JPH0568589A - Production of optically active carboxylic acid and its enantiomer ester - Google Patents

Abstract

PURPOSE:To obtain the subject compound in an extremely good yield in a short time by treating the racemate of a carboxylate ester with a transformed microorganism containing a DNA fragment coding a specific esterase having excellent thermal stability. CONSTITUTION:A host microorganism is transformed with a recombinant ligated with a DNA fragment comprising a base sequence of formula I originated from Pseudomonas putida MR-2068 (FERN P9677) or its part to obtain a transformant. The cultured solution of the transformed microorganism or its cells (treated product) is acted on the racemate of a carboxylate ester of formula II (R1 is alkyl, aryl, etc.; R2, R3 are alkyl; (n) is 1, 2) preferably at a pH of 5-8 at a reaction temperature of >=40 deg.C to obtain the objective compound of formula III.

Description

Detailed Description of the Invention

[0001]

The present invention has the general formula

[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), a host microorganism is transformed with a plasmid DNA into which a DNA fragment encoding an esterase that asymmetrically hydrolyzes a racemate of a carboxylic acid ester (hereinafter referred to as a carboxylic acid ester [I]) is incorporated. And a method for producing an optically active carboxylic acid and its enantiomer ester, which comprises asymmetric hydrolysis using the transformed microorganism described above.

[0004]

[Prior Art]

 General formula

[0005]

[Chemical 4]

The optically active carboxylic acid represented by the formula (wherein R _1, R _{2, and} n are the same as above) and its enantiomer ester are useful as raw materials for synthesizing various physiologically active substances. The present inventors have already proposed for the first time a method of asymmetrically hydrolyzing a racemate of a carboxylic acid ester represented by the general formula [I] using an enzyme or a microorganism (for example, JP-A-60-12992). , 60-12993, etc.).

The present inventors have further proposed, as this esterase-producing strain having a high asymmetric hydrolysis activity, Pseudomonas putida microscopic research institute No. 9677 obtained by isolation from soil (Japanese Patent Laid-Open No. 1-222798). See). On the other hand, at present, recombinant DNA technology is often used as a means for obtaining a microorganism having a higher enzyme activity, but it is intended to improve the production capacity of esterase involved in the above reaction. As a DNA fragment, Pseudomonas fluorescens (Pseudomonas fl
Uorescens) A method is known in which a DNA fragment of a gene encoding an esterase derived from IFO3018 is prepared, and a transformed microorganism obtained by transforming a host microorganism with a plasmid DNA incorporating the DNA fragment is used (Japanese Patent Application Laid-Open No. HEI-H10 (1998)).
1-67190 publication).

[0008]

When carrying out asymmetric hydrolysis of the above-mentioned carboxylic acid ester [I], 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. In order to carry out an efficient reaction in this method, not only the enzyme activity of the bacterial cells is high, but also the ability of the enzyme itself to produce a carboxylic acid with high optical purity and the reaction conditions such as temperature and pH. It needs to be highly stable to the conditions. Especially for enzymes with poor thermal stability, heat deactivation occurs over time, making it difficult to improve the reaction rate by increasing the reaction temperature or to recover and reuse the enzyme. It is necessary to obtain bacterial cells that produce good enzymes.

As a method for increasing the enzyme activity of the bacterial cells, recombinant DN is disclosed in Japanese Patent Laid-Open No. 67190/1989.
A technology is effective. On the other hand, the property of the enzyme itself essentially corresponds to the DNA encoding the enzyme, and if an enzyme having high stability to the above-mentioned conditions is to be obtained, first, the enzyme has excellent stability to the reaction conditions. It is necessary to find a DNA fragment encoding the enzyme.

However, as described above, Japanese Patent Laid-Open No. 1-6
Pseudomonas fluorescens (Ps
eudomonas fluorescens) plasmid DNA incorporating a DNA fragment encoding an esterase derived from IFO 3018
However, the esterase of this strain is inferior in thermostability and has a problem that it is inactivated in several hours at a temperature of 45 ° C. or higher, for example.

Therefore, an object of the present invention is to prepare a DNA fragment encoding an esterase having excellent thermostability when asymmetrically hydrolyzing a carboxylic acid ester [I], and to contain the DNA fragment and to achieve good expression. This is to efficiently perform the asymmetric hydrolysis using a transformed microorganism having a property.

[0012]

Means for Solving the Problems As a result of intensive studies on the method of using an enzyme source effective for asymmetric hydrolysis of the above-mentioned carboxylic acid ester [I], the present inventors have
The present inventors have isolated from the soil, by using the cells of the transforming microorganism containing the esterase gene cloned from Pseudomonas putida Microbiology Research Institute No. 9677, the cells have high enzymatic activity, The present inventors have found that the enzyme can perform the reaction in an extremely high yield and in a short time because heat deactivation does not occur with time even at a high temperature reaction of 50 ° C. or higher, and the present invention has been completed.

That is, the present invention has the general formula

[0014]

[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) in the racemic form of the carboxylic acid ester represented by
A general formula, characterized in that a culture medium, cells or treated product of transformed microorganisms containing a recombinant plasmid to which NA fragment is ligated is applied.

[0016]

[Chemical 6]

(Wherein R _1, R ₁ and n are the same as above) and a method for producing an optically active carboxylic acid (hereinafter referred to as an optically active carboxylic acid [II]) and its enantiomer ester Is. Substituents of the above general formulas [I] and [II]
In R ₁ , examples of the alkyl group include a methyl group and an ethyl group, examples of the aralkyl group include a benzyl group, examples of the aryl group include a phenyl group, and examples of the substituent R ₂ or R ₃ include an alkyl group. Is
Examples thereof include a methyl group and an ethyl group. Then, as the carboxylic acid ester [I], for example, methyl β-acetylthio-α-methylpropionate, S-acetyl-β
-Methyl mercaptoisobutyrate, methyl S-acetyl-γ-mercapto-α-methyl-n-butyrate, methyl S-benzoyl-β-mercaptoisobutyrate and methyl S-phenylacetyl-β-mercaptoisobutyrate.

The nucleotide sequence of SEQ ID NO: 1, which is a characteristic of the DNA fragment according to the present invention, is determined from the region essential for activity and the SD sequence after investigating the entire nucleotide sequence of the recombinant plasmid obtained in Example 1. The details of the determination method are as described in Example 1. As a DNA fragment donor according to the present invention, Pseudomonas putida (Pse
udomonas putida) Microorganisms Research Institute No. 9677 is suitable. Cloning of a DNA fragment containing an esterase gene from this strain, preparation of a recombinant plasmid, and introduction of the recombinant plasmid into a microorganism can be carried out, for example, by the method described in Examples.

The microorganism transformed with the esterase gene having excellent thermostability of the present invention has the same enzymatic property as that of the parent strain Pseudomonas putida which is a DNA donor. In addition, by ligating to a multi-copy plasmid, it expresses much higher activity than the parent strain. This transformed microorganism is used in the reaction as a culture solution containing the same, cells, or a treated product of cells.

Cultivation of these transformed microorganisms is usually carried out by liquid culture, but can also be carried out by solid culture. As the medium, for example, LB medium is used. Culturing is performed at a temperature of 10 to 50 ° C. and a pH range of 2 to 11. Aeration and agitation may be carried out to promote the growth of microorganisms. When carrying out the hydrolysis reaction, the carboxylic acid ester [I] may be added to the medium at the beginning or during the culture, or the carboxylic acid ester [I] may be added to the culture medium after culturing the microorganism in advance. Good. Alternatively, the cells of the grown microorganism may be collected by centrifugation or the like and added to the reaction medium containing the carboxylic acid ester. 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 disrupted cells, cell extracts, etc. A treated product of bacterial cells can also be used. As the reaction medium, for example, ion-exchanged water or a buffer solution is used. The concentration of carboxylic acid ester in the reaction medium or culture solution is 0.01 to 50.
Weight percent is preferred. The carboxylic acid ester can also 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 2 to 11, preferably 5
The range is from 8 to 8. As the reaction proceeds, the pH of the reaction solution decreases due to the optically active carboxylic acid formed. In this case, it is preferable to maintain the pH at an optimum level with a suitable neutralizing agent. Regarding the reaction temperature, since the enzyme produced by the transformed microorganism of the present invention has extremely good thermal stability,
The reaction is possible at 80 ° C, but it is preferably 40 ° C or higher in order to utilize the characteristics of this strain-forming enzyme. A high temperature reaction is possible while maintaining the enzyme activity.

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 1 1) Cloning of a DNA fragment encoding an esterase gene From the cultured bacterial cells of Pseudomonas putida (Microtechnology Research Institute, No. 9677), the method of Marmur et al. [J. Marmur, J. of Molecular
Biology, 3 , 208, (1961)]
A was prepared. This was partially digested with the restriction enzyme EcoRI and DN
A fragment was obtained. On the other hand, the vector plasmid pUC19 was cut with the same enzyme. PUC19 linearized by EcoRI digestion
And the DNA fragment from Pseudomonas putida are mixed together, and T4
Hybrid DN by ligating ends with DNA ligase
A was formed. Escheric of the host microorganism, which was previously treated with CaCl ₂ to make it easier to accept foreign DNA
It was introduced into the JM105 strain, which is one of the hia coli K-12 strains. pU
When the JM105 strain containing C19 was grown on LB agar medium containing ampicillin, IPTG and XGal, β expressed in the JM105 strain was expressed.
-Galactosidase activity cleaved XGal to form blue colonies. To select a transformant expressing the esterase gene derived from Pseudomonas putida by utilizing the fact that when a foreign DNA fragment is inserted into the multiple cloning site of pUC19, the β-galactose activity is not expressed and the colony becomes colorless. In addition, colorless colonies were selected on LB agar medium containing ampicillin, IPTG and XGal, and among them, a strain expressing esterase activity was screened. Esch with an esterase gene
The search for erichia coli JM105 strain was performed as follows. Ten
The colonies were transferred to a paper impregnated with mM Tris-HCl (pH 7.5), 0.01% bromocresol purple, and 100 ppm (±) -β-acetylthio-α-methylmethylpropionate and left at room temperature for several hours. Colonies with esterase activity produced acid and the pH around the colonies decreased. Bromocresol purple, which is a pH indicator, changed from blue-purple to yellow, so that a strain having an esterase gene could be obtained by visual observation.

From this transformant, a plasmid DNA is again obtained.
Of the plasmid pPE101, pPE110, pPE11 having smaller DNA fragments.
1, pPE112, pPE113, pPE114, pPE115 and pPE116 were prepared. The position of the esterase gene could be known depending on the presence or absence of esterase activity of the strain transformed with E. coli JM105 by these plasmids (see FIG. 1).

E. coli JM105 (pPE116), which was obtained by transforming E. coli JM105 with plasmid pPE116, was deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as MR-2101, and the deposit number is Micromachine Research Institute Bacteria No. 12232. No. 2) Determining the nucleotide sequence of the esterase gene and the predicted amino acid sequence The entire nucleotide sequence of the DNA fragment derived from the parent strain contained in pPE116 was determined using the dideoxy chain term using the M13 phage vector.
inater method (F. Sanger. Science., 214 , 1205 (1981))
Determined by As a result, DN of about 1.2 kb derived from the parent strain
The base sequence of the A fragment was as shown in SEQ ID NO: 2. Examining the nucleotide sequence of SEQ ID NO: 2 and judging from the fact that there is only one open reading frame that covers the region essential for activity, and that the SD sequence exists upstream of the start codon number base, SEQ ID NO: 1 It was revealed that the nucleotide sequence shown in 1 is a structural gene for esterase. The amino acid sequence of esterase predicted from this base sequence is as shown in the lower part of the sequence corresponding to the 302nd to 1132nd bases of SEQ ID NO: 2. 3) Asymmetric hydrolysis of methyl (±) -β-acetylthio-α-methylpropionate E. coli JM105 (pPE116) containing 50 μg / ml ampicillin 50
After culturing with shaking in 0 ml of LB medium at 37 ° C. overnight, cells were obtained by centrifugation. The total amount of the cells was suspended in 200 ml of 5% (±) -β-acetyl-α-methylpropionate, and the reaction was carried out at 45 ° C for 3 hours while controlling the pH to 7.0 with 0.1N NaOH. After completion of the reaction, cells were removed by centrifugation, and unreacted methyl β-acetylthio-α-methylpropionate was extracted and removed from the supernatant with ethyl acetate. Next, after lowering the pH of the aqueous layer of the extraction residual liquid to 2.0 or less with dilute sulfuric acid,
-Acetylthio-α-methylpropionic acid was extracted with ethyl acetate. Then, anhydrous sodium sulfate was added to the extract for dehydration, and then the solvent was removed by evaporation to obtain an oily substance. After taking a part and diluting with water, β-acetylthio-α-methylpropionic acid was quantified by HPLC. In addition, a part of it was dissolved in chloroform and an automatic polarimeter (Union Giken PM-101
The optical rotation was measured with a (type). As a result, the desired product is 1.
5g was obtained, and the specific rotation was

[0024]

[Equation 1]

[0025] In the parent strain (Pseudomonas putida
9677) and the specific rotation of the product was almost equal to -58.3. Example 2 1) Preparation of pPE117 pPE116 was completely digested with PstI, 3.4 kb and 0.4 kb fragments were recovered and ligated with ligase. This recombinant plasmid was transformed into competent cells of E. coli C600, a strain showing esterase activity was selected in the same manner as in Example 1, and the plasmid possessed by the strain was named pPE117. This pPE117 is a Pseudomonas pu contained in pPE116.
It is a DNA derived from tida lacking the 0.2 kb fragment of PstI-SmaI. 2) Measurement of esterase activity Using the cultured bacterial cells of the above transformant, an asymmetric hydrolysis reaction of methyl (±) -β-acetylthio-α-methylpropionate was carried out in the same manner as in Example 1. Of these, the enzyme activity in the initial 1 hour was measured and compared with the parent strain.

[0026]

[Table 1]

As shown in Table 1, E. coli C600 (pPE117)
Showed about 200-fold higher esterase activity than the parent strain P. putida.

[0028]

INDUSTRIAL APPLICABILITY The recombinant plasmids pPE101, pPE116, pPE117 obtained by the present invention and the Escherichia coli transformant transformed with these contain an esterase gene which asymmetrically hydrolyzes carboxylic acid ester (I). .. In addition, the esterase of the transformant strain is characterized by being as excellent in thermostability as that of the parent strain. Then, the esterase activity of the transformed strain could be dramatically increased as compared with that of the parent strain. Furthermore, the optically active carboxylic acid [II] and its enantiomer ester can be efficiently obtained by allowing the microbial cell of this transformant or a treated product thereof to act on the carboxylic acid ester [I] at a high temperature of 45 ° C or higher. It was

[0029]

[Sequence list]

SEQ ID NO: 1 (1) Sequence length: 831 (2) Sequence type: Nucleic acid (3) Number of strands: Double strand (4) Topology: Linear (5) Type of sequence: genomic DNA (6) ) Origin (a) Organism name: Pseudomonas put
ida) (b) Strain name: MR-2068 Mikken Kenkyubo No. 9677 (7) Sequence features: 1-831 P CDS (8) Sequence: ATGAGCTATG TAACCACGAA GGACGGCGTACAGATCTTCT ACAAGGACTG GGGCCCGCGC 60 GATGCGCCGG TCATCCACTT CCACCACGGC TGCGCGCTCA GTGC CAGATGCTGT TCTTCCTCGC CCACGGTTAC CGCGTGGTCG CCCACGACCG CCGCGGCCAT 180 GGCCGCTCCA GCCAGGTATG GGACGGCCAC GACATGGACC ACTACGCCGA CGACGTAGCC 240 GCAGTGGTGG CCCACCTGGG CATTCAGGGC GCCGTGCATG TCGGCCACTC GACCGGTGGC 300 GGTGAGGTGG TGCGCTACAT GGCCCGACAC CCTGCAGACA AGGTGGCCAA GGCCGTGCTG 360 ATCGCCGCCG TACCGCCGTT GATGGTGCAG ACTCCCGATA ATCCCGGTGG CCTGCCCAAA 420 TCCGTTTTCG ACGGCTTCCA GGCCCAGGTC GCCAGCAACC GCGCGCAGTT CTACCGGGAT 480 GTGCCGGCAG GGCCGTTCTA CGGCTACAAC CGCCCCGGTG TCGACGCCAG CGAAGGCATC 540 ATCGGCAACT GGTGGCGCCA GGGCATGATC GGTAGCGCCA AGGCCCATTA CGATGGCATC 600 GTGGCGTTTT CCCAGACCGA CTTCACCGAA GACCTGAAGG GCATTACCCA GCCGGTGCTG 660 GTGATGCATG GCGACGACGA CCAGATCGTG CCGTATGAGA ACTCCGCGCT GCTGTCGGCC 720AAAGCTGCTCT GCATGG CATGCCGACC 780 ACCCATGCCG ATGTGATCAA TGCGGATTTG CTGGCGTTTA TCCGTAGCTG A SEQ ID NO: 2 (1) Sequence length: 1329 (2) Sequence type: Nucleic acid (3) Number of strands: Double strand (4) Topology: Linear (5) ) Sequence type: genomic DNA (6) Origin (a) Organism name: Pseudomonas putida
ida) (b) Strain name: Mikken Kenkyukai No. 9677 (7) Sequence characteristics: 302-1132 P CDS (8) sequence 10 20 30 40 50 60 CCCGGGCCGTGAGCGATGCCATCCTCGGTGACGACGACCTGCTGGCGCTATATCAAGGCA 70 80 90 90 110 110 120 TCGACAACGGCCGCTTCCCCGACCGCGCCGACCTGCTGG. 170 180 AGGCCTGGTACCGGATGCGCGACCGCGCCTGATCGCCTGGCACCGCTCCTACACGGCGCC 190 200 210 220 230 240 GGGCAGGCCGGAAGCATGGTGCAAGCCCACTGCAGTGCAGTCACCACAAATTCCGGCGCC 250 260 270 280 290 300 AAGCAAAATTCCTCCTATTCTCAATAGCTCACTTCGCTTCCTGCACACAGGAGACCCGAC 310 320 330 340 350 360 CATGAGCTATGTAACCACGAAGGACGGCGTACAGATCTTCTACAAGGACTGGGGCCCGCG MetSerTyrValThrThrLysAspGlyValGlnIlePheTyrLysAspTrpGlyProArg 370 380 390 400 410 420 CGATGCGCCGGTCATCCACTTCCACCACGGCTGGCCGCTCAGTGCCGACGACTGGGACGC AspAlaProValIleHisPheHisHisGlyTrpProLeuSerAlaAspAspTrpAspAla 430 440 450 460 470 480 GCAGATGCTGTTCTTCCTCGCCCACGGTTACCGCGTGGTCGCCCACGACCGCCGCGGCCA GlnMetLeuPhePheLeuAlaHisGlyTyrArgValValAlaHisAspArgArgGlyHis 490 500 510 520 530 540 TGGCCGCTCCAGCCAGGT ATGGGACGGCCACGACATGGACCACTACGCCGACGACGTAGC GlyArgSerSerGlnValTrpAspGlyHisAspMetAspHisTyrAlaAspAspValAla 550 560 570 580 590 600 CGCAGTGGTGGCCCACCTGGGCATTCAGGGCGCCGTGCATGTCGGCCACTCGACCGGTGG AlaValValAlaHisLeuGlyIleGlnGlyAlaValHisValGlyHisSerThrGlyGly 610 620 630 640 650 660 CGGTGAGGTGGTGCGCTACATGGCCCGACACCCTGCAGACAAGGTGGCCAAGGCCGTGCT GlyGluValValArgTyrMetAlaArgHisProAlaAspLysValAlaLysAlaValLeu 670 680 690 700 710 720 GATCGCCGCCGTACCGCCGTTGATGGTGCAGACTCCCGATAATCCCGGTGGCCTGCCCAA IleAlaAlaValProProLeuMetValGlnThrProAspAsnProGlyGlyLeuProLys 730 740 750 760 770 780 ATCCGTTTTCGACGGCTTCCAGGCCCAGGTCGCCAGCAACCGCGCGCAGTTCTACCGGGA SerValPheAspGlyPheGlnAlaGlnValAlaSerAsnArgAlaGlnPheTyrArgAsp 790 800 810 820 830 840 TGTGCCGGCAGGGCCGTTCTACGGCTACAACCGCCCCGGTGTCGACGCCAGCGAAGGCAT ValProAlaGlyProPheTyrGlyTyrAsnArgProGlyValAspAlaSerGluGlyIle 850 860 870 880 890 900 CATCGGCAACTGGTGGCGCCAGGGCATGATCGGTAGCGCCAAGGCCCATTACGATGGCAT IleGlyAsnTrpTrpArgGlnGlyMetIleGlySerAlaLysAlaHisTyrAspGlyIle 910 920 930 940 950 960 CGTGGCGTTTTCCCAGACCGACTTCACCGAAGACCTGAAGGGCATTACCCAGCCGGTGCT ValAlaPheSerGlnThrAspPheThrGluAspLeuLysGlyIleThrGlnProValLeu 970 980 990 1000 1010 1020 GGTGATGCATGGCGACGACGACCAGATCGTGCCGTATGAGAACTCCGGGCTGCTGTCGGC ValMetHisGlyAspAspAspGlnIleValProTyrGluAsnSerGlyLeuLeuSerAla 1030 1040 1050 1060 1070 1080 CAAGCTGCTGCCCAATGGCACACTGAAGACCTACCAGGGCTACCCGCATGGCATGCCGAC LysLeuLeuProAsnGlyThrLeuLysThrTyrGlnGlyTyrProHisGlyMetProThr 1050 1100 1110 1120 1130 1140 CACCCATGCCGATGTGATCAATGCGGATTTGCTGGCGTTTATCCGTAGCTGATGTGATCG ThrHisAlaAspValIleAsnAlaAspLeuLeuAlaPheIleArgSer *** 1150 1160 1170 1180 1190 1200 CCTGCACCGGCCTCTTCGCGGGCACTGGCAACACACCTCCCCCAGGATTACCATGTCACG 1210 1220 1230 1240 1250 1260 CTTCTAGTGCGGCCCTTTGCCGCCCCTTGCCTCCCTGCCTGCCAAAACCCCATGCCCTTC 1270 1280 1290 1300 1310 1320 GAACTCACCGTAGAACCCCTCACCCTGCTGATCCTGGCCCTGGTCGCCTTCGTCGCCGGT TTCATCGAT

[Brief description of drawings]

FIG. 1 shows a deletion plasmid prepared from pPE101 and esterase activity.

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[Procedure amendment]

[Document name] Contract number change notification

[Submission date] April 23, 1992

[Former name of depositary institution] Ministry of International Trade and Industry, Institute of Industrial Technology, Institute of Microbial Technology

[Old consignment number] Microtechnology Research Institute, Microbiology No. 12232 (F
ERM P-12232)

[Old consignment number] Microtechnology Research Institute Microbiology No. 12416 (F
ERM BP-1246 6)

[Name of new depositary institution] Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology

[New consignment number] Micro Engineering Research Institute No. 3838 (FE
RM BP-3838)

[New consignment number] Mikori Kenjoyori No. 3835 (FE
RM BP-3835)

Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 15/55 C12R 1:40)

Claims (3)

[Claims] 1. A general formula: (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 racemic carboxylic acid ester is represented by the base of SEQ ID NO: 1. A compound of the general formula: ## STR2 ## characterized in that a culture solution of transformed microorganisms containing a recombinant plasmid in which a DNA fragment consisting of a sequence or a part thereof is ligated, a microbial cell or a treated product of the microbial cell is allowed to act. (In the formula, R _1, R ₂ and n are the same as the above), and a process for producing an optically active carboxylic acid and its enantiomer ester. 2. The method for producing an optically active carboxylic acid and its enantiomer ester according to claim 1, wherein the transformed microorganism is Escherichia coli. 3. The method for producing an optically active carboxylic acid and its enantiomer ester according to claim 1, which is operated at a temperature of 45 ° C. or higher.