base is used, the reaction rate and stereoselectivity may improve.

The reaction is usually carried out with stirring or shaking, and the reaction time is preferably several hours to 120 hours although it varies depending on the substrate concentration and the amount of growing cells. Preferably, the reaction is terminated when the mass of the residual group is 50% of the initial substrate concentration by analysis such as gas chromatography. The decrease in pH due to the optically active carboxylic acid produced along with the stereoselective ester hydrolysis reaction during the reaction is controlled by a weakly basic aqueous solution of an alkali metal compound or a suspension thereof, or an aqueous ammonia solution, so that the reaction pH It is possible to effectively obtain the optically active carboxylic acid ester of high optical purity and the optically active carboxylic acid by controlling the pH of the microorganism at the optimum pH of the microorganism or its microbial cell-producing enzyme. The weakly basic alkali metal compound is an alkali metal carbonate, an alkali metal hydrogencarbonate, an ammonium carbonate, or an ammonium hydrogencarbonate. Specific examples thereof include aqueous solutions or aqueous suspensions of aqueous ammonia, sodium carbonate, potassium carbonate, ammonium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, ammonium hydrogen carbonate, and calcium carbonate.

Thus, the optically active carboxylic acid alkyl ester and the optically active carboxylic acid remaining in the reaction solution can be recovered and purified by a general method. For example, after removing the bacterial cells from the reaction solution by centrifugation, the supernatant is extracted with a solvent such as ethyl acetate, and the ethyl acetate phase is fractionated with the optically active carboxylic acid alkyl ester, and the aqueous phase is fractionated with the optically active carboxylic acid. Can be extracted. Further, the supernatant after removing the bacterial cells is concentrated by an evaporator to remove the aqueous phase, and the solvent is replaced with ethyl acetate or the like. After dehydrating the solution with anhydrous magnesium sulfate, the solvent is removed under reduced pressure to obtain the optically active carboxylic acid alkyl ester and the crude syrup of the optically active carboxylic acid. Further, it may be purified by distillation or column chromatography using silica gel.

[0014]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. In addition,% in the examples represents (w / v) unless otherwise specified. Example 1 1.0 each of peptone, yeast extract and glycerol
% Of a nutrient medium (pH 7.2) containing 2.5 L was sterilized at 121 ° C. for 15 minutes in a 5 L jar fermenter (incubator).
The Enterobacter sp. DS-S-75 strain, which is the test bacterium, was preliminarily prepared by culturing the nutrient medium (pH 7.2) containing peptone, yeast extract, and glycerol in an amount of 1.0% each at 30 ° C for 16 hours with shaking. The above medium was aseptically inoculated with 2% (v / v).
And temperature 30 ℃, ventilation rate 0.5L / min, rotation speed 5
Aeration-agitation culture was carried out for about 24 hours under the condition of 00 rpm. After the completion of the culture, the culture solution was taken out, collected by centrifugation, washed with 20 mM phosphate buffer (pH 7.2) containing 2 mM magnesium sulfate three times, and 100 g of resting cells were prepared. 50 g of the resting cells are 2500 ml of the above buffer solution
Suspended in the same 5 L jar fermenter (turbidity 9.5 OD (turbidity at 660 nm)), 200 g of racemic 3-hydroxybutanoic acid ethyl ester was added,
The optical resolution reaction was carried out for 4 hours with stirring. Note that p
5% calcium carbonate (25 g) was added to the reaction as an H neutralizer.

The 3-hydroxybutanoic acid ethyl ester remaining in the reaction solution at that time was subjected to gas chromatography.
(Column carrier: PEG20M, 60-80 mesh) As a result of analysis,
The residual rate was 48%. After completion of the reaction, bacterial cells were removed and the mixture was extracted twice with an equal amount of ethyl acetate. After dehydrating the ethyl acetate layer with anhydrous magnesium sulfate, the solvent was removed under reduced pressure to obtain 77 g of syrup containing 3-hydroxybutanoic acid ethyl ester. The substance was identified and quantified by the same gas chromatography, and confirmed by the retention time with the original compound. After trifluoroacetic acid conversion of 3-hydroxybutanoic acid ethyl ester in this syrup with trifluoroacetic anhydride, a capillary column G-TA (0.25 mm x 30) manufactured by Astec Co.
The optical isomers were analyzed by gas chromatography using m). As a result, it was found that the recovered 3-hydroxybutanoic acid ethyl ester was the S form with an optical purity of 99.1% ee.

On the other hand, 3-hydroxybutanoic acid remaining in water was identified and quantified by the same gas chromatography as the retention time with the original compound. At that time, the pH of the aqueous solution was adjusted to 4 with hydrochloric acid. As a result, it was found that 2.2% (w / v) of 3-hydroxybutanoic acid remained. The aqueous solution was removed by an evaporator to obtain 44 g of 3-hydroxybutanoic acid syrup. Then, put 50 mg of this syrup in a 30 ml Erlenmeyer flask, and cool with ice, 122 mg of 4-dimethylaminopyridine and 10 m of dichloromethane.
l, 100 μl of hydrochloric acid saturated ethanol, 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride
After adding 15 mg and stirring for about 10 minutes, the mixture was capped and stirred overnight at room temperature. The organic layer was washed twice with 1N hydrochloric acid, then the solvent was removed with a desiccator, and converted to 3-hydroxybutanoic acid ethyl ester. This was trifluoroacetic acid similarly to the above-mentioned 3-hydroxybutanoic acid ethyl ester, and then a capillary column G-TA manufactured by Astec Co.
The optical isomer was analyzed by gas chromatography using (0.25 mm × 30 m), and the ester conversion product was the R form with an optical purity of 98.2% ee. As a result, it was found that the 3-hydroxybutanoic acid remaining in the water was the R form. Optical purity analysis conditions: column temperature; 90 ° C., nitrogen flow rate; 0.3 ml / min,
Split ratio: 100: 1, retention time R
Body: 7.8 minutes, S-body: 11.9 minutes

Example 2 Reaction was carried out in the same manner as in Example 1 except that racemic 3-hydroxybutanoic acid ethyl ester was changed to racemic lactic acid ethyl ester, and the amount of the product was determined in the same manner as in Example 1. In addition, the optical purity was analyzed. As a result, from 200 g of racemic lactic acid ethyl ester, 56
g of R-form lactic acid ethyl ester was obtained. Its optical purity is 9
It was 8.7% ee. The lactic acid obtained from water was 90 g. The lactic acid was converted into the ethyl ester form by the same method as in Example 1 and analyzed for optical purity. The optical purity was 65.1% ee of S form. The retention times are R-type; 5.2 minutes and S-type; 5.4 minutes, respectively.

Example 3 The reaction was performed in the same manner as in Example 1 except that the racemic 3-hydroxybutanoic acid ethyl ester was changed to racemic 2-hydroxybutanoic acid ethyl ester, and in the same manner as in Example 1. The product was quantified as well as analyzed for optical purity. As a result, 75 g of R-form 2-hydroxybutanoic acid ethyl ester was obtained from 200 g of racemic 2-hydroxybutanoic acid ethyl ester. Its optical purity was 99.2% ee. The amount of 2-hydroxybutanoic acid obtained from water was 47 g. The carboxylic acid was converted into an ethyl ester form in the same manner as in Example 1 and analyzed for optical purity. The optical purity was 95.1% ee of S form. Retention times are R bodies; 9.
4 minutes, S-body; 10.8 minutes.

Example 4 The same method as in Example 1 except that the racemic 3-hydroxybutanoic acid ethyl ester was changed to racemic 4-phenyl-2-hydroxybutanoic acid ethyl ester and the addition amount was changed to 50 g. With Example 1, and Example 1
The product was quantified in the same manner as in. 50 g of racemate 4-
From phenyl-2-hydroxybutanoic acid ethyl ester, 4.8 g of ethyl ester and 30.2 g of carboxylic acid were obtained. The optical purity of the obtained ethyl ester compound was analyzed by high performance liquid chromatography (HLPC) using CHIRALEL OD (25 cm x 0.46 cm) manufactured by Daicel. The analysis conditions at that time were: developing solvent: hexane / isopropanol (100: 1), flow rate: 0.5
ml, column temperature; 25 ° C., detection: UV 250 nm. As a result, the optical purity of the ethyl ester compound was 9
The R form was 8.1% ee. Further, the produced carboxylic acid was converted into an ethyl ester form by the same method as in Example 1, and then the optical purity was analyzed in the same manner as described above. Its optical purity was S-form with 19.4% ee. The retention times are S body; 37.5 minutes and R body, 61.3 minutes, respectively.

A table summarizing Examples 1-4 is shown below.

[Table 1]

Examples 5-12 The microorganisms to be used were selected from Enterobacter sp. Strain DS-S-75 and Citr.
bacterium freundii DS-S-13 strain or Citrobacter freu
The reaction was performed in the same manner as in Examples 1 to 4 except that the strain was changed to the ndii DS-K-40 strain. 50 g of each of racemic 3-hydroxybutanoic acid ethyl ester, racemic lactic acid ethyl ester, racemic 2-hydroxybutanoic acid ethyl ester and racemic 4-phenyl-2-hydroxybutanoic acid ethyl ester used as substrates were used. The resting cells used were 50 g (wet weight) each.
And Quantification of the product and analysis of the optical purity were carried out in Example 1
Alternatively, the same procedure as in Example 4 was performed. The produced carboxylic acids were converted into ethyl ester form in the same manner as in Example 1 and analyzed for optical purity.

[0022]

[Table 2]

[0023]

[Table 3]

Example 13 Peptone, yeast extract and glycerol were each added to 1.0
% Liter fermenter (incubator) containing 2.5 L of nutrient medium (pH 7.2) containing 100% was sterilized at 121 ° C. for 15 minutes. The test bacteria, Enterobacter sp. DS-S-75 strain, were preliminarily prepared with peptone, yeast extract and glycerol 1.0% each.
An inoculum was prepared by culturing with shaking in a nutrient medium (pH 7.2) at 30 ° C. for 16 hours, and 2% (V / V) of the above medium was aseptically inoculated. And temperature 30 ℃, ventilation rate 0.5L / mi
The culture was carried out with aeration and agitation for about 24 hours under the conditions of n and rotation speed of 500 rpm. After completion of the culture, the culture solution is taken out and collected by centrifugation to obtain 20m containing 2mM magnesium sulfate.
It was washed three times with M phosphate buffer (pH 7.2) to prepare 100 g of resting cells. 100 g of the resting cells were suspended in the same 5 L jar fermenter containing 2500 ml of the above-mentioned buffer solution (cell turbidity 19 OD (turbidity at 660 nm)), and 1
00 g of racemic tetrahydrofuran-2-carboxylic acid methyl ester was added, and an optical resolution reaction was carried out for 24 hours while stirring at 30 ° C. 5% calcium carbonate (25 g) was added to the reaction as a pH neutralizer. Tetrahydrofuran-2-carboxylic acid methyl ester remaining in the reaction solution at that time was subjected to gas chromatography (column carrier: PE
G20M, 60-80 mesh), the residual rate was 40%. In addition, racemic tetrahydrofuran-
2-Carboxylic acid methyl ester was synthesized by refluxing racemic tetrahydrofuran-2-carboxylic acid and 5 equivalents of methanol in the presence of a catalytic amount of sulfuric acid at 80 ° C. for 5 hours. After completion of the reaction, the obtained tetrahydrofuran-2-carboxylic acid methyl ester was extracted with ethyl ether to obtain a tetrahydrofuran-2-carboxylic acid methyl ester standard product.

After the division reaction was completed, the bacterial cells were removed and the mixture was extracted 3 times with an equal amount of ethyl acetate. The ethyl acetate layer was dehydrated with anhydrous magnesium sulfate and the solvent was removed under reduced pressure to obtain 36 g of syrup containing tetrahydrofuran-2-carboxylic acid methyl ester. Identification and quantification of the methyl ester was carried out by the same gas chromatography, and confirmed from the retention time with the original compound. The tetrahydrofuran-2-carboxylic acid methyl ester in this syrup was analyzed for optical isomers by gas chromatography using a capillary column G-TA (0.25 mm x 30 m) manufactured by Astec. as a result,
The recovered tetrahydrofuran-2-carboxylic acid methyl ester was found to be the R form with an optical purity of 99.1% ee. The retention times of the R form and the S form were 8.0 minutes and 9.0 minutes, respectively.

On the other hand, the tetrahydrofuran-2-carboxylic acid remaining in water was identified and quantified by the same gas chromatography as the retention time with the original compound. At that time, adjust the pH of the aqueous solution with hydrochloric acid.
It was performed after setting to 4. The aqueous solution was removed by an evaporator to obtain 57 g of tetrahydrofuran-2-carboxylic acid syrup. For the analysis of optical purity, the obtained tetrahydrofuran-2-carboxylic acid was converted into methyl ester according to the method of Example 1 and then analyzed by the above-mentioned method.
As a result, it was found that the produced tetrahydrofuran-2-carboxylic acid was an S-isomer having an optical purity of 65.1% ee. Optical purity analysis conditions Column temperature 120 ° C, nitrogen flow rate 0.3 ml / mi
n, split ratio 100: 1, retention time
R form; 8.0 minutes, S form; 9.0 minutes

Example 14 A reaction was carried out in the same manner as in Example 13 except that the substrate was changed from racemic tetrahydrofuran-2-carboxylic acid methyl ester to tetrahydrofuran-2-carboxylic acid ethyl ester. Then, in the same manner as in Example 13, the product was quantified and the optical purity was analyzed. as a result,
From 100 g of racemic tetrahydrofuran-2-carboxylic acid ethyl ester, 35.2 g of R-form tetrahydrofuran-2-carboxylic acid ethyl ester was obtained. Its optical purity was 98.7% ee. Moreover, the obtained tetrahydrofuran-2-carboxylic acid was 51 g. According to the method of Example 1, the carboxylic acid was converted into a methyl ester and analyzed. The optical purity was 65.1% ee of S form. The retention times of the ethyl ester were R-form 8.2 minutes and S-form 9.2 minutes, respectively.

Example 15 An optical resolution reaction was carried out in the same manner as in Example 13 except that the microorganism used was changed to Citrobacter freundii DS-S-13 strain or Citrobacter freundii DS-S-40 strain. Then, in the same manner as in Example 13, the product was quantified and the optical purity was analyzed. As a result, 32.1 g and 33.1 g of R-form tetrahydrofuran-2-carboxylic acid methyl ester were obtained from 100 g of racemic tetrahydrofuran-2-carboxylic acid methyl ester, respectively. The optical purities were 98.5% ee and 98.2% ee, respectively. Further, the obtained tetrahydrofuran-2-carboxylic acid was converted into methyl ester according to Example 1 and then measured, and as a result, the optical purity was 67.3% ee and 6 respectively.
The S form was 5.1% ee.

Example 16 An optical resolution reaction was carried out in the same manner as in Example 15 except that the substrate was changed from racemic tetrahydrofuran-2-carboxylic acid methyl ester to racemic tetrahydrofuran-2-carboxylic acid ethyl ester. From 100 g of racemic tetrahydrofuran-2-carboxylic acid ethyl ester, Citrobacter freundii DS-S-13 strain or Citrob
33.2 g and 3 for acter freundii DS-S-40 strain, respectively.
4.5 g of ethyl ester and 67.1 g and 62.6 g
Carboxylic acid was obtained. The optical purity of the obtained ethyl ester form was found to be R form with 99.1% ee and 98.5% ee, respectively. Further, after converting the produced carboxylic acid into the methyl ester form according to the method of Example 1,
The optical purity was measured. As a result, each is 66.4% e
e and 67.2% ee S isomer.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, microorganisms belonging to the genus Enterobacter (for example, Enterobacter sp. DS-S-75 strain) and microorganisms belonging to the genus Kitrobacter (for example, Citrobacter
freundii DS-S-13 strain, Citrobacter freundii DS-K-40
Strain) is used to efficiently produce optically active hydroxycarboxylic acid alkyl ester having high optical purity and optically active hydroxycarboxylic acid in the optical resolution reaction of racemic hydroxycarboxylic acid alkyl ester [1] by ester hydrolysis reaction. be able to. Further, in the optical resolution reaction of racemic tetrahydrofuran-2-carboxylic acid alkyl ester, the optically active tetrahydrofuran-2-carboxylic acid and the optically active tetrahydrofuran-2-carboxylic acid having high optical purity can be efficiently produced.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (72) Inventor Atsushi Nakagawa 1-10-8 Edobori, Nishi-ku, Osaka City, Osaka Prefecture Daiso Corporation (72) Invention Motoko Higami 1-10-8 Edobori, Nishi-ku, Osaka-shi, Osaka Prefecture F-term in Daiso Corporation (reference) 4B064 AD09 AD21 AD27 AD64 AD75 AE45 CA02 CB03 CD10 CD27 CE08 DA01 DA11

Claims (15)

[Claims] 1. The following formula [1] R ¹ — (CH ₂ ) _n —CHOH— (CH ₂ ) _m —COOR ² [1] (In the formula, R ¹ is a methyl group or a benzyl group, and R ² is An alkyl group, and when m is 0, n is 0 or 1
And when m is 1, n is 0. ) In the racemic hydroxycarboxylic acid ester, or racemic tetrahydrofuran-2-carboxylic acid alkyl ester, belonging to the genus Enterobacter (Enterobacter) or the genus Citrobacter (Citrobacter), a microorganism having carboxylic acid ester degrading activity or its production An optically active hydroxycarboxylic acid alkyl ester or an optically active hydroxy compound, characterized in that a living organism is allowed to act thereon to optically resolve the ester, and one remaining optically active carboxylic acid alkyl ester or the other optically active carboxylic acid produced is collected. Process for producing carboxylic acid, optically active tetrahydrofuran-2-carboxylic acid alkyl ester or optically active tetrahydrofuran-2-carboxylic acid. 2. The following formula [1] R ¹ — (CH ₂ ) _n —CHOH— (CH ₂ ) _m —COOR ² [1] (In the formula, R ¹ is a methyl group or a benzyl group, and R ² is An alkyl group, and when m is 0, n is 0 or 1
And when m is 1, n is 0. ), A racemic hydroxycarboxylic acid ester, a microorganism belonging to the genus Enterobacter or the genus Chitrobacter and having a carboxylic acid ester degrading activity or a product thereof is allowed to act, and the ester is optically resolved, and one of the remaining optical activities A method for producing an optically active hydroxycarboxylic acid alkyl ester or an optically active hydroxycarboxylic acid, which comprises collecting the carboxylic acid alkyl ester or the other optically active carboxylic acid produced. 3. A racemic tetrahydrofuran-2-carboxylic acid alkyl ester is allowed to react with a microorganism belonging to the genus Enterobacter or the genus Chitrobacter and having a carboxylic acid ester-decomposing activity or a product thereof, and the ester is optically resolved to remain. A method for producing an optically active tetrahydrofuran-2-carboxylic acid alkyl ester or an optically active tetrahydrofuran-2-carboxylic acid, which comprises collecting one optically active alkyl carboxylic acid ester or the other optically active carboxylic acid produced. 4. The process according to claim 1, wherein the S-form 3-hydroxybutanoic acid methyl or ethyl ester is collected by using racemic 3-hydroxybutanoic acid methyl or ethyl ester as a substrate. 5. The method according to claim 1, wherein the R-form 2-hydroxybutanoic acid methyl or ethyl ester is collected by using racemic 2-hydroxybutanoic acid methyl or ethyl ester as a substrate. 6. The method according to claim 1, wherein the R-form methyl lactate or ethyl ester is collected using racemic methyl lactate or ethyl ester as a substrate. 7. The method according to claim 1, wherein R-form tetrahydrofuran-2-carboxylic acid methyl or ethyl ester is collected by using racemic tetrahydrofuran-2-carboxylic acid methyl or ethyl ester as a substrate. 8. The method according to claim 1, wherein R-form 3-hydroxybutanoic acid is collected using racemic 3-hydroxybutanoic acid methyl or ethyl ester as a substrate. 9. The method according to claim 1, wherein S-form 2-hydroxybutanoic acid is collected using racemic 2-hydroxybutanoic acid methyl or ethyl ester as a substrate. 10. The method according to claim 1, wherein S-form lactic acid is collected using racemic methyl lactate or ethyl ester as a substrate. 11. The method according to claim 1, wherein the S-form tetrahydrofuran-2-carboxylic acid is collected using racemic tetrahydrofuran-2-carboxylic acid methyl or ethyl ester as a substrate. 12. The method according to claim 1, wherein the microorganism used is a microorganism belonging to the genus Enterobacter. 13. The method according to claim 1, wherein the microorganism used is a microorganism belonging to the genus Kitrobacter. 14. The microorganism used is Enterobacter (E
nterobacter) sp. DS-S-75 strain (international deposit number FERM
BP-5494), The manufacturing method in any one of Claims 1-12. 15. The microorganism to be used is a Citrobacter freundii DS-S-13 strain (international deposit number FERM BP-5492) or a Chitrobacter freundii DS-K-40 strain (international deposit number FERM). BP-5493).
11. The method according to any one of 11 or 13.