JP3151893B2 - Ester asymmetric hydrolase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene encoding an ester asymmetric hydrolase, ie, an esterase, an expression plasmid containing the gene, and a transformant carrying the same. By expressing the gene obtained according to the present invention in a microbial cell, the industrially produced enzyme is asymmetrically hydrolyzed with an ester of chrysanthemic acid or a chrysantic acid derivative which is an intermediate of a pyrethroid insecticide, It can be applied to a bioprocess for producing optically active chrysanthemic acid or a derivative thereof.

[0002]

2. Description of the Related Art In recent years, many attempts have been made to apply reactions utilizing microorganisms or enzymes to organic synthesis.
In particular, like pharmaceuticals, agricultural chemicals and their synthetic intermediates,
This is a very effective method when it is necessary to obtain an optically active compound having a complicated structure because the enzyme can utilize the high stereoselectivity of the enzyme.

[0003] In particular, regarding the production of an optically active compound, one isomer is obtained from a racemic ester compound by a stereoselective hydrolysis reaction, or a chiral compound is obtained from a prochiral compound by a regioselective hydrolysis reaction. Esterases and lipases have been used for the reaction for producing Escherichia coli, or for the stereoselective production of esters utilizing their reverse reaction.

For example, an esterase derived from pig liver is used in such a reaction as described in Laumen et al. (Tetrahedro
n Lett .26, 407-410, (1985)), and Wang et al. (J. Am, Che
m. Soc. 106, 3695 (1984)). On the other hand, as an example of utilizing a microorganism-derived esterase for an organic synthesis reaction, Bacillus subtilis NRRL-B-558 was used for the synthesis of a cephalosporin derivative (Appl Microbiol. 30, 413-419 (19)
75)).

[0005]

However, the enzyme of animal origin has a limited supply, is unstable and difficult to handle, and is economically difficult to use for industrial use. When using an esterase derived from a microorganism, it is difficult to culture the bacterial strain of a strain obtained by separating a large number of microorganisms from nature and selecting one having high optical selectivity for the intended reaction, and the reaction efficiency is low. There were problems such as not being sufficiently high and the productivity of the enzyme being not high.

[0006] For these reasons, the isolated strain is cultured and used for an industrial process, or the high-performance asymmetric hydrolase is produced inexpensively and in large quantities by culturing the strain, thereby producing Application to the field was not easy.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have made intensive efforts to obtain esterases having a wide range of application to organic synthesis reactions, particularly ester asymmetric hydrolysis reactions. Arthrobacter showing selectivity
Globiformis (Artblobglob)
iformis) (IFO-12958). (JP-A-1-181788) As a result of further research, a high esterase-producing strain Arthrobacter-SC-6-98-28 (FERM BP-36) was developed.
5 8) was obtained. (Japanese Patent Application No. 3-474) Furthermore, a gene encoding the enzyme was successfully obtained.

[0008] This makes it possible to cause the microorganism to produce the enzyme in large quantities by using the recombinant DNA technique.
It is possible to dramatically increase the catalytic ability of microorganisms as compared to conventional ones, and to use recombinant microorganisms or esterases,
It can be used as a bioreactor for ester asymmetric hydrolysis in a free state or immobilized on a carrier.

Hereinafter, the present invention will be described in more detail. A first object of the present invention is to provide a gene encoding an esterase. Esterase gene of the present invention, a microorganism for example A Le having an ester asymmetrically hydrolyzing <br/> scan Arthrobacter SC-6-98-28 (FERM BP-
36 5 8). The gene encoding the esterase is a chromosome DN cut to an appropriate length.
The A fragment is obtained by, for example, using a phage vector λgtl.
Alternatively, a chromosomal DNA library can be prepared by inserting it into pUC19 or the like, which is a plasmid vector, and can be obtained by a screening method. Regarding the selection of DNA, various methods such as an immunological method using an antibody against esterase, a hybridization method using a synthetic DNA corresponding to the partial amino acid sequence of the protein as a probe, and a screening method using esterase activity are used. Can be used. Further, when only a part of the esterase DNA is obtained by the above-mentioned method, a complete DNA can be obtained again using this as a probe. A preferable base sequence of the esterase gene thus obtained is a gene containing a base sequence corresponding to the following amino acid sequence.

[Image Omitted] SEQ ID NO: 1

[0010] More preferred are genes represented by the following nucleotide sequences.

[Image Omitted] SEQ ID NO: 2

A second object of the present invention is to provide an expression plasmid containing a base sequence encoding an esterase. An expression vector contains a replicable genetic information in a host cell, and is capable of autonomous propagation,
Those which can be easily isolated and purified from a host and have a detectable marker are preferable. Various vectors are commercially available, and restriction enzymes used for cutting the vector are also commercially available. Methods of using these are known. For example, for expression in E. coli, an expression vector containing a promoter such as lac, tac, or trp (these are commercially available from Pharmacia PL as an expression vector or as a promoter cartridge) can be used. A preferred expression plasmid is a DNA capable of self-propagation in a host cell, that is, a DNA containing a nucleotide sequence necessary for self-propagation, and particularly preferred is an expression plasmid pAGE into which the gene represented by the nucleotide sequence has been cloned. -201, pAG
E-202 and pAGE-203.

It is a third object of the present invention to provide a microorganism produced in an esterase cell. Depending on the expression plasmid, examples of microorganisms that produce esterase include Escherichia coli, Bacillus subtilis, lactic acid bacteria, and mold.
Preferably, Escherichia coli is used. Particularly preferred microorganisms are Escherichia coli JM109 (pAGE-201), JM109 (pAGE-2
02), and JM109 (pAGE-203) and Escherichia coli JM
105 (pAGE-201), JM105 (pAGE-202), and JM105 (pAGE-203)
It is. A known method is used for transforming the microorganism with the expression plasmid. By culturing this microorganism, it has become possible to produce esterase in large quantities.

The thus produced recombinant cells producing esterase can be used as a bioreactor for asymmetric hydrolysis of esters, for example, for producing useful compounds such as optically active compounds which are intermediates of pharmaceuticals and agricultural chemicals. Is possible. In addition, by culturing recombinant cells, the esterase can be mass-produced and used as an enzyme reactor.

[0014]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples, and ordinary changes in the technical field of the present invention can be made. . Examples Isolation of esterase DNA clones Preparation of chromosomal DNA Arthrobacter SC-6-98-28 (FERM B
The P-36 5 8) strain, the culture medium for the preculture 5 ml (soluble starch 3.0%, polypeptone 0.7%, _0.5% yeast _{extract, KH 2 PO 4 0.5%,} pH5.0) in , 3
After shaking culture at 0 ° C. for 24 hours, the obtained culture solution was mixed with 500 ml of a main culture medium (soluble starch 6.0%,
Polypeptone 1.0%, Yeast extract 0.2%, KH ₂ P
O ₄ 0.5%, were inoculated into pH 5.0), and cultured with shaking at 30 ° C.. At that _time, when _{the OD 660} reached 0.25, was added to the penicillin G final concentration of 300 units / ml culture _medium, the culture was continued until the _{OD 600} reached 1.0.

The cells are recovered by centrifugation, and 45 ml of 150
mM NaCl, 15 mM sodium citrate 10 mM, EDTA
After suspending in 27% sucrose, egg white lysozyme was added to a final concentration of 5 mg / ml and incubated at 37 ° C for 30 minutes. Next, 10 ml of 10% SDS was added, and protease K was further added to a final concentration of 200 μg / ml.
And incubated at 37 ° C. for 4 hours. Then, add 2 volumes with an equal volume of 0.1 M tris-saturated phenol.
After extraction twice with ether, twice the volume of ethanol was added to the aqueous layer to precipitate nucleic acids, which were wound around a glass rod in a thread form to collect DNA.

After drying the obtained nucleic acid, 5 ml of Tris EDTA
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and treated with RNaseA at 37 ° C. for 2 hours at a final concentration of 100 μg / ml. Then, an equal volume of phenol / chloroform (1: 1)
(V / V)), and the DNA was precipitated by adding twice the volume of cold ethanol to the aqueous layer. Obtained DNA
Is washed with 80% ethanol and then dried. Tris / EDTA
Dissolved in buffer. About 5.8 mg of chromosomal DNA was obtained.

Preparation of Chromosomal DNA Library The chromosomal DNA obtained in 1 was digested with KpnI. On the other hand, the vector pUC19 (Takara Shuzo Co., Ltd.) was digested with KpnI, treated with alkaline phosphatase, mixed with the chromosomal DNA fragment obtained above, and ligated using T ₄ DNA ligase. Next, a competent cell of JM109, one of the E. coli K-12 strains (Takara Shuzo Co., Ltd.), and this ligase reaction solution were mixed, and plasmid DNA was introduced into the cells.

When the JM109 strain containing pUC19 is grown on an LB agar medium containing ampicillin, IPTG, and X-Gal, the X-Gal is cleaved by the activity of β-galactosidase expressed in the JM109 cells, so that the colony grows. It has a blue color. However, when a foreign DNA fragment is inserted into the multicloning site of pUC19, β-galactosidase activity is not expressed, and the colony becomes colorless. By the above method, a colony in which the gene was inserted into pUC19 was selected, and the DNA sequence was determined.

First, white colonies were selected from the colonies on the plate, then a mixed DNA probe corresponding to the purified N-terminal amino acid sequence of esterase was synthesized, and colony hybridization was carried out according to the following method. That is, the white colonies spread on the plate were transferred to a nylon membrane or planted on a nylon membrane with a bamboo skewer, and the membrane was placed on an LB ampicillin plate and cultured for several hours. When the colonies grew on the membrane, the operation of immersing the membrane in 0.5 N NaOH was performed twice, the colonies on the membrane were lysed, and the membrane was washed twice with 1 M Tris-HCl (pH 7.5) and neutralized twice. did. The DNA extracted from the colonies was fixed on a membrane at 80 ° C under vacuum.
To dry the membrane for 2 hours.

Next, this membrane was placed in 6 × SSC, 1
% SDS, 10x Denhardt (0.2% Ficoll,
0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin) After incubating at 55 ° C. for 1 hour, 6 × S
The reaction was performed in the order of SC, 1% SDS, 10 × Denhardt, 100 μg / ml salmon testis DNA, 55 ° C., 4 hours. On the other hand, the mixed DNA was 5 ′ using [γ ³² P] ATP.
The ends were labeled and purified by column. Hybridization was performed by adding a solution of the membrane to a plastic bag, adding labeled DNA at about 5 × 10 ⁵ cpm per membrane, and overnight at 55 ° C.

The filter subjected to hybridization was 6 × SSC, 55 ° C., 15 minutes, 6 × SSC,
55 ° C., 30 minutes, 6 × SSC, 1% SDS, 55 ° C.,
After reacting in the order of 30 minutes, the mixture was air-dried, applied with an X-ray film (Fuji RX) and an intensifying screen, and an autoradiogram was taken. As a result, a pK-12 strain giving a positive signal was obtained.

The pK-12 strain was not long enough to encode the full length of the esterase. Therefore, after determining the nucleotide sequence of pK-12 by the dideoxy method, screening by colony hybridization was performed again using a part of the sequence as a DNA probe. At this time, a chromosome DNA was digested with EcoRI, and a library prepared by ligating to a vector pUC19 similarly digested with EcoRI was used. As a result, a clone giving a positive signal, pEH-16 strain, was obtained.

Furthermore, the inserted fragment of the pK-12 strain was
And inserted into the EcoRI site of the pEH-16 strain to construct a plasmid pAGE-1 containing the entire translation region of esterase. The construction method is shown in FIG.

A restriction map of the esterase gene and
Determination of base sequence For the esterase DNA clone obtained by the above-described method, a restriction enzyme map of the inserted fragment was prepared.

Escherichia coli JM109 strains into which plasmids pK12 and pEH16 have been introduced are each cultured, and the plasmid DN is prepared by the method of Birnboim-Doly.
A was prepared. Plasmid DNA was digested with various restriction enzymes, and the digested DNA fragments were analyzed by 1% agarose gel and 5% polyacrylamide gel electrophoresis. A restriction map was obtained by comparing the chain lengths of the various DNA fragments that appeared.

Determination of Esterase DNA Base Sequence and Amino Acid Sequence The base sequence of a total of 2,175 base pairs of the inserted fragment of pAGE-1 was
Primer DNA in both forward and reverse directions of C plasmid (Takara Shuzo Co., Ltd.), sequentially synthesized primer DNA, and 7
-Deaza Determined by the dideoxy method using a sequencing kit (Takara Shuzo Co., Ltd.).

The total 2,1 of the determined DNA fragments inserted into pAGE-1
The base sequence of 74 base pairs is shown in FIGS. As a result of searching for an open reading frame from the nucleotide sequence, as shown in FIGS. 1 to 4, it was found that from GTG at the base number 211 to 1335 was the only open reading frame. As the reading frame of protein, the other two reading frames in which the bases are shifted one by one can also be considered.
In these, a stop codon appears many times in the middle of the base sequence,
It was not thought that it could encode an estera with a molecular weight of 43,000 estimated by SDS-PAGE.

Further, since the amino acid sequence at the amino terminus of the purified esterase was identical to the amino acid residue below the start codon of this open reading frame, it was clear that this open reading frame encodes an esterase. became. As a result, the esterase was found to be a protein consisting of 375 amino acid residues and having a molecular weight of 39,836.

Construction of Esterase High Expression Plasmid Expression vector pKK223-3 (Boyer et al., Prot. Natl. Acad. Sci. U.
SA, 80, 21 to 25 and (1983) Pharmacia) was partially digested with BamHI, then blunted by T ₄ DNA polymerase, to produce a pKK223-4 which was deleted one place missing a BamHI site by performing a ligation

On the other hand, in order to convert the DNA sequence 5 ′ from the start codon of the esterase gene,
NA fragment was applied to Applied DNA Synthesizer Model 3
Synthesized using 80A. Synthesized pH-21, ES-01, ES-11,
The 5'-ends of the ES-21 and ES-13 fragments were phosphorylated and ligated and annealed to the untreated fragments, respectively, to prepare the following three types of double-stranded DNA fragments.

PH-22

[Image Omitted] SEQ ID NO: 3

PH-21

[Image Omitted] SEQ ID NO: 4

ES-01

[Image Omitted] SEQ ID NO: 5

ES-02

[Image Omitted] SEQ ID NO: 6

ES-13

Embedded image SEQ ID NO: 7

ES-11

[Image Omitted] SEQ ID NO: 8

ES-12

[Image Omitted] SEQ ID NO: 9

ES-21

[Image Omitted] SEQ ID NO: 10

ES-22

Embedded image SEQ ID NO: 11 The 5 ′ end of each of the synthesized pH-21, ES-01, ES-11, ES-21, and ES-13 fragments is phosphorylated, and ligated and annealed to the untreated fragments, respectively. Thus, the following three types of double-stranded DNA fragments were prepared.

Embedded image Both ends of the prepared double-stranded DNA fragment were phosphorylated. On the other hand, the translation region of the esterase was cut out by digesting pAGE-1 with Nsp (7524) V and HindIII, and the synthesized DNA fragment and the vector pKK223-4 digested with BamHI and treated with alkaline phosphatase were used. Ligation was performed. In this manner, three types of Escherichia coli expression plasmids pAGE-201, pAGE-202, and pAGE-2 having a modified 5 'DNA sequence downstream of the toc promoter.
Got 03.

Chrysanthemum ester asymmetric by recombinant Escherichia coli
Production of hydrolase Each of the three expression plasmids was introduced into E. coli JM109, and M9 medium (10.5 g of K ₂ HPO ₄ , 4.5 g of KH ₂ PO ₄ , (NH ₄ ) ₂ SO _4
4 1.0 g, sodium citrate 0.5 g, MgSO ₄ 7H ₂ O 0.2 g,
The cells were cultured at 37 ° C. using 2.0 g of glucose, thiamine hydrochloride, 2 mg / L), and IPTG (isopropylthio-β-D-galactoside) was added during the logarithmic growth phase to a final concentration of 1 mM, and esterase was added. Expression was induced.

After completion of the culture, the cells were collected by centrifugation, and a part of the cells was subjected to SDS-PAGE. As a result, esterase was recognized as a main band at the position of molecular weight 40,000, and all of the enzymes were high in E. coli. It turned out that it was expressed.

Ethyl chrysanthe by recombinant Escherichia coli
Asymmetric hydrolysis of these compounds Using these cells, a racemic compound, ethyl ester of chrysanthemic acid (2,2-dimethyl-3-isobutenylcyclopropane-1-carboxylic acid) (cis-form: trans-form = 10: 9
(0, (+) / (-) = 50/50) (hereinafter abbreviated as KCE in the present specification). That is, cells equivalent to 100 ml of the culture solution were transferred to 50 ml of 200 mM glycine.
Suspend in sodium hydroxide buffer (pH 10.0)
g of the above chrysanthemic acid ethyl ester was added and reacted at 37 ° C. with stirring at 1,000 rpm. After 6 hours,
After adding 5.0 ml of 35% hydrochloric acid to terminate the reaction, the produced chrysanthemic acid (hereinafter abbreviated as KCA) and unreacted KCE were extracted with methyl isobutyl ketone (hereinafter abbreviated as MIBK) and extracted. The extract is subjected to gas chromatography (column: Sh
inchrom F55 (5%) + H 3 PO 4 (1%), 2.6m, 1
(90 ° C.) and the hydrolysis rate was calculated from the area percentage.

Next, 20 ml of 0.01N NaoH was added to the above-mentioned extract to extract only KCA as a sodium salt into an aqueous layer, and KCA was again extracted by acid precipitation with 35% hydrochloric acid and MIBK. The extract was concentrated and dehydrated, a part of which was added with dicyclohexylcarbodiimide and 3.5-dichloroaniline, left at room temperature for 3 hours, and then subjected to high performance liquid chromatography (column: SUMIPAX OA-2100 × 2, mobile phase: Isomer analysis was performed with n-hexane: 1,2-dichloromethane (17: 3 V / V), flow rate 1.5 ml / min, detection: 254 mm). The results are shown in Table 1.

[0044]

[Table 1] 1) Hydrolysis rate: It represents the molar fraction of (+)-trans-KCA obtained relative to the number of moles of (+)-trans-KCE in the raw material.

Chrysanthemic acid ethyl ester by recombinant Escherichia coli
Of introducing asymmetric hydrolysis three expression plasmids to each E. coli JM105, cultured at 37 ° C. using a M9 medium, IPTG was added to the logarithmic growth phase to a final concentration of 1 mM, Estella - Ze Expression was induced. Using these cells, racemic, ethyl ester of chrysanthemic acid (2,2-dimethyl-3-isobutenylcyclopropane-1-carboxylic acid) (cis-form: trans-form = 10: 90, (+) / (-) = 50 /
The hydrolysis reaction of 50) was performed. That is, 100 ml of culture solution
The corresponding bacterial cells were suspended in 50 ml of 200 mM glycine / sodium hydroxide buffer (pH 10.0), and 1.0 g of the above-mentioned ethyl chrysanthenate was added thereto.
The reaction was performed at ℃. Six hours later, 5.0 ml of 35% hydrochloric acid was added to the reaction solution to terminate the reaction, and then the produced chrysanthemic acid (hereinafter referred to as K) was treated with methyl isobutyl ketone (hereinafter abbreviated as MIBK).
CA), and unreacted KCE was acid precipitated and extracted. The extract was subjected to gas chromatography (column: Shinchrom F.
55 (5%) + H ₃ PO ₄ (1%), 2.6 m, 190 ° C.), and the hydrolysis rate was calculated from the area percentage. Table 2 shows the results.

[Table 2]

Conventionally, Arthrobacter globiformis (IFO-1295
When 8) was used, the expression level of esterase in the cells was extremely low, so that 20 μg of the purified enzyme (corresponding to a 2.5 L culture solution) was used and a 90 KCE concentration of 2 w / v% at 24 ° C. was used.
When the reaction was carried out for a time, the hydrolysis rate was 6.4% (see JP-A-1-181788). According to the present invention, the gene was cloned into Escherichia coli, and the vicinity of the promoter and initiation codon was changed to a sequence suitable for Escherichia coli. By the conversion, the expression level of the enzyme in the cells is dramatically increased, and as a result, (+)-
It has become possible to obtain trans KCA specifically and in high yield.

[Sequence list]

SEQ ID NO: 1 Sequence length: 375 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence SEQ ID NO: 2 Length of sequence: 1125 SEQ types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism: A Le Surobakuta globiformis (Art
hrobacterglobiformis) Ltd. name: SC-6-98-28 (FERM BP- 36 5
8) Characteristic of sequence Symbol indicating characteristic: CDS Location: 1. . 1125 Method Determined Features: E Sequence SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 4 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 5 Sequence length: 44 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 6 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 7 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 8 Sequence length: 44 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 9 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 10 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence SEQ ID NO: 11 Sequence length: 33 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence from the 1st to the 570th in the inserted DNA fragment of clone pAGE-1, and the amino acid sequence of the esterase deduced therefrom.

FIG. 2 shows the nucleotide sequence from position 571 to position 1002 of the inserted DNA fragment of clone pAGE-1, and the amino acid sequence of the esterase deduced therefrom. The N-terminal DNA probe used for the screening corresponds to base numbers 211 to 230.

FIG. 3 shows the nucleotide sequence at positions 1003 to 1695 of the inserted DNA fragment of clone pAGE-1, and the amino acid sequence of the esterase deduced therefrom. The translated region of the esterase corresponds to nucleotides 210 to 1335.

FIG. 4 shows the nucleotide sequence at positions 1696 to 2175 of the inserted DNA fragment of clone pAGE-1.

FIG. 5 shows positive clones PK-12 and pEH16 obtained by colony hybridization, and clones containing the full length of the esterase translation region constructed based on these clones.
3 shows a restriction map of pAGE-1. In FIG. globifor
The DNA derived from mis indicates the esterase translated region.

FIG. 6 shows a construction strategy of an esterase high expression plasmid pAGE-201-3. In the figure, c represents the synthetic DNA, and the thick arrow represents the tac promoter.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/00

Claims (12)

(57) [Claims] 1. An ester asymmetric hydrolase gene having the asymmetric hydrolytic ability of an ester of chrysanthemic acid or a chrysantic acid derivative and encoding an enzyme represented by the following amino acid sequence: Number 1 2. The ester asymmetric hydrolase gene according to claim 1, wherein the gene is specified by the following nucleotide sequence: 3. A plasmid comprising the gene according to claim 1 or 2. 4. A method comprising a promoter that functions in Escherichia coli,
A plasmid having the gene according to claim 1 or 2 downstream thereof. 5. A microorganism which is characterized by retaining the claims 3 or 4 Symbol mounting plasmids 6. The microorganism according to claim 5, which is Escherichia coli. 7. The microorganism according to claim 6 , wherein the Escherichia coli is JM109 strain. 8. The microorganism according to claim 6 , wherein the Escherichia coli is strain JM105. 9. A process for producing an ester asymmetric hydrolase, which comprises culturing the microorganism according to claim 5, 6, 7, or 8. 10. A method for producing a plasmid, comprising incorporating the gene according to claim 1 into a plasmid capable of self-propagation in a host cell. 11. A method for producing a recombinant microorganism, which comprises transforming a microorganism with the plasmid according to claim 3 or 4. 12. A method comprising contacting a culture or cells of the microorganism of claim 5, 6, 7 or 8 with an ester of chrysanthemic acid or a derivative of chrysantic acid, and subjecting the ester to asymmetric hydrolysis. For producing optically active chrysanthemic acid or chrysanthemic acid derivative