MXPA99007259A - Novel carbonyl reductase, gene that encodes the same, and method of utilizing these - Google Patents

Abstract

An enzyme having the activity of asymmetrically reducing a carbonyl compound to an optically active alcohol, a DNA which encodes the enzyme, a plasmid having the DNA, cells transformed by the plasmid, and a process for producing optically active alcohols with the enzyme and/or the transformed cells.

Description

NOVEDOSA CARBONILO REDUCTASE. GENE THAT CODIFIES IT. AND METHOD FOR ITS USE TECHNICAL FIELD The present invention relates to an enzyme having carbonyl reduction activity, which reduces a carbonyl compound asymmetrically, to produce an optically active alcohol (this enzyme is hereinafter referred to as the CRD enzyme), a DNA that encodes such an enzyme, a plasmid having such DNA, a transformant, which is a cell transformed with such a plasmid, and a method of producing an optically active alcohol, which uses the enzyme and / or the transformed cell. The resulting optically active alcohol, for example, the (S) -4-halo-3-hydroxy-butyric ester, is a useful compound as a raw material for the synthesis of medicines, agricultural chemicals, and the like.

PREVIOUS TECHNIQUE A number of CRD enzymes are known (see Yuki Gosei Kagasu, 49, 52 (1991) and Eur. J. Biochem., 184, i (1981)). Among such CRD enzymes, those that act on the 4-haloacetoacetic ester, to produce the (S) -halo-3-hydroxy-butyric ester, which are derived from microbes, and which have reported characteristics, are only an enzyme derived from Geotrichum candidum (Enzyme Microb. Technol. (1992), Vol 14, 731) and an enzyme derived from Candida parapsilosis (Enzi and Microb Technol. (1993), Vol. 15, 950). However, no information has been reported on genes that code for these two types of enzymes. The reduction of the 4-haloacetoacetic ester with the use of these enzymes only proceeds at a low substrate concentration. Therefore, it is not practical to analyze the (S) -4-halo-3-hydroxy-butyric ester, with the use of such enzymes as catalysts. In addition to the above reaction, which uses the various types of enzymes, a number of reactions using microbial bodies and the products of such reactions are known to carry out the asymmetric reduction of the 4-haloacetoacetic ester (Japanese patent No. 1723728, Japanese publications, open to the public, Nos. 6-209782 and 6-38776, etc.). However, such reactions are not carried out with a high concentration of the substrate and thus it can not be asserted that a practical production method has been established. See, for example, a reaction method using a two-phase system with an organic solvent (Japanese Patent No. 2566962. One method, which uses an optically active phosphine complex, and ruthenium, as a catalyst, has been reported (Japanese publication, open to the public, No. 1-211551) However, this method has many problems to solve, such as the requirement of a high-pressure reaction vessel and the need for an expensive catalyst. above, the development of a practical enzyme has been desired for use in the asymmetric reduction of a carbonyl compound, such as the 4-haloacetoacetic ester, to produce an optically active alcohol, such as the ester (S) -4 -halo-3-hydroxy-butyric A CRD enzyme requires a reduction-type coenzyme for the reaction, Conventionally, when a carbonyl compound is to be reduced using a body of microbes and the like, which has a CRD enzyme, it is it adds a saccharide, such as glucose, to the reaction system, to activate a group of enzymes of the regeneration system, to change an oxidized coenzyme to a reduced type, whereby the coenzyme is regenerated in order to be used for the reduction. Such a group of enzymes of the regeneration system are probably blocked or damaged by substrates and reduced products. This has been considered to be one of the main reasons why the reduction proceeds only when the concentration of substrates or products is low. It is known that the amount of an expensive coenzyme, used during the reduction, can be greatly reduced, by combining an enzyme, which has the ability to regenerate a coenzyme of which a CRD enzyme depends, with the CRD enzyme during the reaction, (Japanese Patent No. 2566960 and Enzyme Microb. Technol. (1933), Vol. 15, 950, for example). In this case, however, it is required to prepare a source of enzymes to regenerate the coenzyme separately from the preparation of the CRD enzyme before the regeneration enzyme is added to a reaction system. The inventors of the present application have discovered a novel CRD enzyme derived from the Candida gene, and found that an optically active alcohol can be efficiently produced from a carbonyl compound using this CRD enzyme. They also found that an optically active alcohol can be produced efficiently using a transformed cell containing a gene for an enzyme, which has the ability to regenerate a coenzyme (eg, a glucose dehydrogenase gene) concurrently. Thus, the present invention, which will be described in the specification, can advantageously provide a novel CRD enzyme, a DNA encoding this enzyme, a plasmid having this DNA, a transformant, which is a cell transformed with this plasmid, and a method of producing an optically active alcohol, with the use of the enzyme and / or the transformed cell above.

EXPOSITION OF THE INVENTION Carbonyl reductase, according to the present invention, has the physical and chemical properties (1) to (4) of: (1) action: it acts on ethyl 4-chloroacetoacetate, which uses NADPH as a coenzyme, to produce the (S) -4-chloro-3-hydroxybutyrate of ethyl; (2) substrate specificity: it exhibits a strong activity to ethyl 4-chloroacetoacetate, while it has substantially no activity to ethyl acetoacetate; (3) Optimal pH: 5.5 to 6.5; and (4) optimal temperature of action: 50 to 552C.

In one embodiment, the carbonyl reductase has the additional physical and chemical properties (5) to (7) of: (5) heat stability: being stable up to about 402C, when processed at a pH of 7.0, for 30 minutes; (6) inhibition: being inhibited by mercury ions and quercetin; and (7) molecular weight: approximately 76,000 by gel filtration analysis and approximately by 32,000 SDS polyacrylamide electrophoretic analysis.

The carbonyl reductase, according to the present invention, has an amino acid sequence of SEQ ID NO: 1, of the Sequence List, or an amino acid sequence with one or several amino acids being deleted, substituted or added in the sequence of amino acids of SEQ ID N0: 1 of the Sequence List, or part of the amino acid sequences of SEQ ID N0: 1 of the Sequence Listing, and having an activity of reducing the ethyl 4-chloroacetoacetate asymmetrically, to produce the (S) ethyl 4-chloro-3-hydroxybutyrate. In one embodiment, the enzyme is obtained from a microbe, which belongs to the genus of Candida. In a preferred embodiment, the enzyme is obtained from Candida magnoliae. In a more preferred embodiment, the enzyme is obtained from Candida magnoliae IFO 0705. The DNA, according to the present invention, codes for the above enzyme. In one embodiment, the DNA has a nucleotide sequence of SEQ ID NO: 2 of the Sequence List. The plasmid, according to the present invention, has the above DNA sequence. In one embodiment, the plasmid is pNTSl. The transformed cell, according to the present invention, is a transformant, which is a cell transformed with the previous plasmid. In one embodiment, the transformed cell is E. coli. In a preferred embodiment, the transformed cell is E. coli HB101 (pNTSl). Plasmid, according to the present invention, has a DNA encoding an enzyme that has the activity of asymmetrically reducing ethyl 4-chloroacetoacetate to produce ethyl (S) -4-chloro-3-hydroxybutyrate and a DNA which encodes an enzyme that has the ability to regenerate a coenzyme, on which the enzyme depends (eg, glucose dehydrogenase). In one embodiment, glucose dehydrogenase is derived from Bacillus megaterium. In a preferred embodiment, the plasmid is pNTSIG. The transformed cell, according to the present invention, is a transformant, which is a cell transformed with the previous plasmid. In another embodiment, the transformed cell is E. coli. In a preferred embodiment, the transformed cell is E. COli HB101 (pNTSl). - The transformed cell, according to the present invention, is a transformant, which is a cell transformed with a first plasmid, having a DNA encoding an enzyme having an asymmetric activity to reduce ethyl 4-chloroacetoacetate to produce the < S) ethyl 4-chloro-3-hydroxybutyrate, and a second plasmid, which has a DNA encoding an enzyme, which has the ability to regenerate a coenzyme of which the enzyme depends (eg, glucose dehydrogenase) . In one embodiment, the transformed cell is a transformant which is a cell transformed with the plasmid pNTSl, and a plasmid having a DNA encoding the glucose dehydrogenase derived from Bacillus megaterium. In a preferred embodiment, the transformed cell is E. coli. The production method for obtaining an optically active 3-hydroxy-butyric ester, according to the present invention, includes the steps of: reacting with an ester of 3-oxo-butyric an enzyme, having an activity of asymmetrically reducing an ester 3-oxo-butyric, to produce an optically active 3-hydroxy-butyric ester or a culture of a microbe that has the ability to produce the enzyme or a processed product from the culture; and collecting an optically active 3-hydroxy-butyric ester. The production method for obtaining an optically active 3-hydroxybutyric ester, according to the present invention, includes the steps of: reacting a transformant, which is a transformed cell, with a plasmid, having a DNA encoding an enzyme, which has an activity of asymmetrically reducing a 3-oxo-butyric ester, to produce an optically active 3-hydroxy-butyric ester with a 3-oxo-butyric ester; : and collect an optically active 3-hydroxy-butyric ester. The production method for obtaining an optically active alcohol, according to the present invention, includes the steps of: reacting with a carbonyl compound a transformant, which is a cell transformed with a plasmid, having a DNA encoding an enzyme with an activity of asymmetrically reducing a carbonyl compound, to produce an optically active alcohol, and a DNA encoding an enzyme having the ability to regenerate a coenzyme, on which it depends on the enzyme; and collecting an optically active 3-hydroxy-butyric ester. The production method for obtaining an optically active alcohol, according to the present invention, includes the steps of: reacting with a carbonyl compound a transformant, which is a cell transformed with a first plasmid, having a DNA encoding a an enzyme having an activity of asymmetrically reducing a carbonyl compound, to produce an optically active alcohol, and a second plasmid, having a DNA encoding an enzyme that has the ability to regenerate a coenzyme, on which the enzyme depends; and collecting an optically produced active alcohol.

In one embodiment, the carbonyl compound is a 3-oxo-butyric ester, represented by the general formula: 0 II and the resulting optically active alcohol is an optically active 3-hydroxy-butyric ester represented by the general formula: In a preferred embodiment, in the above general formulas, R? and R 2 are, independently, halogen, azide, benzylamino or hydrogen, one of Ri and R 2 is hydrogen and R 3 is an alkyl group, substituted or unsubstituted or an aryl group. In a more preferred embodiment, in the above general formulas, Rx is chloro, R2 is hydrogen and R3 is ethyl.

In a preferred embodiment, in the above general formulas, Ri and R2 are, independently, an alkyl group, a hydroxyl group or hydrogen, one of Rx and R is hydrogen, and R3 is an alkyl group, substituted or unsubstituted, or an aryl group. In a more preferred embodiment, in the above general formulas, i is a hydroxyl group, R 2 is hydrogen and R 3 is ethyl.

SEQ ID NO: 8 is a view showing the above sequence and an estimated amino acid sequence. Figure 1 is a view illustrating a method for constructing a recombinant pNTSIG plasmid.

THE BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail. Purification of the CRD enzyme The organism used as a source of the CRD enzyme, according to the present invention, is not specifically restricted, but can be a yeast of the Candida genus, for example. An especially preferred example is Candida magnoliae IFO 0705, which is a microbe originally deposited at Centraalbureau voor Schimmel-cultures (CBS, Oosterstraat 1, Postbus 273, NL-3740 AG Baarn, The Netherlands) under the number OPS166 and of which the isolation and characteristics they are described in "The Yeasts, a Taxonomic Study, 3β Ed. (1984), pp. 731. The microbe capable of producing the enzyme, according to the present invention, can be a wild strain or a mutant strain. A microbe derived by a genetic technique, such as cell fusion or genetic manipulation, can also be used, for example, a microbe derived by the genetic manipulation that produces the enzyme, according to the present invention, can be obtained by a method which includes the steps of: isolating and / or purifying such an enzyme, to determine part or all of the amino acid sequence of the enzyme, obtaining a DNA sequence of a DNA coding for the to enzyme based on the amino acid sequence obtained; introduce the DNA into another microbe, to obtain a recombinant microbe; and cultivate this recombinant microbe to obtain the enzyme, according to the present intention. The means for culturing a microbe and obtaining the enzyme, according to the present invention (or a microbe used in the production method of the (S) -4-halo-3-hydroxy-butyric ester, according to the present invention) it is not specially restricted, as long as the microbe can grow. For example, a normal liquid nutrient medium, containing a carbon source, a nitrogen source, an inorganic salt, an organic nutrient, and the like, can be used. The "microbial culture", as used herein, means a body of microbes or a liquid culture containing the body of microbes, and "its processed product" means a product obtained by extraction and purification, as described below, by example. A method of extracting and purifying enzymes, normally used by those skilled in the art, can be used to extract and purify an enzyme from the resulting culture. For example, the culture is centrifuged to separate the bodies of microbes and the resulting microbe bodies are suspended in an appropriate regulator. These bodies of microbes in the suspension are destroyed or dissolved by the use of a physical technique, such as by using glass globules or a biochemical technique, such as by using an enzyme. The solids in the solution are then removed by centrifugation, to obtain a crude enzyme solution. Alternatively, such a crude enzyme solution can be obtained from the culture by a purification method, similar to that described above. The above crude enzyme solution can then be purified using a method normally employed by those skilled in the art, such as the precipitation of ammonium sulfate, dialysis and chromatography, alone or in combination. For chromatography, various types of chromatography, such as hydrophobic chromatography, ion exchange chromatography (e.g., DEAR Sepharose), and gel filtration, can be used alone or in combination, to obtain the enzyme, in accordance with the present invention. For example, a CRD enzyme can be isolated from Candida magnoliae IFO 0705 in the following manner. First, the above yeast is cultured in an appropriate medium, and the bodies of microbes are collected from the resulting culture by centrifugation. The microbial bodies are destroyed by the Dyno mill (manufactured by Dyno-Mill), for example, and centrifuged to remove cell debris and thus obtain a cell-free extract. This cell-free extract is then subjected to a process, such as salting (for example, the precipitation of ammonium sulfate and the precipitation of sodium phosphate), the precipitation of solvent (a precipitation method of protein fractionation, which use acetone, ethanol, or the like), dialysis, gel filtration, ion exchange, column chromatography, such as reverse phase chromatography, and ultrafiltration, alone or in combination, to purify the enzyme. The activity of the CRD enzyme can be determined by measuring a reduction in absorption at 340 nm at 300C, for 100 mM of a phosphate buffer (pH 6.5) with 1 mM ethyl 4-chloroacetoacetate, as a substrate, 0.1 mM of NaDPH, as a coenzyme, and the enzyme there added, or 200 mM of a phosphate buffer (pH of 7.0) with 0.2 mM of ethyl 4-chloroacetoacetate as a substrate and 0.32 mM of NADPH as a coenzyme there added. Under these reaction conditions, the oxidation of 1 μmol of NADPH in NADP in one minute is defined as a unit of enzymatic activity. The expression that an enzyme is "stable", as used here, means that, after being processed at a pH of 7.0 and 40 & amp;; C, for 30 minutes, the system holds an activity of 90% or more of that before the process. The molecular weight of the enzyme was measured by gel filtration using a TSK-G3000SW column (f 0.75 x 60 cm, manufactured by Tosoh Corporation). As an eluent, 0.1M phosphate buffer (pH 7.0) containing 0.1 M Na 2 S0 and 0.05% NaN3 is used. The molecular weight of a subunit was determined by carrying out the electrophoresis with 10% SDS-polyacrylamide gel, under reducing conditions (reducing agent: 2 V / V% of 2-mercaptoethanol) and calculating the relative mobility of a standard protein . For example, a CRD enzyme, having an amino acid sequence of SEQ ID NO: 1, according to the present invention, has the physical and chemical properties (1) to (4) of: (1) action: acts in the Ethyl 4-chloroacetoacetate, which uses NADPH as a coenzyme, to produce ethyl (S) -4-chloro-3-hydroxybutyrate; (2) substrate specificity: it exhibits a strong activity to ethyl 4-chloroacetoacetate, while it has substantially no activity to ethyl acetoacetate; (3) Optimal pH: 5.5 to 6.5; and (4) optimal temperature of action: 50 to 55SC.

In one embodiment, the carbonyl reductase has the amino acid sequence of SEQ ID NO: 1, according to the present invention, has the additional physical and chemical properties (5) to (7) of: (5) heat stability: being stable up to about 40CC, when processed at a pH of 7.0, for 30 minutes; (6) inhibition: being inhibited by mercury ions and quercetin; and (7) molecular weight: approximately 76,000 by gel filtration analysis and approximately by SDS polyacrylamide electrophoretic analysis.

An enzyme, which has substantially identical properties as the enzyme, according to the present invention, can be a natural enzyme or a recombinant enzyme. For example, a recombinant enzyme can be obtained in the following manner: An amino acid or several amino acids in the amino acid sequence of an enzyme derived from Candida magnoliae IFO 0705, are substituted, deleted, inserted or added, to produce the recombinant enzyme, and the activity of the enzyme is measured. Preparation of the synthetic oliaonucleotide probe The purified CRD enzyme, obtained in the above manner, is denatured (for example, with 8M urea), and then digested with the endopeptidase (for example, the lysyl endopeptidase). The amino acid sequence of the resulting fragment of the peptide was determined by the Edman method. A DNA probe is synthesized based on the determined amino acid sequence. Such a probe can be labeled with 32P, for example. Creation of the Aene Collection A chromosomal DNA of a microbe, which produces the CRD enzyme, according to the present invention, or its cDNA, is partially digested with an appropriate restriction enzyme, for example Sau3AI. A DNA fragment, having an appropriate size (for example 23 kb to 20 kb) of the digested product is inserted into a compatible restriction enzyme of a phage vector. This resulting recombinant phage vector is packaged in vitro and then E. coli is allowed to be infected with it, to create a collection of genes. Cloning of the semen of the CRD enzyme from the collection of genes The collection of genes, thus created, can be classified by. a CRD enzyme gene by a plaque hybridization method, using a synthetic DNA probe, labeled with 3 P (Science, 196, 180 (1977)). The analysis of the basic sequence of the resulting DNA can be determined by the dideoxy sequence method, a dideoxy chain termination method, or the like. Such sequence determination can be performed using the ABI PRISM Dye Terminator Cycle Sequencng Ready Reaction Kit (manufactured by Perkin Elmer) and the ABI 373A DNA Sequencer sequencer (manufactured by Applied Biosystems). The resulting DNA fragment can be amplified by the PCR method or the like, and cloned.

Construction of the recombinant plasmid, which includes the CRD enzyme sene A CRD enzyme gene is introduced into a host microbe and expressed there using a vector DNA. As a vector DNA, any vector DNA can be used as long as it can express the gene of the CRD enzyme within an appropriate host microbe. Examples of such vector DNA include a plasmid vector, a phage vector, and a cos-bound vector. A shuttle type vector, which allows the exchange of genes between different host strains can be used. Such vector DNA may have a control element, such as a promoter (for example, the lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter and pL promoter) and an enhancer element, linked there Operably. For example, pUCNT (WO94 / 03613) and the like, can preferably be used. Plasmid pUCNT is preferable, since it has insertion sites, such as Ndel and EcoRI, downstream of a lac promoter.

Construction of the recombinant plasmid that includes both the CRD enzyme gene and the enzyme gene that has the ability to regenerate the coenzyme of which the CRD enzyme depends As enzymes that have the ability to regenerate a coenzyme, hydrogenase, format dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase and the like, can be used. Preferably the glucose dehydrogenase is used. More specifically, a glucose dehydrogenase derived from Bacillus megaterium (hereinafter abbreviated as GDH) is used. Plasmid pGDA2 (J. Biol. Chem. (1989), 264, 6381) includes a GDH gene derived from Bacillus megaterium.

A fragment of the GDH gene is cut and separated from this plasmid and inserted into a plasmid that includes a CRD enzyme gene upstream or downstream of a CRD enzyme gene, to produce a recombinant plasmid that has both the enzyme gene CRD as the CDH gene.

Transformation The resulting recombinant plasmid, having a CRD enzyme gene or a recombinant plasmid having both the CRD enzyme gene and the GDH gene, can be introduced into a host cell by a conventional method. Alternatively, a recombinant plasmid having a CRD enzyme gene and a recombinant plasmid having a GDH gene can be introduced into a host cell simultaneously at different times, to obtain a transforming strain, which has been transformed with these two plasmids. As such host cell, a bacterium, a yeast, a filamentous fungus, a plant cell, an animal cell and the like, can be used. Especially preferred is the use of E. coli. A plasmid can be introduced into a host by a method known in the art, such as a method that includes the step of mixing a host cell in a competent state and a recombinant plasmid, and a method that includes the step of transfecting (introducing) a plasmid that uses an assistant plasmid, for the transmission of conjugation. The plasmid introduced into a host can be duplicated autonomously as an episome. Alternatively, all or part of the plasmid can be incorporated into a chromosome and duplicated along with this chromosome. The GDH activity of the transformed cell can be determined by measuring an increase in absorption at 340 nm and at 252C by a 1M tris hydrochloric acid buffer (pH 8.0), with 0.1 M glucose as a substrate, 2 mM NADP as a coenzyme and the enzyme added there.

Acquisition of the optically active alcohol The optically active 4-halo-3-hydroxy-butyric ester, which is a type of optically active alcohol, was purchased in the following manner, for example. As a substrate, the 4-haloacetoacetic ester can be used, which is represented by the following general formula: 0 • I (wherein Ri is a halogen, R2 is hydrogen, and 3 is an alkyl group or an aryl group, substituted or unsubstituted). When 3 is an alkyl group, this is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, or the like. When R3 is an aryl group, this is, for example, a phenyl group, a tolyl group, or the like. When R3 is a substituted aryl group, this is, for example, a fluorophenyl group, a chlorophenyl group, or the like. Preferably Ra is chloro or bromo and R is an alkyl group having 1 to 4 carbon atoms. More preferably, the substrate is methyl 4-chloroacetoacetate, ethyl 4-chloroacetoacetate, methyl 4-bromoacetoacetate or ethyl 4-bromoacetoacetate. Alternatively, ethyl 4-iodoacetoacetate, ethyl 4-hydroxyacetoacetate, ethyl 2-chloro-3-oxobutyrate, ethyl 2-methyl-3-oxobutyrate, ethyl 4-azide-acetoacetate and the like can be used as a substrate. . The above 4-haloacetoacetic ester can be prepared by the method described, for example, in Japanese publication, open to the public, No. 61-146191. For example, the 4-haloacetoacetic ester can be prepared by a method where the diketene is used as a starting material and reacts with a halogen to obtain the 4-haloacetoacetate halide, which is then reacted with alcohol. Alternatively, the 4-haloacetoacetic ester can be prepared by a method where an acetoacetic ester is used as a starting material and its quaternary position is directly halogen. The 4-haloacetoacetic ester, as a substrate, is added to an appropriate solvent, together with NADPH, as a coenzyme and a culture of the transforming microbe or its processed product and the like, and stirred while adjusting the pH. This reaction is carried out at a pH of 4 to 10, at a temperature of 10 to 700C. Although the prepared concentration of the substrate varies between 0.1 to 90% (weight / volume), the substrate can be added continuously. The reaction is carried out in a batch or continuous manner. The processed product of a microbe and the like, mentioned above, refers to a crude extract, bodies of cultured microbes, a lyophilized organism, an organism dried in acetone, homogenates of such microbial bodies, and the like. Such processed products and the like may be used in the state of being immobilized as they are, ie, as enzymes or bodies of microbes, by known means. The immobilization can be performed by a method known to those skilled in the art (for example, by an entanglement method, a physical absorption method, and a trapping method). In the reaction, the amount of an expensive coenzyme, used in the reaction, can be greatly reduced by the use of a NADPH regeneration system in combination. For example, a method using GDH and glucose, which are typical NADPH regeneration systems, can be employed. The reaction conditions are as follows, although they depend on the enzyme, the microbe or its processed product, the concentration of the substrate, and the like, which are to be used: the concentration of the substrate varies from approximately 0.1 to 90% by weight, the reaction temperature is from 10 to 50oc, the pH from 5 to 8, and the reaction time is from 1 to 36 hours. The above reaction can be enhanced using a culture of a transformed microbe or its processed product, obtained by introducing either a CRD enzyme gene or an enzyme gene (eg, GDH), which has the ability to regenerate a coenzyme in which the CRD enzyme gene depends on a host microbe. In this case, further preparation of a source of enzymes required for the regeneration of a coenzyme is not necessary, and the (S) -4-halo-3-hydroxy-butyric ester can be produced at a lower cost.

The 4-halo-3-hydroxy-butyric ester, produced by the reaction, can be purified by a conventional method. For example, the 4-halo-3-hydroxy-butyric ester is subjected to centrifugation, filtration and other processes, as required, in the case where microbes are used, to remove suspended substances, such as the bodies of microbes. The resulting product is subjected to extraction with an organic solvent, such as ethyl acetate and toluene, and dehydrated with a dehydrating agent, such as sodium sulfate. The organic solvent is removed under decompression. The resulting product is then subjected to decompression evaporation, chromatography (e.g., silica gel column chromatography), and the like, for purification. The quantification of the 4-halo-3-hydroxy-butyric ester can be carried out by gas chromatography. For example, the quantification of ethyl 4-chloro-3-hydroxybutyrate can be performed by chromatography, using a glass column (internal diameter 3 mm x 1 mm) filled with PEG-20M Chromosorb WAWDMCS 10% 80/100 mesh (manufactured by GL Science Co., Ltd.) at 150GC and stopping with FID. The optical purity of (S) -4-halo-3-hydroxybutyrate can be measured by HPLC chromatography, using an optical isolation column CHIRALCEL OB (manufactured by Daicel Chemical Industries, Co., Ltd.) Thus, as described above , the present invention enables mass production of the CRD enzyme. In addition, using this enzyme, an efficient production method of optically active alcohol, such as the (S) -4-halo-3-hydroxy-butyric ester, is provided. Next, the present invention will be described in detail in the form of illustrative but not restrictive examples. The details of the manipulation method, in relation to the recombination DNA technique employed in the examples, are described in the following texts.

(I) Molecular Cloning, 2nd Edition (Cold Spring Harbor Laboratory Press, 1989) (II) Current Protocols in Molecular Biology (Greene Publishing Associates and .Wiley internscience) Example i; Purification of the CRD enzyme A CRD enzyme, which has the ability to reduce the 4-haloacetoacetic ester asymmetrically of Candida magnoliae IFO 0705, to produce the (S) -4-halo-3-hydroxy-butyric ester, was purified from the following way, in order to move as a single band electrophurically.

A liquid medium, 8000 ml, of the following composition was prepared, and a 400 ml portion was distributed in 2000 ml Sa aguchi flasks and steam sterilized at 120BC for 20 minutes.

Composition of 5% Glucose Medium Polipeptone 0.5% KH2P04 0.2% K HP0 0.1% MgSO -7H20 0.02% Water of the pH key of 6.5 The previous medium was inoculated with a culture of Candida magnoliae IFO 0705, which had been previously cultured in the medium, for 5 ml / flask and cultivated for three days at 30 ° C, with shaking. The microbial bodies were collected from the resulting culture by centrifugation and then cleaned twice with a saline solution, whereby 230 g of wet bodies of microbes were obtained. Among these moist microbial bodies, 180 g were suspended in 360 ml of a 50 mM phosphate buffer (pH 7.0) and then the microbial bodies were destroyed by a Dyno mill (manufactured by Dyno-Mill). The bodies of destroyed microbes were centrifuged to remove cellular debris and thus obtain 760 ml of a cell-free extract. Ammonium sulfate was added to, and dissolved in, the cell-free extract, in order to obtain a saturation of 40%. The resulting precipitates were removed by centrifugation and the supernatant was dialysed with a 10 mM phosphate buffer (pH 7.0), containing 0.1 mM DTT. The resulting product was supplied to a column (500 ml) of DEAE Sephacel (manufactured by Pharmacia Biotech), which had been equilibrated with the same regulator, and the column was washed with this same regulator. The active fractions were collected from the eluted solution, which had been passed through the column, and NaCl was added to the active fractions collected, so as to obtain a final concentration of 4 M. The active fractions were supplied to a column (200 ml) of Phenyl Sepharose C1-4B ( manufactured by Pharmacia Biotech), which had been equilibrated with a 10 M phosphate regulator (pH 7.0), containing 4 M NaCl and 0.1 mM DTT, in order to adsorb the enzyme. After the column was washed with the same regulator, the active fractions were eluted using a 10 mM phosphate buffer (pH of 7.0) with a linear gradient of NaCl (from 4M to 0 M) and ethylene glycol (from 0% to 50%). % (weight / volume)). Active fractions, initially eluted, were collected and dialyzed overnight with a 10 mM phosphate buffer (pH of 7.9). The resulting dialysate was delivered to a column (1 ml) of Mono Q HR 5/5 (FPLC system, manufactured by Pharmacia Biotech), which had been equilibrated with a 10 mM phosphate buffer (pH 7.0) containing 0.1 mM of DTT, and washed with the same regulator. The active fractions in the wash solution were collected and concentrated to 200 μl by ultrafiltration. The concentrate was then supplied to a column (24 ml) of Superdex 200 HR 10/30 (manufactured by Pharmacia Biotech) which had been equilibrated with a 10 mM phosphate buffer (pH of 7.0), containing 0.2 M of sodium chloride. sodium and 0.1 mM DTT, and eluted with the same regulator. The active fractions were collected to obtain a purified enzyme specimen.

Example 2: Measurement of enzyme properties The enzymatic properties of the enzyme obtained in Example 1 were examined. Enzymatic activity was determined by, basically, allowing 3 ml of a reaction solution, which includes 0.2 mM of 4-chloroacetoacetate of ethyl as a substrate, 0.32 mM of NADPH as a coenzyme, and 0.1 ml of an enzyme solution in a 200 mM phosphate buffer (pH of 7.0) will react at 30 ° C for one minute and measure a reduction in absorption at 340 nm. (1) Action: The enzyme acted on ethyl 4-chloroacetoacetate with NADPH, as a coenzyme, to produce ethyl (S) -4-hydroxybutyrate, which has an optical purity of 99% or more. (2) Substrate specificity: The enzyme according to the present invention reacted using various carbonyl compounds, shown in the following Table 1, as a substrate, under the same conditions as those used for ethyl 4-chloroacetoacetate. As a result, the enzyme exhibited substrate specificity, as shown in Table 1.

Table% Substrate Q.% m Relative Activity (%) ^ '- C oacetoacetate etiJto? 00 ethyl acetoacetate 0 p-nitrobenzaldehyde 0 o-nitrobenzaldehyde 0 m-nitrobenzaldehyde 0 p-chlorobenzaldehyde 0 o-chlorobenzaldehyde 0 m-chlorobenzaldehyde 0 nicotinaldehyde 0 isonicotinaldehyde 0 benzaldehyde 0 glyoxal 0 methyl-glyoxal 0 * diacetyl 19 chloroacetoaldehyde 0 camphor-quinone 0 2-chloroacetoacetate ethyl 95 methyl 4-chloroacetoacetate 11 methyl 2-chloroacetoacetate 11 4-chloroacetoacetate octyl 36 (3) Optimal pH: The activity of the enzyme was measured in a pH range of 5.0 to 8.5, using a phosphate buffer or a tris-hydrochloric acid regulator. As a result, the optimum pH for the action of the enzyme on ethyl (S) -4-chloro-3-hydroxybutyrate was 5.5 to 6.5. (4) Optimum action temperature: The activity of the enzyme, according to the present invention, was measured using ethyl 4-chloroacetoacetate as a substrate for one minute, in a temperature range of 20 to 60 ° C, to obtain a optimum temperature. As a result, the optimum temperature was 50 to 55ac. (5) Heat Stability: After the enzyme, according to the present invention, was treated at a pH of 7.0 and at 402C, for 30 minutes, the activity of the enzyme was measured using ethyl 4-chloroacetoacetate as a substrate . As a result, the activity was 90% of that before the treatment remained. (6) Inhibition: Various metal ions and inhibitors, with the respective concentrations shown in Table 2 below, were added to the above reaction solution, to measure the activity of ethyl (S) -4-chloro-3-hydroxybutyrate , using the ethyl 4-chloroacetoacetate as a substrate. As a result, the enzyme, according to the present invention, was inhibited by quercetin and mercury ions, as shown in Table 2.

Table 2 Compound Concentration Relative activity f%) of addition (mM) _- Not added Quercetin 0.01 84 0.1 0 Diphenylhydantoin 1 84 Dicumarol 0.1 97 2, 4-dinitrophenol 0.1 86 DTNB 0.05 100 Iodoacetic acid 1 100 NEM 1 100 PMSF 1 93 p -CMB 1 88 EDTA 1 95 Phenylhydrazine 1 97 SnCl 2 1 77 PbCl 2 1 86 CdCl 2 1 91 CuSO 4 1 85 CoCl 2 1 89 MgCl 2 1 83 ZnS04 1 97 HgCl 2 0.1 49 (7) Molecular weight The molecular weight of the enzyme was measured using the TSK-G3000SW column and a 0.1M phosphate buffer (pH 7.0), which contains 0.1 M NaS0 and 0.05% NaN3 as an eluent, and It was found was approximately 76,000. The molecular weight of a subunit of the enzyme was determined by subjecting it to electrophoresis with 10% SDS-polyacrylamide gel in the presence of 2% volume / volume of 2-mercaptoethanol and calculating the relative mobility of a standard protein. As a result, the molecular weight of the enzyme subunit was determined to be approximately 32,000. (8) Resistance to organic solvents: An equivalent amount of ethyl acetate or butyl acetate was added to a phosphate buffer (pH 7.0), which includes the enzyme, according to the present invention, dissolved therein, was shaken at 28 ° C for 30 minutes and then centrifuged. Residual activity of the enzyme in the aqueous phase was measured using ethyl 4-chloroacetoacetate as a substrate. As a result, an activity of 72% remained in the case of the addition of ethyl acetate and an activity of 85% in the case of the addition of butyl acetate.

Example 3: Production of ethyl (S) -4-chloro-3-hydroxybutamate, with the use of the enzyme according to the present invention A 100 mM phosphate buffer (pH of 6.5), 25 ml, containing 50 units of the purified enzyme, according to the present invention, 250 mg of ethyl 4-chloroacetoacetate, 1.56 mg of NADP, 280 mg of glucose and 60 units of glucose dehydrogenase (manufactured by Amano Pharmceutical Co., Ltd.) are stirred at 30 &V for 24 hours. After the reaction, the reaction solution was subjected to extraction with ethyl acetate and, after removal of the solvent, the extract was analyzed. As a result, it was found that ethyl (S) -4-chloro-3-hydroxybutyrate, having an optical purity of 99% or more, had been produced with a 98% yield. The optical purity of ethyl S-4-chloro-3-hydroxybutyrate was measured by HPLC chromatography with the use of an optical isolation column, CHIRALCEL OB (manufactured by Daicel Chemical Industrie, Co., Ltd.). This chromatography was performed using a hexane / isopropanol mixed solvent of 9/1, as a mobile phase, at a flow rate of the mobile phase of 0.8 ml / min.The detection was conducted by measuring the absorption of 215 nm. of ethyl (S) -4-chloro-3-hydroxybutyrate was performed by gas chromatography at 150CC, using a glass column (internal diameter of 3 mm x 1 mm) filled with PEG-20M of Chromosorb WAW DMCS 10% 80/100 mesh (manufactured by GL Science Co., Ltd.) and detected by FID.

Example 4; Production of ethyl (S? -4-bromo-3-hydroxybutyrate, with the use of the enzyme according to the present invention A 100 mM phosphate buffer (pH of 6.5), 2.5 ml, containing 5 units of the purified enzyme, according to the present invention, 25 mg of ethyl 4-bromoacetoacetate, 0.16 mg of NADP, 28 mg of glucose and 6 units of glucose dehydrogenase (manufactured by Amano Pharmceutical Co., Ltd.) was stirred at 30dV for 24 hours After the reaction, the reaction solution was subjected to extraction with ethyl acetate and, after removal of the solvent, the extract was analyzed, as a result, it was found that (S) -4-bromine Ethyl 3-hydroxybutyrate had been produced in 43% yield The quantification of ethyl 4-bromo-3-hydroxybutyrate was performed in substantially the same manner as that for ethyl 4-chloro-3-hydroxybutyrate in Example 2.

Example 5: Production of methyl (S) -4-chloro-3-hydroxybutyrate, with the use of the enzyme according to the present invention. Butyl acetate, 2.5 ml, was added to 2.5 ml of the 100 mM regulator. phosphate (pH of 6.5), containing 5 units of the purified enzyme, according to the present invention, 25 mg of methyl 4-chloroacetoacetate, 0.16 mg of NADP, 28 mg of glucose, and 6 units of glucose dehydrogenase ( manufactured by Amano Pharmaceutical Co., Ltd.) and stirred at 302V for 24 hours. After the reaction, the reaction solution was subjected to extraction with ethyl acetate and, after removal of the solvent, the extract was analyzed. As a result, it was found that methyl (S) -4-chloro-3-hydroxybutyrate had been produced with a yield of 58%. The quantification of methyl 4-chloro-3-hydroxybutyrate was performed in substantially the same manner as that for ethyl 4-chloro-3-hydroxybutyrate in Example 2.

Example 6; Production of ethyl (S) -4-chloro-3-hydroxybutyrate, with the use of the enzyme according to the present invention: continuous addition of the substrate The ethyl 4-chloroacetoacetate, 3.8 ml, was continuously added to 50 ml of the 100 mM phosphate buffer (pH 6.5), containing 100 units of the purified enzyme, according to the present invention, 1.56 mg of NADP, 4.5 g of glucose, 250 units of glucose dehydrogenase (manufactured by Amano Pharmaceutical Co ., Ltd.) and 0.24 g of NaCl, at a rate of 0.23 g per hour, and stirred at 30 ° C for 20 hours, while adjusting the pH using sodium hydroxide. After the reaction, the reaction solution was subjected to extraction with ethyl acetate and, after removal of the solvent, the extract was analyzed. As a result, it was found that ethyl (S) -4-chloro-3-hydroxybutyrate, with an optical purity of 100%, had been produced with a yield of 58%. The quantification of ethyl 4-chloro-3-hydroxybutyrate was performed substantially in the same manner as that for Example 2.

Example 7; Cloning of the CRD enzyme gene (Creation of the chromosomal DNA collection) A chromosomal DNA was extracted from a microbial body cultured from Candida magnoliae IFO 0705, according to the method described by Hereford (Cell, 18, 1261 (1979) ). The resulting chromosomal DNA was partially digested with Sau3AI, and a DNA fragment having a size of 23 kb to 20 kb, of the resulting digestion was inserted into the BamHI site of the phage vector EMBL3 (manufactured by Stratagene). The resulting recombinant phage vector was packaged in vitro using Gigapack II Gold (manufactured by Stratagene), and then E. coli NM415 was allowed to be infected with it, thereby creating a chromosomal DNA collection composed of about 20,000 ADNS. Preparation of the synthetic oligonucleotide probe The purified CRD enzyme, obtained as described in Example 1, was denatured in the presence of 8M urea, and then digested with lysyl endopeptidase, derived from Ac? RomoJbacter (manufactured by Wako Puré Chemical Industries, Ltd.). The amino acid sequence of the resulting peptide fragment was determined by the Edman method.

Based on the resulting amino acid sequence, the DNA probes, which have the following sequence, were synthesized.

Probe 1: 5 • -GCNCAYACNAARAAYGA-3 '(SEQ ID NO: 3) Probe 2: 5-AAYGTNGARTAYCCNGC-3 '(SEQ ID NO: 4) Probe 3: 5-CTRGTYCTRCTRCTRTT-3' (SEQ ID NO: 5) Probes 1, 2 and 3 were labeled with 32P using Megalabel (manufactured by Takaer Shuzo Co., Ltd.) and labeled probes were used in the following experiments.

Cloning of the CRD Enzyme Gene from the DNA Collection of Chromosomes The DNA collection of chromosomes, created as described above, has been classified for phage plaques that include a CRD enzyme gene by a plaque hybridization method (Science, 196 , 180 (1977)) using the synthetic DNA probes labeled with 32P. As a result, a positive plate was obtained. Then, a recombinant phage DNA, obtained from the positive plate, was digested twice with EcoRI and HindIII, and the resulting DNA was analyzed by the Southern blot (J. Mol. Biol., 98, 53 (1975)). As a result, it was found that a digested fragment of about 1.3 kb, generated by double digestion with EcoRI and HindIII, had been hybridized with the above synthetic DNA probes. Based on this fact, the approximately 1.3 kb DNA fragment was inserted into the Eco-RI-HindIII site of plasmid püC19 (manufactured by Takara Shuzo Co., Ltd.), to constitute the recombinant plasmid pUC-HR and selected as the chromosome DNA clone, which includes the CRD enzyme gene. This plasmid was named pUIC-HE. Determination of base sequence A variety of restriction enzymes reacted with the previous recombinant plasmid pUC-HE, and the digested fragments, produced during the reaction, were analyzed to create a cleavage map of restriction enzymes. Then, several DNA fragments obtained during the analysis were inserted into multi-cloning sites of the plasmid pUC19, to obtain recombinant plasmids. Using these recombinant plasmids, the basic sequences of the respective inserted fragments were analyzed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by Perkin Elmer) and the ABI 373A DNA Sequencer sequencer (manufactured by Applied Biosystems). As a result, the entire base sequence of the DNA fragment of about 1.3 kb, which was expected to include a CRD enzyme gene was determined. Figure 1 shows the base sequence thus determined. An estimated amino acid sequence of the base sequence for the portion of the gene structure of the base sequence is also shown under the corresponding base sequence in Figure 1. The amino acid sequence was compared to a partial amino acid sequence of a digested peptide fragment of lysyl endopeptidase of the purified CRD enzyme. As a result, it was found that the partial amino acid sequence of the purified CRD enzyme exists in the estimated amino acid sequence of the base sequence and completely matches it (indicated as the portion underlined in the amino acid sequence in Figure 1), except due to the lack of methionine in the N-terminal. The methionine in the N-terminus is considered to be removed by the modification, after the synthesis of the protein.

Example 8: Construction of plaspide rf «ff > m? ante which includes the CRD enzyme gene In order to express a CRD enzyme in E. coli, a recombinant plasmid used for transformation was constructed. First, a double-stranded DNA, having an Ndel site added to an initiation codon portion of a structural gene of the CRD enzyme and an EcoRI site added immediately after its stop codon, was purchased in the following manner. A sizing of the N-terminal DNA, which has an Ndel site added to the initiation codon portion of the structural gene of the CRD enzyme and a DNA sizing of the C-terminus, which has an EcoRI site added immediately after the codon of Completion of the structural gene of the CRD enzyme was synthesized. The base sequences of these two sizes are as follows.

Sizing terminal DNA N 5 • -TAGTCGTTAACCATATGGCTAAGAACTTCTCCAAC-3 • (SEQ ID NO: 6) Sizing C-terminal 5 '-TCTGAGTTAACGAATTCTTAGGGAAGCGTGTAGCCACCGT-3' DNA (SEQ ID NO: 7) Using the two synthetic DNA primers above, a double-stranded DNA was synthesized by pumping the plasmid pUC-HE, obtained in Example 7 as a standard . The resulting DNA fragment was digested with Ndel and EcoRI, and inserted into the Ndel and EcoRI sites downstream of a lac promoter from the plasmid pUCNT (WO94 / 03613), to obtain the recombinant plasmid pNTSl.

Example 9: Production of the recombinant plasmid including both the CRD enzyme gene and the GDH gene Plasmid pGDA2 (J. Biol. Chem. (1989), 264, 6381) was digested twice with EcoRI and PstI, for isolate a DNA fragment of approximately 0.9 kb, which includes a GDH gene derived from Bacillus megaterium. This DNA fragment was inserted into an EcoRI-PstI site of plasmid PSL301 (manufactured by Invitrogen), to construct the recombinant plasmid pSLG. This recombinant plasmid pSLG was then digested twice with EcoRI and Xhol, to isolate a DNA fragment of about 0.9 kb, which includes a GDH gene derived from Bacillus megaterium. This DNA fragment was inserted into an Eco-RI-SalI site (located downstream of the CRD gene) of the pNTSl constructed in Example 8, to obtain the recombinant plasmid pNTSIG. The construction method and structure of pNTSlG are illustrated in Figure 2.

Example 10; Construction of recombinant E. coli E. coli HB101 (manufactured by Takara Shuzo CO., Ltd.) was transformed using the recombinant plasmid pNTSl, obtained in Example 8, and the recombinant plasmid pNTSlG, obtained in Example 9, to obtain HB101 (pNTSl) from recombinant E. coli and HB101 (pNTSlG), respectively. The transformants, thus obtained, HB101 (pNTSl) and HB101 (pNTSlG) of E. coli, were deposited in the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology, under the deposit numbers respective FERM BP-5834 and FERM BP-5835, on February 24, 1997. In addition, as in Example 9, plasmid pGDDA2 (J. Biol. Chem. (1989), 264, 6381) was digested in double form with EcoRI and PstI, to isolate a DNA fragment of approximately 0.9 kb, which includes a GDH gene derived from Bacillus megaterium. This DNA fragment was inserted into an EcoRI-PstI site of plasmid pSTV28 (manufactured by Takara Shuzo Co., Ltd.) to construct the recombinant plasmid pSTVG. The HBlOl (pNTSl) from E. coli, which had been made competent in advance by a calcium chloride method, was transformed with pSTVG to obtain HB101 (pNTSl, pSTVG) from E. coli.

Example 11: Expression of the CRD enzyme in e. recombinant coli Determination of the activity of the CRD enzyme in recombinant E. coli The HBlOl (pNTSl) of recombinant E. coli, obtained in Example 10, cultured in a 2 x YT medium, containing 50 μg / ml of MPIXILIN, was collected , was suspended in a 100 mM phosphate buffer (pH 6.5) and subjected to an ultrasonic treatment, to obtain a cell-free extract. The activity of the CRD enzyme of the cell-free extract was measured in the following manner. That is, 1 mM of ethyl 4-chloroacetoacetate, as a substrate, 0.1 mM of NADPH as a coenzyme, and the enzyme were added to a 100 mM phosphate buffer (pH of 6.5) per reaction, and the reduction in the absorption at 340 nm, was measured at 30 amps; C. With these reaction conditions, the oxidation of 1 μmol of NADPH in NADP in one minute was defined as a unit of enzymatic activity. The activity of the CRD enzyme, thus measured, in the cell-free extract, was represented as the specific activity and compared with that of a transformant using only a vector plasmid. Similarly, the activity of the CRD enzyme in a cell-free extract of Candida magnoliae IFO 0705, prepared substantially in the same manner as that described in Example 1, was obtained for comparison. The results are shown in the following Table 3. The HBlOl (pNTSl) of E. coli exhibited a definite increase in the activity of the CRD enzyme as compared to the HBlOl (pUCNT) of E. coli which was transformed using only one plasmid of vector, and exhibited an activity about 8.5 times as large as that of Candida magnoliae IFO 0705 Table 3 Name of the strain Specific activity CRD (U / mg) "HB101 (pUCNT) < 0.01 HB101 (pNTSl) 16.0 Candida magnoliae IFO 0705 1.89 Comparison of the N-terminal sequence The N-terminal amino acid sequence of each CRD enzyme purified from the cell-free extract, obtained in substantially the same manner as that in the expression experiment, described above, and a free extract of Candida magnoliae IFO 0705 cells were determined in 30 residues by the Edman method. The resulting N-terminal amino acid sequences were compared and found to completely correspond to each other in this range.

Example 12: Simultaneous expression of the enzyme CRD and GDH in recombinant E. coli The GDH activity of a cell-free extract, obtained by the process of HB101 (pNTSlG) of E. coli and HB101 (pNTSl, pSTVG) of E coli, obtained in Example 10, in the manner described in Example 11, was measured as follows. That is, 0.1 M of glucose as a substrate, 2 mM of NADP as a coenzyme and the enzyme, were added to 1 M of the tris-hydrochloric acid buffer (pH of 8.0) for the reaction and an increase in absorption to 340 nm was measured at 25oc. Under these reaction conditions, the reduction of 1 μmol of NADP in NADPH in one minute was defined as a unit of the enzymatic activity. The activity of the CRD enzyme was also measured as in Example 10. The activity of the CRD enzyme, thus measured, and the activity of the GDH enzyme in the cell-free extract, was represented as the specific activities and compared with those of HB101 (pNTSl) from E.COli, HB101 (pNTSl, pSTVG) and an HBlOl transformant (pUCNT), using only one vector. The results are shown in the following Table 4. HB101 (pNTSlG) and HB101 (pNTSl, pSTVG) from E. coli exhibited a defined increase in CRD enzyme activity and GDH activity, as compared to HBlOl (pUCNT) of E. coli, which was transformed using only one vector plasmid.

Table 4 Name of the strain Activity specific to CRD-specific activity (U / mg) GDH (U / mg) HB101 (pUCNT) < 0. 01 < 0. 01 HB101 (pNTSl) 16. 0 < 0. 01 HB101 (pNTSlG) 8.03 62. 6 HB101 (pNTSl, pSTVG) 13 .5 1. 6 EXAMPLE 131 Synthesis of the ester (B> 4-halo-3-hydroxybutyric ester of 4-haloacetoacetic ester, with the use of recombinant E. coli, which has an introduced CRD enzyme gene HB101 (pNTSl) of recombinant E. coli , obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask and cultivated, with shaking, at 372C for 13 hours.GDH (manufactured by Amano Pharmaceutical CO., Ltd .) 1250 U, 5.5 g of glucose and 1.6 mg of NADP were added to 50 ml of the resulting culture.The culture was stirred at 30 ° C, while adjusting the pH to 6.5 with a 5M solution of sodium hydroxide. Ethyl 4-chloroacetoacetate is added to the culture, in 250 mg portions, every 15 minutes In this way, a total of 5 g of ethyl 4-chloroacetoacetate were added and the reaction was carried out for five hours. reaction, the reaction solution was subjected to extraction using ethyl acetate, and after Remove the solvent, the extract was analyzed. As a result, it was found that ethyl (S9-4-chloro-3-hydroxybutyrate, which has an optical purity of 100%, had been produced with a yield of 90%.) The quantification of 4-chloro-3-hydroxybutyrate ethyl was performed by gas chromatography, using a glass column (internal diameter 3 mm x 1 mm) filled with PEG-20M Chromosorb WAW DMCS 10% 80/100 mesh (manufactured by GL Science Co., Ltd.) a 150BC and detected by FID The optical purity of ethyl (S) -4-chloro-3-hydroxybutyrate was measured by HPLC chromatography using an optical isolation column, CHIRLCEL OB (manufactured by Daicel Chemical Industries, CO., Ltd. This chromatography was performed using a hexane / isopropanol mixed solvent of 9/1, as a mobile phase at a flow rate of the mobile phase of 0.8 ml / min.The detection was conducted by measuring the absorption of 215 nm.

Example 14: synthesis of tB) -4-halo-3-hydroxy-butyric ester from the 4-haloacetoacetic ester, using recombinant E. coli with the enzyme CRD and GDH expressed simultaneously HB101 (pNTSlG) of E. coli , obtained in the Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 372C for 13 hours. The glucose, 5.5 g and 3.2 mg of NADP were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while the pH was adjusted to 6.5 with a 5M solution of sodium hydroxide. While stirring, ethyl 4-chloroacetate is added to the culture in 250 mg portions, every 15 minutes. In this way, a total of 5 g of the ethyl 4-chloroacetoacetate were added and the reaction was carried out for five hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, and after removing the solvent the extract was analyzed. As a result, ethyl (S) -4-chloro-3-hydroxybutyrate was found to have an optical purity of 100% and had been produced in 92% yield. The quantification and measurement of the optical purity of the ethyl 4-chloro-3-hydroxybutyrate were performed in substantially the same manner as in Example 13.

Example 15: Synthesis of ethyl (8) -4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate, using the recombinant E. coli with the enzyme CRD and GDH expressed simul t »« »'" "fc HB101 (pNTSlG) of recombinant E. coli, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37BC for 13 hours. Glucose, 19.2 g and 2.5 mg of NADP were added to 40 ml of the resulting culture. The culture was stirred at 30 ° C while the pH was adjusted to 6.5 with a 5M solution of sodium hydroxide. While stirring, a total of 16.1 g of ethyl 4-chloroacetate was continuously added to the culture at a rate of 2 g per hour. The reaction was carried out for 24 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel column chromatography, to obtain 15.6 g of the (S) -4- ethyl chloro-3-hydroxybutyrate. The optical purity of ethyl (S) -4-chloro-3-hydroxybutyrate was analyzed by the HPLC chromatography method and found to be 100% lH-NMR (CDC13) d (ppm): 1.33 (3H, t), 2.65 (2H, d), 3. 31 (1H, d), 3.60 (2H, d >, 4.2 (3H, m); Column: Chiralcel OB (0.46 x 25 cm), manufactured by Daicel Chemical Industries, CO., Ltd .; Column temperature: 00C, Eluent: n-hexane / 2-propanol of 9/1, flow rate: 0.8 ml / min, Detection: 215 nm, Elution time, 19.2 minutes for (S), 17.0 minutes for (R).

Example 16: Synthesis of ethyl (B) -4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate, using recombinant E. coli with the enzyme CRD and GDH expressed simultanously HBlOl (pNTSlG) from E. coli The recombinant, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37dC for 13 hours. The glucose, 9.6 g, was added to 40 ml of the resulting culture. The culture was stirred at 30 ° C while the pH was adjusted to 6.5 with a 5M solution of sodium hydroxide. While stirring, a total of 8.1 g of ethyl 4-chloroacetate is added to the culture at a rate of about 2 g per hour. The reaction was carried out in a total of 24 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, and after removing the solvent the concentrate was analyzed. As a result, ethyl (S) -4-chloro-3-hydroxybutyrate was found to have an optical purity of 100% and had been produced in 96% yield.

Example 17: Synthesis of ethyl (S) -4-bromo-3-hydroxybutyrate from ethyl 4-bromoacetoacetate, using recombinant E. coli with the enzyme CRD and GDH expressed simultaneously. The HBlOl (pNTSlG) of recombinant E. coli, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500-ml Sakaguchi flask. mi, and was cultivated with agitation at 37dC for 13 hours. Glucose, 1.3 g and 3.2 mg of NADP were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while adjusting the pH at 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 900 mg of the (S) -4-bromo- Ethyl 3-hydroxybutyrate. The optical purity of ethyl (S) -4-bromo-3-hydroxybutyrate was analyzed and found to be 100%. That is, the sample was converted to a carbamate using the phenyl isocyanate under the presence of the pyridine and the optical purity of the carbamate was measured by a method of HPLC chromatography. lH-NMR (CDC13) d (ppm): 1.38 (3H, t), 2.75 (2H, m), 3.28 (1H, br), 3.51 (2H, m), 4.18 (3H, q), 4.25 (1H, m); Column: Chiralcel OJ (0.46 x 25 c) manufactured by Daicel Chemical Industries CO. , Ltd .; Column temperature: 25fiC; Eluent: n-hexane / 2-propanol of 9/1; Flow rate: 0.8 ml / min .; Detection: 254 nm: Elution time: 24.2 minutes for (S), 27.8 minutes for (R).

Example 18: Synthesis of ethyl (8) -4-budo-3-hydroxybutyrate from ethyl 4-odoacetoacetate, using recombinant E. coli with the enzyme CRD and GDH expressed simultaneously HBlOl (pNTSlG) from E. coli The recombinant, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37 ° C for 13 hours. The glucose, 0.5 g and 3.2 mg of NADP and then 0.5 g of ethyl 4-iodoacetoacetate, were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while adjusting the pH at 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 900 mg of the (S) -4-iodine Ethyl 3-hydroxybutyrate. The optical purity of ethyl (S) -4-iodo-3-hydroxybutyrate was analyzed and found to be 91.6%. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide, to obtain the ethyl 4-cyano-3-hydroxybutyrate, which was then changed to the benzoic ester, using the benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the method of HPLC chromatography. NMR (CDC13) d (ppm): 1.28 (3H, t), 2. 65 (2H, d), 3.31 (3H, m), 4.00 (1H, m), 4.20 (2H, q); Column: Chiralcel AS (0.46 x 25 cm) manufactured by Daicel Chemical Industries CO., Ltd .; Column temperature: 25fiC; Eluent: n-hexane / ethanol 95/5; Flow rate: 1 ml / min .; Detection: 254 nm: Elution time: 19.6 minutes for (S), 21.3 minutes for (R).

Example 19: Synthesis of methyl (Bl-4-chloro-3-hydroxybutyrate from methyl 4-chloroacetoacetate, using the Recombinant E. coli with the enzyme CRD and GDH expressed simultaneously The HBlOl (pNTSlG) of recombinant E. coli, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and was cultivated with shaking at 372C for 13 hours. Glucose, 7.2 g and 3.2 mg of NADP and then 4 g of methyl 4-chloroacetoacetate were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while adjusting the pH at 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 24 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 3.85 g of the (S) -4-chloro- Methyl 3-hydroxybutyrate. The optical purity of methyl (S) -4-bromo-3-hydroxybutyrate was analyzed and found to be 100%. That is, the sample was converted to a carbamate using the phenyl isocyanate under the presence of the pyridine and the optical purity of the carbamate was measured by a method of HPLC chromatography. lH-NMR (CDC13) d (ppm): 2.65 (2H, m), 3.20 (1H, br), 3.63 (2H, m), 3.73 (3H, S), 4.28 (1H, m), Column: Chiralcel OJ (0.46 x 25 cm) manufactured by Daicel Chemical Industries CO ., Ltd .; Column temperature: 25SC; Eluent: n-hexane / 2-propanol 8/2; Flow rate: 1 ml / min .; Detection: 254 nm: Elution time: 19.2 minutes for (S), 22.6 minutes for (R).

Example 20: Synthesis of ethyl (B) -4-azide-3-hydroxybutyrate from ethyl 4-azide-acetoacetate, using the recombinant E. coli with the enzyme CRD v GDH expressed simultaneously. HB101 (pNTSlG) of recombinant E. coli, obtained in Example 10, was inoculated into 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37 ° C for 13 hours. The glucose, 3.1 g and 3.2 mg of NADP and then 2 g of ethyl 4-azideacetoacetate, were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while adjusting the pH at 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 1.6 g of the (S) -4-azide. Methyl 3-hydroxybutyrate. The optical purity of ethyl (S) -4-azide-3-hydroxybutyrate was analyzed by the HPLC chromatography method and found to be 90%. lH-NMR (CDC13) d (ppm): 1.25 (3H, t), 2.55 (2H, d), 3.30-3.35 (3H, m), 4.20 (3H, m). Column: Chiralcel OB (0.46 x 25 cm) manufactured by Daicel Chemical Industries CO., Ltd.; Column temperature: 25 £ iC; Eluent: n-hexane / 2-propanol of 9/1; Flow rate: 1 ml / min .; Detection: 254 n: Elution time: 16.2 minutes for (S), 19.6 minutes for (R).

Example 2l? Synthesis of (8> -3.4-ethyl dihydroxybutyrate from ethyl 4-hydroxy-acetoacetate, using recombinant E. coli with the enzyme CRD v GDH expressed simultaneously.) HBlOl (pNTSlG) from recombinant E. coli, obtained in Example 10, it was inoculated into 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37CC for 13 hours, glucose, 7.4 g and 3.2 mg of NADP were added and then 4 g of the ethyl 4-hydroxyacetoacetate, 50 ml of the resulting culture The culture was stirred at 30 ° C while adjusting the pH to 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 18 hours. , the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of (S) -3,4-dihydrate was analyzed ethyl oxybutyrate and was found to be 100%. The analysis was performed as follows. The sample was reacted with sodium cyanide in ethane, at room temperature, to obtain 4-cyano-3-hydroxyethyl butyrate, which was then changed to a benzoic ester, using benzolyl chloride under the presence of pyridine The optical purity of the benzoic ester was measured by the method of HPLC chromatography. lH-NMR (CDC13) d (ppm): 1.30 (3H, t), 2.55 (2H, m), 3.18 (1H, br), 3.55 (1H, d), 3.68 (1H, d), 4.15 (1H, s), 4.29 (2H, q); Column: Chiralcel AS (0.46 x 25 cm) manufactured by Daicel Chemical Industries CO. , Ltd .; Column temperature: 252C; Eluent: n-hexane / ethanol 95/5; Flow rate: 1 ml / min .; Detection: 254 nm: Elution time: 19.6 minutes for (S), 22.3 minutes for (R).

Example 22: Synthesis of ethyl 3-hydroxy-2-methylbutyrate from ethyl 2-methyl-3-oxoacetate, using the recombinant E. coli with the enzyme CRD and GDH expressed simultaneously. The HBlOl (pNTSlG) of recombinant E. coli, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37CC for 13 hours. The glucose, 7.5 g and 3.2 mg of NADP and then 4 g of ethyl 2-methyl-3-oxoacetate, were added to 50 ml of the resulting culture. The culture was stirred at 30 ° C while adjusting the pH at 6.5 with a 5M solution of sodium hydroxide, to allow the reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 3.5 mg of the 3-hydroxy-2-methylbutyrate. ethyl. The optical purity of ethyl 3-hydroxy-2-methylbutyrate was analyzed as follows and found to be 91.6%. The analysis was performed as follows. The sample was reacted with sodium cyanide in dimethyl sulfoxide, at room temperature, to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to the benzoic ester using benzoyl chloride, in the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1 H-NMR (CDC13) d (ppm): 1.17 (3H, t), 1.22 (2H, t), 1.28 (3H, t), 2.46 (1H, m), 2.82 (1H, br), 3.90 (1H, m), 4.18 (2H, q). Example 23: Synthesis of ethyl 2-chloro-3-hydroxybutyrate by the reduction of ethyl 2-chloro-3-oxoacetate, using the recombinant E. coli with the enzyme CRD and GDH expressed simultaneously "?" T? Fc. HBlOl (pNTS? G) of recombinant E. coli, obtained in Example 10, was inoculated in 100 ml of a 2 x YT medium, sterilized in a 500 ml Sakaguchi flask, and cultivated with shaking at 37 ° C for 13 hours. The glucose, 6.5 g and 3.2 mg of NADP and then 4 g of the ethyl 2-chloro-3-oxoacetoacetate were added to 50 ml of the resulting culture.The culture was stirred at 302C while the pH was adjusted to 6.5 with a 5M solution. of sodium hydroxide, to allow the reaction for 18 hours After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression and the concentrate was purified by silica gel chromatography, to obtain 3.8 g of ethyl 2-chloro-3-hydroxybutyrate.1H-NMR (CDC13) d (ppm): 1.35 (6H, m), 2.55 (1H, br), 4. 15 (1H, d), 4.25 (1H, m), 4.30 (2H, q). INDUSTRIAL APPLICABILITY Using the novel CRD enzyme, optically active alcohols, such as (S) -4-halo-3-hydroxy-butyric ether, useful as synthetic intermediates for medicines and the like, can be produced efficiently. By cloning the CRD enzyme gene and analyzing its basic sequence, a transformant, which has a high capacity to produce the CRD enzyme, can be obtained. A transformant was also obtained that has high capacity to produce the enzyme CRD and GDH, simultaneously. Using the above transformants, it is possible to carry out synthesis of optically active alcohols, such as the (S) -4-halo-3-hydroxy-butyric ester, from carbonyl compounds, such as the 4-haloacetoacetic ester , more efficiently.

Claims (2)

1. CLAIMS 1. A carbonyl reductase, which has the physical and chemical properties (i) to (iv) of: (i) action: acts on ethyl 4-chloroacetoacetate, which uses NADPH as a coenzyme, to produce the ( S) ethyl 4-chloro-3-hydroxybutyrate; (ii) substrate specificity: exhibits a strong activity to ethyl 4-chloroacetoacetate, while having substantially no activity to ethyl acetoacetate; (iii) Optimum pH: 5.5 to 6.5; and (iv) optimal temperature of action: 50 to 55fiC.
2. 2. A carbonyl reductase, according to claim 1, which also has the physical and chemical properties (v) to (vii) of: (v) heat stability: being stable up to about 40 & C, when processed at a pH of 7.0, for 30 minutes; (vi) inhibition: being inhibited by mercury and quercetin ions; and (vii) molecular weight: approximately 76,000 by gel filtration analysis and approximately 32,000 by electrophoretic analysis of polyacrylamide in SDS. 9. A plasmid, which has a DNA according to claims 7 or 8. 10. A plasmid, according to claim 9, in which this plasmid is pNTSl. 11. A transformant, which is a cell transformed with a plasmid, according to claims 9 or 10. 12. A transformed cell, according to claim 11, wherein the transformed cell is E. coli. 13. A transformed cell, according to claim 11, wherein the transformed cell is from HB101 (pNTSl) of E. coli. 14. A production method of the ester (S) -4-halo-3-hydroxy-butyric, represented by the general formula: (wherein Rx denotes a halogen atom, R 2 denotes hydrogen, and R 3 denotes an alkyl group or an aryl group, substituted or unsubstituted), this method comprises the step of: reacting a 4-haloacetoacetic ester, represented by the formula general: 0 II and an enzyme, according to any one of claims 1 to 3, or a culture of a microbe, which has the ability to produce the enzyme or a processed product of the culture. 15. A method, according to claim 14, wherein the halogen atom is chlorine or bromine and R3 is an alkyl group having 1 to 4 carbon atoms. 16. A method, according to claim 15, wherein the substrate is methyl 4-chloroacetoacetate, ethyl 4-chloroacetoacetate, methyl 4-bromoacetoacetate or ethyl 4-bromoacetoacetate. 17. A method, according to any of claims 14 to 16, wherein the microbe is a microbe belonging to the genus of Candida. 18. A method, according to claim 17, wherein the microbe is Candida magnoliae. 19. A method, according to claim 18, wherein the microbe is Candida magnoliae IFO 0705. 20. A method, according to any of claims 14 to 16, wherein the microbe is a transformed cell, according to any of claims 11 to 13. 21. A method of producing an optically active 3-hydroxy-butyric ester represented by the general formula: this method comprises the steps of: reacting a transformant, which is a cell transformed with a plasmid, having a DNA encoding an enzyme, having an activity of asymmetrically reducing a 3-oxo-butyric ester, represented by the general formula : 0 N to produce an optically active 3-hydroxy-butyric ester represented by the general formula: OH with the 3-oxo-butyric ester, represented by the general formula: OR II and collecting an optically active 3-hydroxy ester ester, represented by the general formula: OH 22. One method, according to the claim 21, in which in the general formulas Ri and R2 are independently halogen, azide, benzylamino or hydrogen, one of Ri and R2 is hydrogen, and R3 is an alkyl group or an aryl group, substituted or unsubstituted, or in the formulas general, Rx and R2 are, independently, an alkyl group, a hydroxide group, or hydrogen, one of R? and R2 is hydrogen, and R3 is an alkyl group or an aryl group, substituted or unsubstituted.23. One method, according to the claim 22, in which, in the general formulas, R. is a hydroxyl group, R is hydrogen and R3 is ethyl. 24. A method, according to claim 22, wherein, in the general formulas, ^ is chlorine, R is hydrogen, and R3 is ethyl. 25. A method, according to any of claims 21 to 24, wherein the transformed cell is a transformed cell according to any of claims 11 to 13. 26. A plasmid, which has a DNA according to claims 7 or 8, and a DNA encoding glucose dehydrogenase. 27. A plasmid, according to claim 26, wherein the glucose dehydrogenase is derived from Bacillus megaterium. 28. A plasmid, according to claim 27, wherein the plasmid is pNTSIG. 29. A transformant, which is a cell transformed with a plasmid, according to any of claims 26 to 28. 30. A transformed cell, according to claim 29, wherein the transformed cell is E.coli. 31. A transformed cell, according to claim 30, wherein the transformed cell is from HBlOl (pNTSlG) of E. coli. 32. A method for the production of an optically active alcohol, comprising the steps of: reacting a transformant, which is a cell transformed with a plasmid, having a DNA encoding an enzyme, having an activity of asymmetrically reducing a carbonyl compound, to produce an optically active alcohol, and a DNA encoding an enzyme, which has the ability to regenerate a coenzyme, on which the enzyme depends, with a carbonyl compound; and collect the optically produced active alcohol. 33. A method, according to claim 32, wherein the enzyme having the ability to regenerate a coenzyme is the glucose dehydrogenase. 34. A method, according to claim 32, wherein the transformed cell is a transformed cell according to any of claims 29 to 31. 35. A method, according to any of claims 32 to 34, wherein the carbonyl compound is a 3-oxo-butyric ether, represented by the general formula: 0 II and the resulting optically active alcohol is an optically active 3-hydroxy-butyric ester represented by the general formula: OH 36. One method, according to the claim 35, in which in the general formulas Rx and R2 are independently halogen, azide, benzylamino or hydrogen, one of Rj. and R2 is hydrogen, and 3 is an alkyl group or an aryl group, substituted or unsubstituted, or in the general formulas, R? and R2 are, independently, an alkyl group, a hydroxide group, or hydrogen, one of. and R2 is hydrogen, and R3 is an alkyl group or an aryl group, substituted or unsubstituted. 37. One method, according to the claim 36, in which Ri is chlorine, R2 is hydrogen, and R3 is ethyl. 38. A transformant, which is a cell transformed with a first plasmid, having a DNA according to claims 7 or 8, and a second plasmid, having a DNA encoding the glucose dehydrogenase. 39. A cell, transformed, according to claim 38, wherein the first plasmid is pNTSl and the glucose dehydrogenase is derived from Bacillum megaterium. 40. A transformed cell, according to claim 38 or 39, wherein the transformed cell is E. coli. 41. A method for the production of an optically active alcohol, comprising the steps of: reacting a transformant, which is a cell transformed with a first plasmid, having a DNA encoding an enzyme, with the one activity of asymmetrically reducing a compound carbonyl, to produce an optically active alcohol, and a second plasmid, which has a DNA encoding an enzyme, with the ability to regenerate a coenzyme, on which the enzyme depends, with a carbonyl compound; and collect the optically produced active alcohol. 42. A method, according to claim 41, wherein the enzyme having the ability to regenerate a coenzyme is the glucose dehydrogenase. 43. A method, according to claim 41, wherein the transformed cell is a transformed cell according to any of claims 38 to 40. 44. A method, according to any of claims 41 to 43, wherein the carbonyl compound is a 3-oxo-butyric ether, represented by the general formula: 0 II and the resulting optically active alcohol is an optically active 3-hydroxy-butyric ester represented by the general formula: 45. One method, according to the claim 44, in which, in the general formulas, R? and R2 are, independently, halogen, azide, benzylamino or hydrogen, one of R.}. and R is hydrogen, and R3 is an alkyl group or an aryl group, substituted or unsubstituted, or, in the general formulas, Rj and R2 are, independently, an alkyl group, a hydroxyl group, or hydrogen, one of R? and R 2 is hydrogen, and R 3 is an alkyl group or an aryl group, substituted or unsubstituted. 46. One method, according to the claim 45, in which, in the general formulas, Rj is chloro, R2 is hydrogen, and R3 is ethyl.