# Patent

## Title
Hydrolytic enzyme inhibitors/inactivators and methods for using same

## Abstract

The present invention relates to a DNA fragment containing a nucleotide sequence that encodes an amino acid sequence of esterase, said esterase asymmetrically hydrolyzing carboxylic acid esters represented by the formula (I); ##STR1## (wherein R.sub.1 is alkyl, aralkyl or aryl, R.sub.2 and R.sub.3 are alkyl, and n is 1 or 2) an esterase encoded by the DNA fragment, a recombinant plasmid containing the DNA fragment, a microorganism transformed with the recombinant plasmid and methods of producing optically active carboxylic acids and their enantiomeric esters.

## Background
The present invention relates to a DNA fragment containing a nucleotide sequence encoding esterase which is highly heat stable and asymmetrically hydrolyzes carboxylic acid esters, an esterase encoded by the DNA fragment, a recombinant plasmid containing the DNA fragment, a microorganism transformed with the recombinant plasmid and methods of producing optically active carboxylic acids and their enantiomeric esters.

An optically active carboxylic acid and its enantiomeric ester, which are represented by the general formula (II); ##STR2## (wherein R.sub.1 is alkyl, aralkyl or aryl, R.sub.2 is alkyl, and n is 1 or 2), are useful as a raw material of various physiologically active substances.

The present inventors disclose methods of asymmetrically hydrolyzing a racemic mixture of carboxylic acid esters to produce an optically active carboxylic acid and its enantiomeric ester using an enzyme or a microorganism, the carboxylic acid esters being represented by the general formula (I); ##STR3## (wherein R.sub.1, R.sub.2 and n are the same meaning as described above and R.sub.3 is alkyl) [see Japanese Patent Application KOKAI No. 12992/1985, No. 12993/1985].

The present inventors isolated Pseudomonas putida (FERM BP-3846) from a soil and found that it produces esterase having a potent asymmetrical hydrolysis activity for a racemic mixture of carboxylic acid esters, and disclosed the bacterium in Japanese Patent Application KOKAI No. 222798/1989).

To obtain a microorganism having an enhanced enzymatic activity, the recombinant DNA technology is often utilized these days. There is a method known in the art in which a transformed microorganism is used to increase the production of esterase involved in hydrolysis reaction: a DNA fragment encoding an esterase gene derived from Pseudomonas fluorescens IF03018 is prepared; a host microorganism is transformed with a plasmid having the DNA fragment; esterase is produced from the transformed microorganism (Japanese Patent Application KOKAI No. 67190/1989).

Typically, bacterial cells are directly utilized as "enzyme" rather than utilizing an enzyme purified by a complicated manipulation such as isolation and purification when carboxylic acid esters of the formula (I) are required to be asymmetrically hydrolyzed. In such a method, an efficient reaction relies on several factors such as a potent enzyme activity of bacterial cells, the property of an enzyme that serves to produce carboxylic acids with high optical purity and stability in various conditions such as temperature, pH and the like.

Heat labile enzymes are susceptible to thermal inactivation as reaction time progresses so that it is not easy to increase a reaction rate by raising reaction temperature or to reuse recovered enzyme. Bacterial cells that produce enzyme, which has an excellent heat stability and potent enzymatic activity, are necessary for efficient reaction.

The recombinant DNA technology is an effective method of increasing the enzymatic activity of enzyme produced by bacteria as described in Japanese Patent Application KOKAI No. 67190/1989. The properties of enzyme essentially depends on a DNA sequence that encodes an enzyme. To obtain a highly stable enzyme that satisfies the above requirement, one needs to find a DNA fragment encoding the enzyme.

The Japanese Patent Application KOKAI No. 67190/1989 discloses a microorganism transformed with a recombinant plasmid containing a DNA fragment. The DNA fragment comprises the sequence encoding an esterase gene derived from Pseudomonas fluorescens IF03018. Esterase produced by the microorganism is less heat stable and is inactivated in several hours, for example, at 45.degree. C. or more.

It is a primary object of the present invention to prepare a DNA fragment encoding a highly heat stable esterase for the asymmetrically hydrolysis of carboxylic acid esters of the formula (I) and to efficiently carry out the asymmetrical hydrolysis using a transformant that contains the DNA fragment and efficiently produces the enzyme.

The present inventors have investigated an efficient method of asymmetrically hydrolyzing carboxylic acid esters of the formula (I) using enzyme. We have successfully found that a microorganism transformed with a recombinant plasmid containing an esterase gene derived from Pseudomonas putida (FERM BP-3846), which is isolated from a soil, is useful for obtaining a high yield of reaction products and for carrying out an efficient reaction in a short period of time. The microorganism produces enzyme having potent enzymatic activity and the enzyme is not inactivated with time even at a reaction temperature of 50.degree. C. or more.

In addition, we have isolated a highly heat stable, novel esterase substantially in a pure form from the transformed microorganism and successfully characterized the physical and chemical properties of the enzyme.

The present invention provides a DNA fragment containing a nucleotide sequence that encodes the amino acid sequence of esterase as described in SEQ ID No. 2, said esterase asymmetrically hydrolyzing carboxylic acid esters represented by the formula (I); ##STR4## (wherein R.sub.1 is alkyl, aralkyl or aryl, R.sub.2 and R.sub.3 are alkyl, and n is 1 or 2) to give an optically active carboxylic acid represented by the formula (II); ##STR5## (wherein R.sub.1, R.sub.2, and n are the same meaning as described above).

In addition, the present invention provides an esterase having an amino acid sequence as described in SEQ ID No. 2, an esterase encoded by a DNA fragment as described in SEQ ID No. 3, a recombinant plasmid containing the DNA fragment and a microorganism transformed with the recombinant plasmid.

Furthermore, the present invention provides a method of producing optically active carboxylic acids represented by the formula (II) and their enantiomeric esters, by reacting a racemic mixture of carboxylic acid esters represented by the formula (I) with the culture or bacterial cells of the transformant or bacterial-cell-treated materials.

The method of the present invention efficiently produces optically active carboxylic acids represented by the formula (II), and their enantiomeric esters from a racemic mixture of carboxylic acid esters represented by the formula (I).

The esterase of the present invention has the following physical and chemical properties.

(1) Optimum pH: 7.0 (see FIG. 4)

(2) Stability of esterase in varied pH: 6.0-8.0 (see FIG. 5)

(3) Optimum temperature: 60.degree.-70.degree. C. (see FIG. 6)

(4) Heat stability: up to 70.degree. C. (see FIG. 7)

(5) Molecular weight: about 30,000 daltons (by SDS-PAGE)

(6) Isoelectric point: pH 3.90.+-.0.1 (by isoelectric focusing)

(7) Amino acid sequence: sequence described in SEQ ID No. 2

(8) Specificity: mainly hydrolyzes carboxylic acid esters having 8 or less carbon atoms as well as aromatic alcohols such as acetyl esters

FIG. 1 shows the cloning vector pUC19.

FIG. 2 shows a restriction map of recombinant plasmid pPE101 comprising the DNA fragment that contains the esterase structural gene of the present invention.

FIG. 3 shows deleted DNA fragments and their positions relative to the EcoRI fragment in pPE101, a recombinant plasmid constructed by incorporating the DNA fragment into the pUC19, and esterase activity of microorganisms transformed with the recombinant plasmids.

FIG. 4 shows an optimum pH of the esterase of the present invention.

FIG. 5 shows stability of esterase in varied pH.

FIG. 6 shows an optimum temperature of the esterase of the present invention.

FIG. 7 shows heat stability of the esterase of the present invention.

In the Figures, the symbols are defined as follows: Amp    = ampicillin resistance coding region galactosidase structural gene lac i  = repressor structural gene 1      = region derived from pUC19 2      = region derived from the chromosomal DNA of Pseudomonas putida B      = BglII C      = ClaI EI     = EcoRI EV     = EcoRV P      = PvuII S      = SalI Sm     = SmaI Ps     = PstI ______________________________________

In the general formula (I) and (II), preferable alkyl represented by R.sub.1 is (C.sub.1 -C.sub.6) alkyl, for example, methyl and ethyl, aralkyl is, for example, benzyl, aryl is, for example, phenyl, and preferable alkyl represented by R.sub.2 or R.sub.3 is (C.sub.1 -C.sub.6) alkyl, for example, methyl and ethyl.

Carboxylic acid esters represented by the formula (I), include methyl .beta.-acetylthio-.alpha.-methylpropionate, methyl S-acetyl-.beta.-mercaptoisobutyrate, methyl S-acetyl-.gamma.-mercapto-.alpha.-methyl-n-butyrate, methyl S-benzoyl-.beta.-mercaptoisobutyrate and methyl S-phenylacetyl-.beta.-mercaptoisobutyrate.

The DNA fragment of the present invention includes, for example, a DNA fragment comprising the whole or part of a nucleotide sequence described in SEQ ID No. 1 and a DNA fragment comprising the whole or part of a nucleotide sequence described in SEQ ID No. 3.

In addition, the DNA fragment of the present invention may be obtained by cloning a desirable esterase gene derived from the chromosome of a microorganism or by synthesizing a desirable sequence by a DNA synthesizer.

The nucleotide sequence may be a single-stranded or a double-stranded DNA. Either of them can be used in the present invention.

Microorganisms suitable as a chromosome donor in cloning are, for example, Pseudomonas putida (FERM BP-3846).

The following will illustrate cloning of a DNA fragment containing a whole nucleotide sequence described in SEQ ID No. 1.

Chromosomal DNA is obtained from Pseudomonas putida (FERM BP-3846) by the Marmur et al method [J. Marmur et al., J. Mol. Biol., 3:208, 1961] and is then partially digested with EcoRI. After digestion, restriction fragments are obtained.

The plasmid pUC19 was digested with EcoRI, which cleaves the EcoRI site of a multiple cloning site in the lacZ gene(one of the genes in lac operon of E. coli) to give a linear DNA.

The EcoRI digested chromosomal DNA and pUC19 are ligated using T4 DNA ligase.

The reaction product is used to transform CaCl.sub.2 -treated E. coli JM105 cells (one of the strains of E. coli K-12). The transformation mixture is spread on an LB agar plate containing ampicillin, IPTG (isopropyl .beta.-D-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside), and incubated.

Transformants containing the DNA-fragment-free pUC19 at its EcoRI site become ampicillin resistance and grow to form colonies. When induced by IPTG, these transformants also express .beta.-galactosidase that cut X-Gal to make blue colonies. Conversely, transformants containing the DNA-fragment-containing pUC19 at its EcoRI site become ampicillin resistance and are unable to express .beta.-galactosidase. These transformants make white colonies.

After incubation on a plate, positive transformants are selected by picking white colonies. The positive transformants are then screened for esterase activities.

Screening may be carried out as described below. The colonies on the plate are transferred to a filter paper soaked in a solution of 10 mM Tris-HCl/pH 7.5, 0.01% bromocresolpurple and 100 ppm of methyl DL-.beta.-acetylthio-.alpha.-methylpropionate. The filter is incubated at room temperature for several hours. Transformants having an esterase activity produce carboxylic acid and change the color of bromocresolpurple (pH indicator) from purple to yellow around the colony because of a decrease of pH. Transformants having an esterase gene can be, therefore, observed with the naked eye.

The position of an esterase gene in the recombinant plasmid is determined by the following method.

Plasmids are isolated from the transformant having an esterase activity. EcoRI DNA fragments are then obtained by digesting the plasmid DNA with EcoRI. The EcoRI digest is further digested with various restriction enzymes. The restriction fragments are inserted into the plasmid pUC19 to give recombinant plasmids. E. coli JM105 is transformed with the recombinant plasmids. The transformants are tested for an esterase activity. Among the positive transformants, a transformant comprising a minimum length DNA fragment for an esterase gene expression is selected. The nucleotide sequence of the DNA fragment is determined by various methods and SEQ ID No. 1 is obtained.

Cloning vectors include the pBR and pUC series that are a high-copy-number plasmid stably maintained in E. coli, pHY300PLK used for Bacillus subtilis and vectors derived from RSF1010 that have a wide host range.

Host microorganisms used for transformation include E. coli, yeast, Bacillus subtilis and Actinomycetes.

Microorganisms transformed with a recombinant plasmid containing a esterase gene encoding a heat stable esterase produce enzyme that has similar enzymatic properties to those of a parental strain, Pseudomonas putida (FERM BP-3846). In addition, the transformant has by far higher enzymatic activity than the DNA donor bacterium because the transformant contains a high-copy-number plasmid. The transformant is grown in a culture medium and used in a form such as culture, bacterial cells or bacterial-cell-treated materials.

The transformed microorganism is typically grown in a liquid culture medium or in a solid culture medium. For example, an LB culture medium can be used. Culture is carried out at 10.degree.-50.degree. C., pH2-11. Aeration and agitation of the culture is also utilized to facilitate growth. Hydrolysis may be carried out by adding carboxylic acid esters of the formula (I) to culture at the beginning of culture or in the middle of culture or after completion of culture.

Alternatively, grown bacterial cells are harvested by centrifugation and may be added to a solution containing carboxylic acid esters of the formula (I). When bacterial cells are used, bacterial cells are dried by, for example, lyophilization or spray-drying, or are treated by organic solvents such as acetone or toluene, or are disrupted to give broken cells, or are extracted to give bacterial-cell-treated materials. Solvents used for reaction include deionized water or buffer. The preferable concentration of carboxylic acid esters of formula (I) in a solvent or in a culture medium may be 0.01-50 wt %. Carboxylic acid esters of formula (I) suspended in water may be added to carry out reaction. Solvents such as methanol or acetone may be added to help dissolve esters. The pH of a reaction mixture is 2-11, preferably 5-8. As reaction proceeds, the pH of a reaction mixture decreases due to the increase of the reaction product, optically active carboxylic acids of the formula (II). The pH of a reaction mixture should be adjusted with an appropriate neutralizing agent. An enzyme produced by the transformant of the present invention is highly heat stable so that reaction temperature may be in the range of 5.degree.-80.degree. C. If the property of the enzyme produced by the transformant of the present invention is attempted to be optimized, 45.degree. C. or more is preferable for reaction. At that temperature range, the enzyme can maintain its activity during reaction.

Isolation and purification of a reaction product from a reaction mixture or a culture may be carried out by the method known in the art, for example, extraction, recrystallization, and column chromatography.

The novel esterase of the present invention may be obtained from the culture of the transformant of the present invention by the method known in the art: Enzyme endogenouly produced in the transformant is extracted by any method in the art to give bacterial-cell free extract. The extract is concentrated using ammonium sulfate, loaded on an ion-exchange chromatography column, gel-filtrated or is repetitiously subjected to the procedure described above to give pure enzyme.

Chromosomal DNA was isolated from Pseudomonas putida (FERM BP-3846) by the Marmur et al method [J. Marmur et al., J. Mol. Biol., 3:208, 1961] as described below:

Pseudomonas putida (FERM BP-3846) was inoculated into 100 ml of an LB culture medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl) and incubated at 37.degree. C. overnight. After the incubation, bacterial cells were harvested by centrifugation and about 3 g of bacterial cells (wet weight) was then suspended in 24 ml of TEG buffer [25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH 8.0]. 1 ml of lysozyme (suspended in TEG buffer, concentration: 10 mg/ml) was added to the suspension. The suspension was stirred at 32.degree. C. for 30 minutes. After the stirring, 2 ml of a 12.5% SDS solution was added to the bacterial lysate. The mixture was stirred. 6.75 ml of a 5M sodium perchlorate solution was added to the lysate mixture. The lysate mixture was stirred. 33.8 ml of a chloroform/isoamylalcohol [24:1 (v/v)] mixture was added to the lysate mixture. The mixture was gently stirred for 30 minutes.

After the stirring, the mixture was centrifuged. The supernatant was saved. Two volumes of ice cold ethanol was gently added to the supernatant. DNA was removed from ethanol by winding it with a glass stick. The DNA was dissolved in 9 ml of 0.1.times.SSC (15 mM NaCl, 1.5  mM sodium citrate). 1 ml of 10.times.SSC (1.5M NaCl, 150 mM sodium citrate) was added to the DNA solution. RNaseA was added to the DNA solution to a final concentration of 50 .mu.g/ml. The DNA mixture was gently stirred for 30 minutes. After the stirring, 10.2 ml of a chloroform/isoamylalcohol [24:1 (v/v)] mixture was added to the DNA mixture. The mixture was gently stirred for 30 minutes.

After the stirring, the mixture was centrifuged. The supernatant was saved. 1 ml of an acetic acid/EDTA (3M sodium acetate, 0.01M EDTA, pH 7.0) mixture was added to the supernatant. Isopropylalcohol (total volume: 5.4 ml) was dropwise added to the mixture while stirring the mixture with a glass stick. DNA was removed by winding it with a glass stick. The DNA was dissolved in 3 ml of 1.times.SSC (0.15M NaCl, 15 mM sodium citrate) to give 2 .mu.g/.mu.l of DNA.

EcoRI was added to 5 .mu.l of chromosomal DNA (corresponds to 10 .mu.g of DNA) obtained in 1-(1) to a final concentration of 2 units/.mu.g DNA. The reaction mixture was incubated at 37.degree. C. for 30 minutes according to the method known in the art. After the incubation, EcoRI partially digested DNA was obtained. In the meantime, 1 .mu.g of plasmid pUC19 DNA was digested with EcoRI according to the method known in the art to give a linear plasmid DNA.

The digested chromosome DNA fragments and pUC19 fragments were combined. T4 DNA ligase was added to the DNA mixture and incubated to give a ligation product.

E. Coli JM105 (TAKARA SYUZO Co., LTD) was grown in an LB culture medium at 37.degree. C. for 2-3 hours. After the incubation, bacterial cells were harvested by centrifugation. The bacterial cells were suspended in an ice cold 50 mM CaCl.sub.2 solution. The suspension was then centrifuged. The cell pellet was resuspended in a fresh 50 mM CaCl.sub.2 solution. The suspension was placed on ice for 30 minutes.

After 30 minute incubation, about 100-200 .mu.l of the suspension was transferred to an Eppendorf tube. The ligation product (about 2 .mu.g of DNA) obtained in 1-(2) was added to the tube. The tube was further incubated on ice for 30 minutes. The bacterial cells were then heatshocked at 42.degree. C. for 2 minutes. 1 ml of an LB culture medium was added to the tube, which was incubated at 37.degree. C. for 60 minutes.

After the incubation, the transformation product, about 100 .mu.l each, was spread on an LB agar culture medium plate containing 100 .mu.g/ml of ampicillin, 0.5 mM IPTG, 0.2% X-Gal. The plate was incubated at 37.degree. C. overnight.

Of colonies appeared on the plate, white colonies were tested for esterase activity. Positive transformants that contain recombinant plasmids comprising an esterase gene were selected as described below.

The white colonies on the plate were transferred to a filter paper soaked in a solution of 10 mM Tris-HCl/pH7.5, 0.01% bromocresolpurple and 100 ppm of methyl DL-.beta.-acetylthio-.alpha.-methylpropionate. The filter was incubated at room temperature for several hours. Transformants having an esterase activity produced carboxylic acid, which changed the color of bromocresolpurple (pH indicator) from purple to yellow around the colony because of a decrease of pH. Positive transformants that contain recombinant plasmids comprising an esterase gene were thus obtained.

One of the positive transformants was grown and the plasmid DNA was prepared according to the method described by Birnboim et al., in Nucleic acid Res., 7: 1513-1523, 1979. The restriction map of the plasmid was constructed and is shown in FIG. 2. The recombinant plasmid shown in FIG. 2 was designated as pPE101.

pPE101 was digested with EcoRI according to the method known in the art. The restriction fragments were electrophoresed and a DNA fragment having EcoRI ends (large EcoRI digest) was obtained. The large EcoRI digest was further digested with various restriction enzymes to give smaller DNA fragments. Several restriction maps are shown in FIG. 3.

The smaller DNA fragments were then inserted into a multiple cloning site within the lac Z region of pUC19. The resulting recombinant plasmids were used to transform E. coli JM105. Transformants were tested for esterase activity.

Preparation of plasmid DNA, transformation and measurement of esterase activity were carried out according to the methods described in 1-(2) and (3). When inserting the smaller fragments into the pUC19, a linker was synthesized and used if it was necessary.

The esterase activity of the transformant is also shown in FIG. 3. According to the esterase activity, about 1.3 kb ClaI/SmaI DNA fragment and about 1.2 kb ClaI/PstI DNA fragment were found to contain an esterase gene.

E. coli JM105 transformed with the plasmid pPE116 was designated as E. coli JM105 (pPE116). E. coli JM105 (pPE116) was deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, under the name of MR-2101, and assigned the accession number FERM BP-3838.

E. coli C600 transformed with the plasmid pPE117 was designated as E. coli C600 (pPE117). E. coli C600 (pPE117) was deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, under the name of MR-2103, and assigned the accession number FERM BP-3835.

The whole nucleotide sequence of the ClaI/SmaI DNA fragment obtained in 1-(4) was determined by the dideoxy chain termination method (F. Sanger, Science., 214: 1205, 1981) using a phage vector. The nucleotide sequence was then analyzed and the positions of an SD sequence and a DNA sequence essential for an enzyme activity were located. Based on the analysis, an open reading frame and a corresponding amino acid sequence were determined. The results are shown in SEQ ID No. 4. Sequences

The whole nucleotide sequence of the DNA fragment in pPE117 was determined by the dideoxy chain termination method (F. Sanger, Science., 214: 1205, 1981) using M13 phage vector. The nucleotide sequence of about 1.2 kb DNA fragment derived from the chromosomal DNA of Pseudomonas putida (FERM BP-3846) is the one described in SEQ ID No. 3. The study of the nucleotide sequence described in SEQ ID No. 3 revealed the following: only an open reading frame encompassing the region essential for enzyme activity is present in the nucleotide sequence described in SEQ ID No. 1; an SD sequence situated several bases upstream of the translation initiation codon as well as the open reading frame is present in the nucleotide sequence described in SEQ ID No. 3. The amino acid sequence deduced from the nucleotide sequence described in SEQ ID No. 1 is shown in SEQ ID No. 2. DL-.beta.-acethylthio-.alpha.-methylpropionate

E. coli JM105(pPE116) was cultured with shaking in 500 ml of an LB culture medium containing 50 .mu.g/.mu.l of ampicillin at 37.degree. C. overnight. After incubation, bacterial cells were harvested by centrifugation. The whole bacterial cell was suspended in 200 ml of a 5% methyl DL-.beta.-acetylthio-.alpha.-methylpropionate solution. The reaction was carried out at 45.degree. C. for three hours while adjusting the pH with 0.1N NaOH to pH 7. After the reaction, the bacterial cells were removed by centrifugation. Unreacted methyl .beta.-acetylthio-.alpha.-methylpropionate in the supernatant was removed using ethyl acetate. The pH of the aqueous layer of the resultant extract was adjusted with diluted sulfuric acid to 2.0 or less. The mixture was then extracted with ethyl acetate to give a D-.beta.-acetylthio-.alpha.-methylpropionate extract. Anhydrous sodium sulfate was added to the extract. After dehydration, the solvent was vapored out to give oily substance. Part of the substance was removed and diluted with water. The resulting diluted sample was subjected to a quantitative analysis of .beta.-acetylthio-.alpha.-methylpropionate by HPLC. Additionally, part of the substance was dissolved in chloroform. The optical rotation of the resulting sample was polarimetrically analyzed (PM-101, Union Giken). As a result, 1.5 g of a desired product was obtained. Specific rotation was calculated by the following equation:

The specific rotation was -58.3, which was the same value as that of a sample obtained from Pseudomonas putida (FERM BP-3846).

E. coli C600 (pPE117) cells were tested for asymmetric hydrolysis of methyl DL-.beta.-acetylthio-.alpha.-methylpropionate according to the similar method described in 1-(7).

The enzymatic activity of Pseudomonas putida (FERM BP-3846), E. coli JM105(pPE116) and E. coli C600 (pPE117) during the first one hour was measured and compared. The results are shown in Table 1. ______________________________________ Relative Baterial strain        specific activity* ______________________________________ Pseudomonas putida (FERM BP-3846) 1 E. coli JM105 (pPE116)[FERM BP-3838] 2 E. coli C600 (pPE117)[FERM BP-3835] 200 ______________________________________ Pseudomonas putida (FERM BP3846) is assumed to be 1

As is shown in Table 1, the specific of E. coli C600 (pPE117) [FERM BP-3835] is about 200 times as much as that of Pseudomonas putida (FERM BP-3846).

Pseudomonas fluorescens IF03018, Pseudomonas putida 2068 (FERM BP-3846) and E. coli JM105(pPE116) [FERM BP-3838] were separately grown in the following condition. ml)

Bacterial cells thus obtained were tested for heat stability of esterase by the method described below.

Bacterial cells were harvested. 1.0 g of the cells (wet weight) was suspended in 100 ml of 0.05M phosphate buffer/pH 7.0 containing 5.0 g of methyl DL-.beta.-acethylthio-.alpha.-methylpropionate. The suspension was incubated at 30.degree. C. for enzyme reaction. As the reaction proceeded, 0.1N NaOH was dropwise added to the suspension to maintain the pH at 7.0. The total volume (Q.sub.1) of NaOH used to maintain the pH during first one hour was measured.

1.0 g of the cells (wet weight) was suspended in 100 ml of 0.05M phosphate buffer/pH 7.0. The suspension was incubated for three hours at a temperature selected from the range of 20.degree.-80.degree. C. After incubation, 5.0 g of methyl DL-.beta.-acethylthio-.alpha.-methylpropionate was added to the suspension. The suspension was incubated at 30.degree. C. for enzyme reaction. As the reaction proceeded, 0.1N NaOH was added dropwise to the suspension to maintain the pH at 7.0. The total volume (Q.sub.2) of NaOH used to maintain the pH during first one hour was measured.

Heat unaffected esterase activity is calculated by the following equation;

The results are shown in Table 2. ______________________________________ Incubation  Esterase activity temperature Baterial strain (.degree.C.) a            b      c ______________________________________ 30          100          100    100 40          85           100    100 50          70           100    100 60          30           100    100 70           5            65     67 80           0            0      0 ______________________________________

As is evident from Table 2, the heat stability of the esterase produced by Pseudomonas putida 2068 (FERM BP-3846) and E. coli JM105(pPE116) is higher than that of esterase produced by Pseudomonas fluorescens IF 3018.

E. coli C600 (pPE117)[FERM BP-3835] was grown in a culture medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 2% glucose, PH7.0) in aeration-agitation fermenter at 37.degree. C for 14 hours. Bacterial cells were harvested by centrifugation and 15 g of bacterial cells was obtained. The bacterial cells were suspended in an equal amount of M/20 phosphate buffer. The bacterial cells were sonicated and then centrifuged. The supernatant was saved and concentrated using ammonium sulfate. The concentrate was then put on a DEAE Sephadex D-50 (Pharmacia) column equilibrated with M/20 phosphate buffer. A linear gradient solution, 0-0.5M NaCl, was added to the column to elute esterase. Esterase active fractions were collected and concentrated. The concentrated sample was put on a Sephadex G-100 (Pharmacia) column equilibrated with M/20 phosphate buffer. Esterase was eluted with M/20 phosphate buffer. Esterase active fractions were pooled and concentrated. 500 mg of purified esterase was obtained.

The molecular weight of the esterase was determined by SDS-PAGE and found that it was about 30,000daltons. The isoelectric point of the esterase was determined by the isoelectric focusing (Fast System, Pharmacia) and found that it was pH 3.90.+-.0.1.

pH for optimum esterase activity and stability of the esterase in different pHs are shown in FIGS. 4 and 5. Temperature for optimum esterase activity and heat stability of the esterase are shown in FIGS. 6 and 7.

N-terminal amino acid sequence of the novel esterase was analyzed by amino acid sequence autoanalyzer (SHIMADZU CORPORATION) and found as follows: NH.sub.2 terminus 2 3 4 5 6 7 Ser Tyr  Val  Thr  Thr  Lys  Asp ______________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 4 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 831 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ( A) ORGANISM: Pseudomonas putida (B) STRAIN: MR-2068 (FERM BP- 3846) (ix) FEATURE: (A) NAME/KEY: 1-831 CDS (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGAGCTATGTAACCACGAAGGACGGCGTACAGATCTTCTACAAGGACTGGGGCCCGCGC60 GATGCGCCGGTCATCCACTTCCACCACGGCTGGCCGCTCAGTGCCGA CGACTGGGACGCG120 CAGATGCTGTTCTTCCTCGCCCACGGTTACCGCGTGGTCGCCCACGACCGCCGCGGCCAT180 GGCCGCTCCAGCCAGGTATGGGACGGCCACGACATGGACCACTACGCCGACGACGTAGCC240 GCAGTGGTGGCCCACCTGGGCATT CAGGGCGCCGTGCATGTCGGCCACTCGACCGGTGGC300 GGTGAGGTGGTGCGCTACATGGCCCGACACCCTGCAGACAAGGTGGCCAAGGCCGTGCTG360 ATCGCCGCCGTACCGCCGTTGATGGTGCAGACTCCCGATAATCCCGGTGGCCTGCCCAAA420 T CCGTTTTCGACGGCTTCCAGGCCCAGGTCGCCAGCAACCGCGCGCAGTTCTACCGGGAT480 GTGCCGGCAGGGCCGTTCTACGGCTACAACCGCCCCGGTGTCGACGCCAGCGAAGGCATC540 ATCGGCAACTGGTGGCGCCAGGGCATGATCGGTAGCGCCAAGGCCCA TTACGATGGCATC600 GTGGCGTTTTCCCAGACCGACTTCACCGAAGACCTGAAGGGCATTACCCAGCCGGTGCTG660 GTGATGCATGGCGACGACGACCAGATCGTGCCGTATGAGAACTCCGGGCTGCTGTCGGCC720 AAGCTGCTGCCCAATGGCACACTG AAGACCTACCAGGGCTACCCGCATGGCATGCCGACC780 ACCCATGCCGATGTGATCAATGCGGATTTGCTGGCGTTTATCCGTAGCTGA831 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 276 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas putida (B) STRAIN: MR-2068 (FERM BP- 3846) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetSerTyrValThrThrLysAspGlyValGlnIlePheTyrLysAsp 15 1015 TrpGlyProArgAspAlaProValIleHisPheHisHisGlyTrpPro 202530 LeuSerAlaAspAspTrpAs pAlaGlnMetLeuPhePheLeuAlaHis 354045 GlyTyrArgValValAlaHisAspArgArgGlyHisGlyArgSerSer 5055 60 GlnValTrpAspGlyHisAspMetAspHisTyrAlaAspAspValAla 65707580 AlaValValAlaHisLeuGlyIleGl nGlyAlaValHisValGlyHis 859095 SerThrGlyGlyGlyGluValValArgTyrMetAlaArgHisProAla 100 105110 AspLysValAlaLysAlaValLeuIleAlaAlaValProProLeuMet 115120125 ValGlnThrProAspAsnProGlyGly LeuProLysSerValPheAsp 130135140 GlyPheGlnAlaGlnValAlaSerAsnArgAlaGlnPheTyrArgAsp 1451501 55160 ValProAlaGlyProPheTyrGlyTyrAsnArgProGlyValAspAla 165170175 SerGluGlyIleIleGlyAsnTrpT rpArgGlnGlyMetIleGlySer 180185190 AlaLysAlaHisTyrAspGlyIleValAlaPheSerGlnThrAspPhe 19520 0205 ThrGluAspLeuLysGlyIleThrGlnProValLeuValMetHisGly 210215220 AspAspAspGlnIleValProTyrGluAsnSerGl yLeuLeuSerAla 225230235240 LysLeuLeuProAsnGlyThrLeuLysThrTyrGlnGlyTyrProHis 245 250255 GlyMetProThrThrHisAlaAspValIleAsnAlaAspLeuLeuAla 260265270 PheIleArgSer 275 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1120 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas putida (B) STRAIN: MR-2068 (FERM BP- 3846) (ix) FEATURE: (A) NAME/KEY: 93-923 CDS (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTGCAGTGCAGTCACCACAAATTCCGGCGCCAAGCAAAATTCCTCCTATTCTCAATAGCT60 CACTTCGCTTCCTGCACACAGGAGACCCGACCATGAGCTATGTAACCACGAAGGACGGCG120 TACAGATCTTCTACAAGGACTGGGGCCCGCGCGATGCG CCGGTCATCCACTTCCACCACG180 GCTGGCCGCTCAGTGCCGACGACTGGGACGCGCAGATGCTGTTCTTCCTCGCCCACGGTT240 ACCGCGTGGTCGCCCACGACCGCCGCGGCCATGGCCGCTCCAGCCAGGTATGGGACGGCC300 ACGACATGGACCACT ACGCCGACGACGTAGCCGCAGTGGTGGCCCACCTGGGCATTCAGG360 GCGCCGTGCATGTCGGCCACTCGACCGGTGGCGGTGAGGTGGTGCGCTACATGGCCCGAC420 ACCCTGCAGACAAGGTGGCCAAGGCCGTGCTGATCGCCGCCGTACCGCCGTTGATGGTGC 480 AGACTCCCGATAATCCCGGTGGCCTGCCCAAATCCGTTTTCGACGGCTTCCAGGCCCAGG540 TCGCCAGCAACCGCGCGCAGTTCTACCGGGATGTGCCGGCAGGGCCGTTCTACGGCTACA600 ACCGCCCCGGTGTCGACGCCAGCGAAGGCATCATCGGC AACTGGTGGCGCCAGGGCATGA660 TCGGTAGCGCCAAGGCCCATTACGATGGCATCGTGGCGTTTTCCCAGACCGACTTCACCG720 AAGACCTGAAGGGCATTACCCAGCCGGTGCTGGTGATGCATGGCGACGACGACCAGATCG780 TGCCGTATGAGAACT CCGGGCTGCTGTCGGCCAAGCTGCTGCCCAATGGCACACTGAAGA840 CCTACCAGGGCTACCCGCATGGCATGCCGACCACCCATGCCGATGTGATCAATGCGGATT900 TGCTGGCGTTTATCCGTAGCTGATGTGATCGCCTGCACCGGCCTCTTCGCGGGCACTGGC 960 AACACACCTCCCCCAGGATTACCATGTCACGCTTCTAGTGCGGCCCTTTGCCGCCCCTTG1020 CCTCCCTGCCTGCCAAAACCCCATGCCCTTCGAACTCACCGTAGAACCCCTCACCCTGCT1080 GATCCTGGCCCTGGTCGCCTTCGTCGCCGGTTTCATCG AT1120 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1329 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas putida (B) STRAIN: MR-2068 (FERM- 3846) ( ix) FEATURE: (A) NAME/KEY: mat-peptide (B) LOCATION: 302..1129 (C) IDENTIFICATION METHOD: dideoxy chain termination method (F. Sanger) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCCGGGCCGTGAGCGATGCCATCCTCGGTGACGACGACCTGCTGGCGCTATATCAAGGCA60 TCGACAACGGCCGCTTCCCCGGTGGC GACCTGCTGGCCGCACCGCTGGAAGCCGCCGCCA120 AGGCCTGGTACCGGATGCGCGACCGCGCCTGATCGCCTGGCACCGCTCCTACACGGCGCC180 GGGCAGGCCGGAAGCATGGTGCAAGCCCACTGCAGTGCAGTCACCACAAATTCCGGCGCC240 AAG CAAAATTCCTCCTATTCTCAATAGCTCACTTCGCTTCCTGCACACAGGAGACCCGAC300 CATGAGCTATGTAACCACGAAGGACGGCGTACAGATCTTCTACAAG346 MetSerTyrValThrThrLysAspGlyValGlnIlePhe TyrLys 151015 GACTGGGGCCCGCGCGATGCGCCGGTCATCCACTTCCACCACGGCTGG394 AspTrpGlyProArgAspAlaProValIleHisPheHis HisGlyTrp 202530 CCGCTCAGTGCCGACGACTGGGACGCGCAGATGCTGTTCTTCCTCGCC442 ProLeuSerAlaAspAspTrpAspAlaGlnMetLeuPhe PheLeuAla 354045 CACGGTTACCGCGTGGTCGCCCACGACCGCCGCGGCCATGGCCGCTCC490 HisGlyTyrArgValValAlaHisAspArgArgGlyHisGl yArgSer 505560 AGCCAGGTATGGGACGGCCACGACATGGACCACTACGCCGACGACGTA538 SerGlnValTrpAspGlyHisAspMetAspHisTyrAlaAspAspV al 657075 GCCGCAGTGGTGGCCCACCTGGGCATTCAGGGCGCCGTGCATGTCGGC586 AlaAlaValValAlaHisLeuGlyIleGlnGlyAlaValHisValGly 80 859095 CACTCGACCGGTGGCGGTGAGGTGGTGCGCTACATGGCCCGACACCCT634 HisSerThrGlyGlyGlyGluValValArgTyrMetAlaArgHisPro 100105110 GCAGACAAGGTGGCCAAGGCCGTGCTGATCGCCGCCGTACCGCCGTTG682 AlaAspLysValAlaLysAlaValLeuIleAlaAlaValProProLeu 115120125 ATGGTGCAGACTCCCGATAATCCCGGTGGCCTGCCCAAATCCGTTTTC730 MetValGlnThrProAspAsnProGlyGlyLeuProLysSerValPhe 130135140 GACGGCTTCCAGGCCCAGGTCGCCAGCAACCGCGCGCAGTTCTACCGG778 AspGlyPheGlnAlaGlnValAlaSerAsnArgAlaGlnPheTyrArg 145 150155 GATGTGCCGGCAGGGCCGTTCTACGGCTACAACCGCCCCGGTGTCGAC826 AspValProAlaGlyProPheTyrGlyTyrAsnArgProGlyValAsp 16016 5170175 GCCAGCGAAGGCATCATCGGCAACTGGTGGCGCCAGGGCATGATCGGT874 AlaSerGluGlyIleIleGlyAsnTrpTrpArgGlnGlyMetIleGly 1 80185190 AGCGCCAAGGCCCATTACGATGGCATCGTGGCGTTTTCCCAGACCGAC922 SerAlaLysAlaHisTyrAspGlyIleValAlaPheSerGlnThrAsp 195 200205 TTCACCGAAGACCTGAAGGGCATTACCCAGCCGGTGCTGGTGATGCAT970 PheThrGluAspLeuLysGlyIleThrGlnProValLeuValMetHis 210 215220 GGCGACGACGACCAGATCGTGCCGTATGAGAACTCCGGGCTGCTGTCG1018 GlyAspAspAspGlnIleValProTyrGluAsnSerGlyLeuLeuSer 22523 0235 GCCAAGCTGCTGCCCAATGGCACACTGAAGACCTACCAGGGCTACCCG1066 AlaLysLeuLeuProAsnGlyThrLeuLysThrTyrGlnGlyTyrPro 240245 250255 CATGGCATGCCGACCACCCATGCCGATGTGATCAATGCGGATTTGCTG1114 HisGlyMetProThrThrHisAlaAspValIleAsnAlaAspLeuLeu 260 265270 GCGTTTATCCGTAGCTGATGTGATCGCCTGCACCGGCCTCTTCGCGGGCACTGGC1169 AlaPheIleArgSer 275 AACACACCTCCCCCAGGATTACCATGTCACGCTTCTAGTGCGGCCCTTTGCC GCCCCTTG1229 CCTCCCTGCCTGCCAAAACCCCATGCCCTTCGAACTCACCGTAGAACCCCTCACCCTGCT1289 GATCCTGGCCCTGGTCGCCTTCGTCGCCGGTTTCATCGAT1329

## Claims

1. Isolated and purified DNA consisting essentially of DNA that encodes the amino acid sequence of esterase as described in SEQ ID No. 2, said esterase asymmetrically hydrolyzing carboxylic acid esters represented by the formula (I): ##STR6## (wherein R.sub.1 is alkyl, aralkyl or aryl, R.sub.2 and R.sub.3 are alkyl, and n is 1 or 2) to give an optically active carboxylic acid represented by the formula (II) ##STR7## (wherein R.sub.1, R.sub.2 and n have the same meaning as described above).

2. Isolated and purified DNA consisting essentially of the nucleotide sequence as described in SEQ ID No. 1 that encodes the amino acid sequence of esterase as described in SEQ ID No. 2.

3. Isolated and purified DNA consisting essentially of the nucleotide sequence as described in SEQ ID No. 3 that encodes the amino acid sequence of esterase as described in SEQ ID No. 2.

4. Isolated and purified DNA consisting essentially of the nucleotide sequence as described in SEQ ID No. 4 that encodes the amino acid sequence of esterase as described in SEQ ID No. 2.

5. A recombinant plasmid comprising the DNA of any one of claims 1, 2, 3 or 4.

6. A transformant characterized by transforming a host microorganism with the recombinant plasmid of claim 5.