CYCLOHEXENECARBOXYLATE ESTER HYDROLASE, AND MUTANT, CODING GENE, EXPRESSION VECTOR, RECOMBINANT BACTERIUM AND USE THEREOF

Abstract

The present invention discloses a cyclohexenecarboxylate ester hydrolase, and a mutant, a coding gene, an expression vector, recombinant bacterium and use thereof. The cyclohexenecarboxylate ester hydrolase AcEst1 and its mutant of the present invention have the function of enantioselectively resolving methyl 3-cyclohexene-1-carboxylate with high efficiency to prepare optically active (S)-3-cyclohexene-1-carboxylic acid. When the substrate concentration is as high as 2000 mM (about 280 g/L), the optical purity of the product is higher than 99%, and the substrate/catalyst is as high as 3500 g/g. As compared with other preparation methods, the product prepared by the method of the present invention has high concentration and high optical purity, the catalytic efficiency is high, the reaction conditions are mild. Furthermore, the method is environmentally friendly, simple in operation and easy for industrial scale-up, thus has a good prospect of application in industry.

Claims

1. A cyclohexenecarboxylate ester hydrolase, wherein the hydrolase is: (a) a protein having an amino acid sequence as shown in SEQ ID NO:2; or (b) a protein having an amino acid sequence modified from the amino acid sequence as shown in SEQ ID NO:2 by means of substitution, deletion, or addition of one or more amino acids, and having the activity of hydrolyzing methyl 3-cyclohexene-1-carboxylate.

2. The cyclohexenecarboxylate ester hydrolase according to claim 1, wherein the hydrolase is obtained by substituting the alanine residue at position 202 with a lysine residue, and the glycine residue at position 326 with an alanine residue in the amino acid sequence as shown in SEQ ID NO:2.

3. The cyclohexenecarboxylate ester hydrolase according to claim 1, wherein the hydrolase is obtained by substituting the phenylalanine residue at position 78 with a valine reside, the alanine residue at position 202 with a lysine residue, and the glycine residue at position 326 with an alanine residue in the amino acid sequence as shown in SEQ ID NO:2.

4. A gene encoding the cyclohexenecarboxylate ester hydrolase according to claim 1.

5. A recombinant expression vector, comprising the gene according to claim 4.

6. A recombinant bacterium expressing the cyclohexenecarboxylate ester hydrolase according to any one of claims 1 to 3 claim 1.

7. Use of the cyclohexenecarboxylate ester hydrolase according to claim 1 in catalyzing methyl 3-cyclohexene-1-carboxylate to produce optically active (S)-3-cyclohexene-1-carboxylic acid.

8. The use according to claim 7, wherein (S)-3-cyclohexene-1-carboxylic acid is produced by catalyzing the enantioselective hydrolysis of methyl 3-cyclohexene-1-carboxylate in a buffer with the cyclohexenecarboxylate hydrolase, and then hydrolyzing into (S)-3-cyclohexene-1-carboxylic acid by heating under an alkaline condition.

9. The use according to claim 8, wherein the buffer is a citrate buffer, a phosphate buffer or a glycine-NaOH buffer, and the buffer has a pH of 5.0 to 10.0.

10. The use according to claim 8, wherein the alkaline condition is a 0.5 to 1.5 M sodium hydroxide solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows the results of expression and purification of the cyclohexenecarboxylate ester hydrolase AcEst1, in which the bands from left to right are respectively standard protein marker, crude enzyme supernatant of AcEst1 and purified AcEst1 protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

Example 1: Cloning of Gene Encoding Cyclohexenecarboxylate Ester Hydrolase AcEst1

[0027] The strain Acinetobacter sp. JNU9335 was cultured in LB medium, and the high-purity, large-segment total genomic DNA was extracted by the CTAB (hexadecyltrimethylammonium bromide) method. An appropriate amount of Acinetobacter sp. JNU9335 was added to and frozen in liquid nitrogen, ground into powder, added with an appropriate amount of 2×CTAB extraction buffer (100 mmol/L Tris-HCl, pH 8.0, 20 mmol/L EDTA, 1.4 mol/L NaCl, 2% (w/v) CTAB, and 40 mmol/L mercaptoethanol), incubated at 65° C. for 10 min, and shaken intermittently. Then an equal volume of chloroform/isoamyl alcohol was added, mixed uniformly by gently turning upside down the centrifuge tube, and centrifuged at 8000 rpm for 10 min at room temperature. The supernatant was transferred to another centrifuge tube, and an equal volume of chloroform/isoamyl alcohol was added, mixed uniformly by gently turning upside down the centrifuge tube, and centrifuged at 8000 rpm for 10 min at room temperature. The upper aqueous phase was added to a new centrifuge tube, and an equal volume of isopropanol was added, mixed uniformly, and allowed to stand at room temperature for 30 min. The solution was centrifuged at 4000 rpm for 10 min, washed twice with 70% ethanol after the supernatant is removed, dried and added to 20 μL of TE buffer (100 mM Tris-HCl, 10 mM EDTA pH 8.0,) to dissolve the DNA, which was then stored at −20° C. for later use. Total DNA was partially enzymatically cleaved by Sau3AI, and the cleaved DNA fragments were purified by electrophoresis. Fragments of approximately 2-6 kb were recovered by a gel extraction kit, and the recovered DNA was dissolved in ddH.sub.2O and stored at −20° C.

[0028] The DNA was connected to the vector pUC118 in the following reaction system:

TABLE-US-00001 TABLE 1 Ligation reaction system Reagent Amount pUC118(BamHI/BAP) 0.1 μg Cleaved total DNA 0.1 μg fragment 10 × ligase buffer 1.0 μL T.sub.4 DNA ligase 0.5 μL Adding ddH.sub.2O to 10.0 μL

[0029] After incubation at 16° C. for 12 h, 10 μL of the enzymatic ligation product was transformed into 200 μL of E. coli DH5a competent cells. The obtained recombinant was induced to express, and then the substrate 3-cyclohexene-1-carboxylic acid methyl ester was added for reaction. Where the product 3-cyclohexene-1-carboxylic acid was produced, high-throughput screening was carried out based on the color changes of bromothymol blue-phenol red dual indicators at different pH values. The colonies with obvious color changes, that is, the colonies changing from brownish green to yellow were further screened, and the product was detected by HPLC. The recombinant with an obvious product peak was shipped to Tianlin Biotechnology Co., Ltd. for sequencing. A nucleotide sequence as shown in SEQ ID NO:1 was obtained. The amino acid sequence deduced from the nucleotide sequence is as shown in SEQ ID NO:2, and the cyclohexenecarboxylate ester hydrolase expressed by this sequence is designated as AcEst1.

Example 2: Production of Recombinant Plasmid and Bacterium Comprising AcEst1, and Recombinant Hydrolase

[0030] Using the forward primer 5′-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3′ (SEQ ID NO:3) and reverse primer 5′-gtggtggtggtggtgctcgagTTA-TTTGGCATTCTTATCCCAAAA-3′ (SEQ ID NO:4), the nucleotide sequence of AcEst1 obtained in Example 1 was amplified by polymerase chain reaction, and the obtained DNA fragment containing the AcEst1 sequence was cleaved with NdeI and XhoI respectively, and then ligated to the plasmid pET-28a(+) that was also cleaved with NdeI and XhoI, to obtain the recombinant plasmid pET-28a(+)-AcEst1.

[0031] The obtained recombinant plasmid pET-28a(+)-AcEst1 was transformed into E. coli BL21, to construct recombinant E. coli containing the cyclohexenecarboxylate ester hydrolase AcEst1. The constructed recombinant E. coli was inoculated into LB medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0) containing 50 μg/mL kanamycin, and incubated overnight at 37° C. with shaking. The cells were inoculated to a 2 L conical flask containing 600 mL LB medium in an amount of 1% (v/v), and incubated on a shaker at 37° C. and 180 rpm. When the OD.sub.600 of the culture medium reached 0.6-2.0, IPTG with a final concentration of 0.2 mmol/L was added as an inducer. After induction at 16° C. for 16 h, the culture medium was centrifuged, and the cells were collected and washed twice with saline to obtain resting cells. The obtained resting cells were suspended in Tris-HCl buffer (20 mM, pH 8.0), homogenized by a high-pressure homogenizer, and freeze-dried to obtain recombinant AcEst1.

Example 3: Production of Mutant A202K/G326

[0032] Mutant A202K/G326A is a random mutant. A random mutant library of AcEst1 was established by error-prone PCR, and the color change of the indicator at different pH values described in Example 1 was used as a means of high-throughput screening. The terminal primers were designed: the forward primer 5′-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3′ (SEQ ID NO:5) and the reverse primer 5′-gtggtggtggtggtgctcgagTTATTTGGCATTCTTATCCCAAAA-3′ (SEQ ID NO:6). PCR system (50 μL): rTaq polymerase 0.25 μL, 10×rTaq Buffer 5 μL, dNTP 5 μL, MgSO.sub.4 2 μL, template plasmid about 100 ng, forward primer 2 μL, reverse primer 2 μL, MnCl.sub.2 (10 mM) 0.5 μL, and ddH.sub.2O making up to 50 μL.

[0033] PCR reaction procedure: (1) pre-denaturation at 98° C. for 5 min; 30 cycles of (2) denaturation at 98° C. for 30 s, (3) annealing at 55° C. for 30 s, and (4) extension at 72° C. for 1 min; and final extension at 72° C. for 10 min. The PCR product was stored at −20° C.

[0034] The PCR fragment containing random mutation sites was cleaved with NdeI and XhoI, then ligated to the pET-28a(+) plasmid with the same cleavage sites, and then transformed into E. coli BL21 (DE3) competent cells. The cells were coated evenly on an LB agar plate containing 50 μg/mL kanamycin. After culturing overnight at 37° C., monoclones were picked to a deep-well plate for culture and induced expression. The mutant library was screened for activity according to the color change of the pH indicator and a mutant with increased activity was obtained, which was shipped to Tianlin Biotechnology Co., Ltd. for sequencing. The sequencing result was aligned with the sequence of the cyclohexenecarboxylate ester hydrolase (AcEst1) gene by the DNAMAN software. The result shows that the alanine at position 202 is mutated to lysine and the glycine at position 326 is mutated to alanine. The obtained mutant is designated as A202K/G326A.

[0035] The mutant protein A202K/G326A obtained by substituting the alanine residue at position 202 with a lysine residue, and the glycine residue at position 326 with an alanine residue in the amino acid sequence as shown in SEQ ID NO:2 has a 3-time increased activity for hydrolyzing 3-cyclohexene-1-carboxylic acid methyl ester. For the same substrate concentration, the mutant A202K/G326A can achieve a conversion rate similar to that of WT only after reaction for 2 h, where the reaction time is shortened by 3 times.

Example 4: Production of Plasmid, Recombinant Bacterium and Mutant A202K/G326A

[0036] The plasmid pET-28a(+)-A202K/G326A obtained in Example 3 was extracted, and transformed into E. coli BL21. The E. coli cells were inoculated into LB medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0) containing 50 μg/mL kanamycin, and incubated overnight at 37° C. with shaking. The cells were inoculated to a 2 L conical flask containing 600 mL LB medium in an amount of 1% (v/v), and incubated on a shaker at 37° C. and 180 rpm. When the OD.sub.600 of the culture medium reached 0.6-2.0, IPTG with a final concentration of 0.2 mmol/L was added as an inducer. After induction at 16° C. for 16 h, the culture medium was centrifuged, and the cells were collected and washed twice with saline to obtain resting cells. The obtained resting cells were suspended in Tris-HCl buffer (20 mM, pH 8.0), homogenized by a high-pressure homogenizer, and freeze-dried to obtain mutant A202K/G326A.

Example 5: Production of Mutant F78V/A202K/G326A

[0037] A202K/G326A was subjected to site-directed mutagenesis, as described in the Site-Directed Mutagenesis Kit (Stratagene, Catalog#200522).

[0038] Degenerate primers F: 5′-TCGCGTAAATTGNDTGATCATCAAATT-3′ (SEQ ID NO:7), and R: 5′-AATTTGATGATCAHNCAATTTACGCGA-3′ (SEQ ID NO:8) were designed,

[0039] where N represents a combination of four bases A, T, C, and G; D represents a combination of three bases A, G, and T; and H represents a combination of three bases A, C, and T.

[0040] PCR reaction system (50 μL): KOD plus Neo 0.25 μL, template 0.5-20 ng, 5 μL 10×KOD plus Neo buffer, 5 μL dNTP (each 2.0 mM), 2 μL MgSO.sub.4 (25 mM), forward primer 2 μL, reverse primer 2 μL, and ddH.sub.2O making up to 50 μL.

[0041] The template was the plasmid pET-28a(+)-A202K/G326A comprising the cyclohexenecarboxylate ester hydrolase mutant obtained in Example 3.

[0042] PCR reaction procedure: (1) pre-denaturation at 98° C. for 5 min; 20 cycles of (2) denaturation at 98° C. for 30 s, (3) annealing at 55° C. for 30 s, and (4) extension at 68° C. for 3.5 min; and final extension at 68° C. for 10 min. The PCR product was stored at −20° C.

[0043] The amplified PCR product was digested with endonuclease DpnI for 2 h at 37° C. and transformed into E. coli BL21 competent cells. Then, the cells were evenly coated on an LB agar plate containing 50 μg/mL kanamycin. After culturing overnight at 37° C., 200 monoclones were picked to a deep-well plate for culture and induced expression. The mutant library was screened for activity according to the color change of the pH indicator and a mutant with increased activity was obtained, which was shipped to Tianlin Biotechnology Co., Ltd. for sequencing. The sequencing result was aligned with the sequence of the cyclohexenecarboxylate ester hydrolase (AcEst1) gene by the DNAMAN software. The result shows that the position 78 is mutated to valine, the position 202 is mutated to lysine and the position 326 is mutated to alanine. The obtained mutant is designated as F78V/A202K/G326A.

[0044] The mutant protein F78V/A202K/G326A obtained by substituting the phenylalanine residue at position 78 with a valine residue, the alanine residue at position 202 with a lysine residue, and the glycine residue at position 326 with an alanine residue in the amino acid sequence as shown in SEQ ID NO:2 has a 6-time increased activity for hydrolyzing 3-cyclohexene-1-carboxylic acid methyl ester. For the same substrate concentration, the mutant F78V/A202K/G326A can achieve a conversion rate similar to that of WT only after reaction for 1 h, where the reaction time is shortened by 6 times.

Example 6: Production of Plasmid and Recombinant Bacterium Comprising Mutant F78V/A202K/G326A, and Recombinant Hydrolase

[0045] The plasmid pET-28a(+)-F78V/A202K/G326A obtained in Example 5 was extracted, and transformed into E. coli BL21. The E. coli cells were inoculated into LB medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0) containing 50 jug/mL kanamycin, and incubated overnight at 37° C. with shaking. The cells were inoculated to a 2 L conical flask containing 600 mL LB medium in an amount of 1% (v/v), and incubated on a shaker at 37° C. and 180 rpm. When the OD.sub.600 of the culture medium reached 0.6-2.0, IPTG with a final concentration of 0.2 mmol/L was added as an inducer. After induction at 16° C. for 16 h, the culture medium was centrifuged, and the cells were collected and washed twice with saline to obtain resting cells. The obtained resting cells were suspended in Tris-HCl buffer (20 mM, pH 8.0), homogenized by a high-pressure homogenizer, and freeze-dried to obtain mutant F78V/A202K/G326A.

Example 7: Catalytic Performance of Recombinant AcESt1

[0046] The cyclohexenecarboxylate ester hydrolase of the present invention was used in the production of optically active (S)-3-cyclohexene-1-carboxylic acid by enzymatic hydrolysis of racemic methyl 3-cyclohexene-1-carboxylate.

[0047] At different temperatures (20-65° C.), sodium phosphate (100 mM, pH 7.0) was used as a buffer, and 1 mM p-nitrophenol cyclohexenecarboxylate was used as a substrate for activity determination. According to the change of absorbance at 405 nm, the activity of the cyclohexenecarboxylate hydrolase AcEst1 was investigated. The results are shown in Table 2. AcEst1 has the highest catalytic activity at 40° C. When the temperature continues to rise, the enzyme activity begins to decrease.

TABLE-US-00002 TABLE 2 Activity of cyclohexenecarboxylate ester hydrolase AcEst1 at different temperatures Temperature (° C.) Relative activity (%) 20 40.9 ± 0.2 25 58.2 ± 0.5 30 73.7 ± 0.6 35 86.9 ± 0.8 40 100.0 ± 0.3  45 97.1 ± 0.7 50 86.6 ± 0.4 55 78.8 ± 0.2 60 71.4 ± 0.2 65 67.8 ± 0.3

[0048] When the reaction temperature was 30° C., 1 mM p-nitrophenol cyclohexenecarboxylate was used as a substrate for activity determination. The activity of AcEst1 in buffers with different pH values was investigated according to the change of absorbance at 405 nm. The buffer systems used: sodium citrate buffer (pH 5.0-6.0); sodium phosphate buffer (pH 6.0-8.0); Tris-HCl buffer (pH 8.0-9.0) and glycine-NaOH buffer (pH 9.0-10.0). The results are shown in Table 3. The optimum pH for AcEst1 is pH 9.0 (Tris-HCl buffer).

TABLE-US-00003 TABLE 3 Activity of the cyclohexenecarboxylate ester hydrolase AcEst1 in buffers with different pH values Buffer pH Relative activity (%) Sodium citrate 4.0  0.1 ± 0.02 5.0  0.2 ± 0.01 6.0  0.6 ± 0.05 Sodium phosphate 6.0  0.8 ± 0.1 7.0 36.3 ± 1.2 8.0 85.5 ± 2.6 Tris-HCl 8.0 95.0 ± 1.7 9.0 100.0 ± 2.1  Glycine-NaOH 9.0 96.5 ± 2.3 10.0 78.2 ± 3.2

Example 8: Catalytic Hydrolytic Resolution of Various 3-Cyclohexene-1-Carboxylate Esters by the Recombinant AcESt1

[0049] Recombinant AcEst1 can catalyze the enantioselective hydrolysis of various 3-cyclohexene-1-carboxylate esters to produce (S)-3-cyclohexene-1-carboxylic acid with high optical purity. The activities of AcEst1 for 3-cyclohexene-1-carboxylic acid methyl ester, 3-cyclohexene-1-carboxylic acid ethyl ester, 3-cyclohexene-1-carboxylic acid isopropyl ester and 3-cyclohexene-1-carboxylic acid butyl ester and the optical purity of the hydrolysis products were investigated respectively. The wild-type AcEst1 obtained in Example 3 was used to catalyze the hydrolysis of different 3-cyclohexene-1-carboxylate esters. 5 mg of crude recombinant AcEst1 enzyme powder was weighed, dissolved in 10 mL of Tris-HCl buffer (200 mM, pH 9.0), added to the substrate 3-cyclohexene-1-carboxylate esters to give a final concentration of 50 mM, and reacted at 30° C. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 The activity of AcEst1 for different 3-cyclohexene-1- carboxylate esters and optical purity of the products Reaction Conversion time rate ee.sub.s ee.sub.p Substrate (h) (%) (%) (%) 3-cyclohexene-1-carboxylic 6 49 73 71 acid methyl ester 3-cyclohexene-1-carboxylic 6 47 64 56 acid ethyl ester 3-cyclohexene-1-carboxylic 10 47 66 59 acid isopropyl ester 3-cyclohexene-1-carboxylic 16 49 19 18 acid butyl ester

Example 9: Catalytic Hydrolytic Resolution of 3-Cyclohexene-1-Carboxylic Acid Methyl Ester by Recombinant AcEst1

[0050] The typical enzymatic hydrolysis and resolution of 3-cyclohexene-1-carboxylate ester was as follows. 16 mg of freeze-dried recombinant AcEst1 enzyme powder was dissolved in 200 mL of Tris-HCl buffer (200 mM, pH 9.0), and added to the substrate 3-cyclohexene-1-carboxylic acid methyl ester to give a final concentration of 200-2000 mM (28-280 g/L), where the corresponding S/C was 350-3500 g/g. The reaction was performed at 30° C. with mechanical stirring at 200 rpm, and the pH was controlled at 9.0 by adding 1.0 M Na.sub.2CO.sub.3, until the e.e. of the substrate was >99%. After the reaction, the pH was adjusted to 12 with 2 M NaOH, and then the solution was extracted three times with dichloromethane. The extracts were combined and dried overnight over anhydrous sodium sulfate. (S)-3-cyclohexene-1-carboxylic acid methyl ester was obtained by removing the solvent by rotary evaporation. The conversion rate and the e.e. value of the hydrolysis product were determined by gas chromatography (chiral capillary column B-DM) Specific analysis conditions: N.sub.2 as carrier gas, inlet temperature 280° C., detector temperature 280° C., initial column temperature 50° C., 2° C./min to 100° C. for 10 min. The results are shown in Table 5. Then (S)-3-cyclohexene-1-carboxylic acid methyl ester was added to 1 M NaOH aqueous solution, reacted for 6 h with stirring under reflux at 50° C., adjusted to pH 5.0 by adding 1 M HCl aqueous solution, and extracted 3 times with an equal volume of dichloromethane. The organic layers were combined, dried over anhydrous Na.sub.2SO.sub.4, filtered, and dried by rotary evaporation to obtain (S)-3-cyclohexene-1-carboxylic acid. The resulting product is a liquid with a special odor. The total yield after separation is 38%, and the optical purity is 99% e.e.

TABLE-US-00005 TABLE 5 Results of catalyzing asymmetric resolution of 3-cyclohexene- 1-carboxylic acid methyl ester by recombinant AcEst1 Substrate Conversion concentration Reaction time rate ee.sub.s (mM) (h) (%) (%)/(conformation) 200 8 61.5 99 500 8 62.8 99 1000 9 62.8 99 2000 20 61.9 99

Example 10: Catalyzing Hydrolytic Resolution of 3-Cyclohexene-1-Carboxylate Methyl Ester by AcEst1 and Mutants Thereof

[0051] The wild-type AcEst1 obtained in Example 3, the mutant A202K/G326A obtained in Example 4, and the mutant F78V/A202K/G326A obtained in Example 5 were respectively used to catalyze the hydrolysis of 3-cyclohexene-1-carboxylic acid methyl ester. 5 mg of crude recombinant AcEst1 or mutant enzyme powder was weighed, dissolved in 10 mL of Tris-HCl buffer (200 mM, pH 9.0), added to the substrate 3-cyclohexene-1-carboxylate to give a final concentration of 50 mM, and reacted at 30° C. From the reaction (as shown in Table 6), it can be seen that the time required for the mutant to achieve a conversion rate of about 50% is significantly reduced.

TABLE-US-00006 TABLE 6 Results of catalyzing asymmetric resolution of 3-cyclohexene-1-carboxylate methyl ester by recombinant AcEst1 and mutants thereof Reaction Conversion time rate ee.sub.s Enzyme (h) (%) (%)/(conformation) AcEst1 6 47.2 75.3/(S) A202K/G326A 2 49.3 73.9/(S) F78V/A202K/G326A 1 46.8 76.6/(S)

Example 11: Production of (S)-3-Cyclohexene-1-Carboxylic Acid

[0052] The reaction was carried out in a 1 L three-necked flask. 200 mL of Tris-HCl buffer at pH 9.0, 16 mg of crude F78V/A202K/G326A enzyme powder prepared in Example 5, and 58.7 g of racemic 3-cyclohexene-1-carboxylic acid methyl ester were added, and reacted at 30° C. under mechanical stirring at 200 rpm, during which 1 M Na.sub.2CO.sub.3 was added by fed-batch to maintain the pH of the reaction solution at 9.0. After reaction for 12 h, the conversion rate reached 61.1%, and the optical purity of (S)-3-cyclohexene-1-carboxylic acid methyl ester was >99%. After the reaction, the pH was adjusted to 12 with 2 M NaOH, and then the solution was extracted three times with dichloromethane. The extracts were combined and dried overnight over anhydrous Na.sub.2SO.sub.4. (S)-3-cyclohexene-1-carboxylic acid methyl ester was obtained by removing the solvent by rotary evaporation. Then (S)-3-cyclohexene-1-carboxylic acid methyl ester was added to 1 M NaOH aqueous solution, reacted for 6 h with stirring under reflux at 50° C., adjusted to pH 5.0 by adding 1 M HCl aqueous solution, and extracted 3 times with an equal volume of dichloromethane. The organic layers were combined, dried over anhydrous Na.sub.2SO.sub.4, filtered, and dried by rotary evaporation to obtain (S)-3-cyclohexene-1-carboxylic acid (22.3 g, yield 38%, GC purity 99.0%, and optical purity 99.5% e.e).

[0053] The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or modifications can be made by those skilled in the art based on the present invention, which are within the scope of the present invention. The scope of the present invention is defined by the appended claims.