JP5005672B2 - Novel carbonyl reductase, gene thereof, and method for producing optically active alcohol using them - Google Patents

Description

  The present invention relates to a novel carbonyl reductase, its gene, and a method for producing an optically active alcohol using them.

Optically active alcohols such as β-hydroxyesters are useful compounds as synthetic raw materials and intermediates for agricultural chemicals and pharmaceuticals. Methods are known in which β-ketoesters are asymmetrically reduced using a reductase to produce optically active β-hydroxyesters (Patent Documents 1 and 2, Non-Patent Documents 1 and 2).
JP-A-8-103269 International Publication No. 2003-093477 Kataoka, M .; Et al., Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells, the expression of the hydrated reductase. Microbiol. Biotechnol. 51, 486-490 (1999). Matsuyama, A .; , Practical applications of biocatalysis for the manufacture of chiral alcohols schu as (R) -1,3-butanediol by stereospecific oxidoid. Asymmetric Catalysis on Industrial Scale (Wiley-VCH Verlag), 217-231 (2004).

  An object of the present invention is to provide a novel carbonyl reductase, a gene thereof, and a method for producing an optically active alcohol using them, which are different from the reductases described in the above-mentioned background art.

(1) One feature of the present invention is the following DNA (a) or (b).
(A) DNA containing the base sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) Hybridizes with DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions, and reduces a compound having a carbonyl group to produce an optically active alcohol. DNA encoding a polypeptide having activity.

  (2) One feature of the present invention is the DNA according to (1), which encodes a polypeptide having an activity for asymmetric reduction of β-ketoesters.

  (3) One feature of the present invention is a polypeptide comprising an amino acid sequence encoded by the DNA according to either (1) or (2).

(4) One feature of the present invention is the following polypeptide (a) or (b).
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, and having an activity of reducing a compound having a carbonyl group to produce an optically active alcohol Having a polypeptide.

  (5) One feature of the present invention is DNA encoding the polypeptide according to (4).

  (6) One feature of the present invention is a vector comprising the DNA according to any one of (1), (2), and (5).

  (7) One feature of the present invention is the vector according to (6), wherein the vector adds an NdeI recognition site to the start codon portion of the gene consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing. A double-stranded DNA having an EcoRI recognition site added immediately after the stop codon and a plasmid pUCNT (International Publication No. WO94 / 03613) digested with NdeI and EcoRI, and ligated to each other. This is a vector that is the replacement plasmid pNTBA.

  (8) One feature of the present invention is the vector according to (6), further comprising DNA encoding a polypeptide having reduced coenzyme regeneration activity.

  (9) One feature of the present invention is a transformant obtained by transforming a host cell with the vector according to any one of (6) to (8).

  (10) One aspect of the present invention is the transformant according to (9), wherein the host cell is Escherichia coli.

  (11) One feature of the present invention is to react the polypeptide according to (3) or (4) or the transformant according to (9) or (10) with a compound having a carbonyl group. Is a method for producing an optically active alcohol.

  (12) One feature of the present invention is that the polypeptide according to (3) or (4) or the transformant according to (9) or (10) is reacted with β-ketoesters. It is the manufacturing method of the optically active (beta) -hydroxyester characterized.

  Other features and effects of the present invention will become apparent from the embodiments and examples described below.

  The present invention provides a novel carbonyl reductase, its gene, and a method for producing a useful optically active alcohol using them.

  Hereinafter, the present invention will be described in detail using embodiments. The present invention is not limited to these.

  Hereinafter, the present invention will be described in detail based on embodiments.

1. Polypeptide The “polypeptide” in the embodiment of the present invention is a polypeptide having an activity of reducing a compound having a carbonyl group to produce an optically active alcohol. In addition, the “polypeptide” of the embodiment of the present invention is a polypeptide having an activity of asymmetrically reducing β-ketoesters to produce β-hydroxyesters. Such a polypeptide can be isolated from organisms such as microorganisms having the activity. Examples of the polypeptide of the present invention include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing encoded by the base sequence shown in SEQ ID NO: 1 in the sequence listing. In addition, the compound having the homology (identity) of a certain value or more with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having a carbonyl group is reduced to produce an optically active alcohol. Or a polypeptide having the activity of asymmetrically reducing β-ketoesters to form β-hydroxyesters is equivalent to the polypeptide and is included in the present invention.

Here, the sequence homology can be determined by, for example, the homology search program FASTA (WR Pearlson, WR & Lipman, DJ, Proc. Natl. Acad. Sci. USA, 85 , 2444-2448 ( 1988)) is used to compare and analyze two amino acid sequences, and is represented by an identity value for the entire sequence. As a polypeptide having a homology of a certain value or more with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, the homology with the polypeptide is 70% or more, preferably 80% or more, more preferably 90 % Or more of the polypeptide.

  Such a polypeptide can be obtained, for example, by ligating DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing to an appropriate vector. It can be obtained by introducing it into a suitable host cell and expressing it.

  In addition, according to a known method described in, for example, Current Protocols in Molecular Biology (John Wiley and Sons (1989)), an amino acid substitution, insertion, or deletion is performed on a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. It can also be obtained by causing loss or addition. The number of amino acids causing substitution, insertion, deletion or addition is not limited as long as the polypeptide after the substitution does not lose the above activity, but is preferably 20 amino acids or less, more preferably 15 amino acids or less, More preferably, it is 10 amino acids or less, and most preferably 5, 4, 3, or 2 or less.

  The microorganism that is the origin of the polypeptide of the present invention is not particularly limited, and examples thereof include bacteria belonging to the genus Paenibacillus, and particularly preferable examples include Paenibacillus albei (NBRC3343). The microorganism can be obtained from the Biological Resource Department of the Biotechnology Headquarters, National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture).

  As a medium for culturing the microorganism that is the source of the polypeptide of the present invention, a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients, etc. may be used as long as the microorganism grows. it can.

  Isolation of the polypeptide from the microorganism that is the source of the polypeptide of the present invention can be carried out by appropriately combining known protein purification methods. For example, it can be implemented as follows.

  First, the microorganism is cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation or filtration. The obtained bacterial cells are crushed by an ultrasonic crusher or a physical method using glass beads or the like, and then the microbial cell residue is removed by centrifugation to obtain a cell-free extract. And salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, ultrafiltration The polypeptide of the present invention is isolated from the cell-free extract by using a technique such as these alone or in combination.

2. DNA
The “DNA” of the present invention is a DNA encoding the polypeptide of the above-described embodiment, and any DNA can be used as long as it can express the polypeptide in a host cell introduced according to the method described below. The untranslated region may be included. If the polypeptide can be obtained, a person skilled in the art can obtain the DNA of the present invention from a microorganism that is the origin of the polypeptide by a known method. For example, the DNA of the present invention can be obtained by the method shown below.

  First, the isolated polypeptide of the present invention is digested with an appropriate endopeptidase, and the resulting peptide fragment is fractionated by reverse phase HPLC. Then, for example, part or all of the amino acid sequences of these peptide fragments are determined by an ABI492 type protein sequencer (Applied Biosystems).

Based on the amino acid sequence information thus obtained, a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized. Next, chromosomal DNA of the microorganism that is the origin of the polypeptide is prepared by a conventional DNA isolation method, for example, the method of Visser et al. (Appl. Microbiol. Biotechnol., 53 , 415 (2000)). Using this chromosomal DNA as a template, PCR is carried out using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The base sequence can be determined using, for example, ABI373A type DNA Sequencer (Applied Biosystems).

If the partial base sequence of DNA encoding the polypeptide is clarified, the entire sequence can be determined by, for example, the i-PCR method (Nucl. Acids Res., 16 , 8186 (1988)). .

  Examples of the DNA of the present invention thus obtained include DNA containing the base sequence shown in SEQ ID NO: 1 in the sequence listing. An optically active alcohol, which is a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing and having a carbonyl group The DNA of the present invention also includes a DNA encoding a polypeptide having the activity of producing a β-ketoester or asymmetrically reducing β-ketoesters to produce a β-hydroxyester.

  A DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing and a DNA that hybridizes under stringent conditions are a colony hybridization method, a plaque hybridization method, or a Southern hybridization. When the method or the like is carried out, DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing refers to DNA that specifically forms a hybrid.

  Here, stringent conditions include, for example, 75 mM trisodium citrate, 750 mM sodium chloride, 0.5% sodium dodecyl sulfate, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1 An aqueous solution composed of 15 mM trisodium citrate, 150 mM sodium chloride, and 0.1% sodium dodecyl sulfate after hybridization at 65 ° C. in an aqueous solution composed of% Ficoll 400 (Amersham Biosciences) The conditions under which cleaning is performed at 60 ° C. are used. Preferably, after hybridization under the above conditions, washing is performed at 65 ° C. using an aqueous solution composed of 15 mM trisodium citrate, 150 mM sodium chloride, and 0.1% sodium dodecyl sulfate. More preferably, the washing is performed at 65 ° C. using an aqueous solution composed of 1.5 mM trisodium citrate, 15 mM sodium chloride, and 0.1% sodium dodecyl sulfate. Unless otherwise specified, the isolation of the above-described DNA described in the present specification and the genetic manipulation such as vector preparation and transformation described below, Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press (1989)), etc. It can be carried out by the method described in the book. Further,% used in the description of the present specification means% (w / v) unless otherwise specified.

  As an example of the DNA of the present invention, in addition to the above DNAs, the base sequence shown in SEQ ID NO: 1 has a base sequence in which a base is substituted, inserted, deleted and / or added, and a carbonyl group. Also included is a DNA encoding a polypeptide having the activity of reducing a compound having an activity to produce an optically active alcohol, or the activity of asymmetrically reducing β-ketoesters to produce β-hydroxyesters. The number of bases to be substituted, inserted, deleted and / or added is not limited as long as the polypeptide encoded by the DNA does not lose the activity, but is preferably 50 bases or less, more preferably 30 bases. Hereinafter, it is more preferably 20 bases or less, most preferably 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less.

3. Vector The “vector” of the present invention is not particularly limited as long as it can express the gene encoded by the DNA in a suitable host cell. Examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host strains can also be used.

  Such vectors usually contain regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter and the like, and are operably linked to the DNA of the present invention. It can be suitably used as an expression vector containing a unit. For example, a plasmid pUCNT that can be easily obtained and prepared by those skilled in the art based on the description in International Publication No. WO94 / 03613 can be preferably used.

  The control factor refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  The term “operably linked” means that a gene is operably linked to various regulatory elements such as promoters and enhancers that regulate the expression of the gene. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host. As an example of the vector of the present invention, a plasmid pNTBA described later in which the DNA shown in SEQ ID NO: 1 is introduced into pUCNT can be mentioned.

4). Host cells The host cells described herein include bacteria, yeasts, filamentous fungi, plant cells, animal cells, etc., but bacteria are preferred from the introduction and expression efficiency, and Escherichia coli is particularly preferred. A vector containing the DNA of the present invention can be introduced into a host cell by a known method. When E. coli is used as the host cell, for example, commercially available E. coli. By using an E. coli HB101 competent cell (manufactured by Takara Bio Inc.), the vector can be introduced into a host cell. The bacteriological properties of Escherichia coli HB101 are described in “BIOCHEMICALS FOR LIFE SCIENCE” (Toyobo Co., Ltd., 1993, pages 116-119) and various other known literatures and are well known to those skilled in the art. It is.

5. Transformant The “transformant” of the present invention can be obtained by incorporating DNA encoding the polypeptide of the present invention into the vector and introducing it into a host cell. The “transformant” of the present invention includes not only cultured cells but also processed products thereof. The treated product as used herein means, for example, a cell treated with a surfactant or an organic solvent, a dried cell, a disrupted cell, a crude cell extract or the like, and those immobilized by a known means. As long as the enzymatic activity of the polypeptide of the present invention remains, it is included. Culture of the transformant of the present invention can be performed using a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like as long as it grows. Examples of the transformant of the present invention include E. coli HB101 (pNTBA). Escherichia coli HB101 (pNTBA) has the same mycological properties as Escherichia coli HB101 except that it can produce a specific enzyme by genetic recombination.

6). Reduction reaction The substrate for the reduction reaction may be any compound containing a carbonyl group that can be reduced by the “polypeptide” of the present invention. Β-ketoesters are preferred. Specifically, for example, methyl acetoacetate, ethyl acetoacetate, t-butyl acetoacetate, methyl 4-chloroacetoacetate, ethyl 4-chloroacetoacetate, octyl 4-chloroacetoacetate, ethyl 4-acetoxyacetoacetate, 4-benzoyloxyacetate Examples thereof include ethyl acetate, methyl 2-oxocyclopentanecarboxylate, ethyl 2-oxocyclopentanecarboxylate, acetoacetanilide, o-acetoacetaniside, p-acetoacetaniside, and the like.

  In the reduction reaction, the substrate may be added all at the beginning of the reaction, or may be added in portions as the reaction proceeds. The temperature during the reaction is usually 10 to 60 ° C., preferably 20 to 40 ° C., and the pH during the reaction is in the range of 2.5 to 9, preferably 5 to 9. The amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates. The substrate concentration in the reaction solution is preferably 0.01 to 50% (W / V), more preferably 0.1 to 30% (W / V). The reaction is usually carried out with shaking or stirring with aeration. The reaction time is appropriately determined depending on the substrate concentration, the amount of enzyme source, and other reaction conditions. Usually, it is preferable to set each condition so that the reaction is completed in 2 to 168 hours.

  In order to promote the reduction reaction, reduced nicotinamide / adenine dinucleotide (hereinafter abbreviated as NADH), reduced nicotinamide / adenine dinucleotide phosphate (hereinafter referred to as NADH) generally required for a reduction reaction by a biological method. The reaction can be promoted by adding a coenzyme such as NADPH). In this case, specifically, these are added directly to the reaction solution.

In order to promote the reduction reaction, it is preferable to carry out the reaction in the presence of an enzyme that reduces NAD ⁺ or NADP ⁺ to the respective reduced form and a substrate for reduction, since an excellent result can be obtained. For example, glucose dehydrogenase as an enzyme that reduces to a reduced form, glucose coexists as a substrate for reduction, or formate dehydrogenase as an enzyme that reduces to reduced form, and formic acid as a substrate for reduction .

  When a transformant introduced with both the DNA of the present invention and a DNA encoding an enzyme having coenzyme regeneration ability is used, there is no need to separately prepare a coenzyme regeneration enzyme. In order to obtain the transformant, two DNAs may be introduced separately, or transformation may be performed with a vector containing both DNAs.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited at all by these Examples. In the following description, “%” means “% by weight” unless otherwise specified.

Example 1 Purification of Polypeptide According to the following method, a polypeptide having the activity of asymmetrically reducing acetoacetanilide was isolated from the above-mentioned Paenibacillus albei NBRC3343 and purified in a single manner. Unless otherwise noted, purification operations were performed at 4 ° C.

  The reduction activity against acetoacetanilide was determined by adding 40 mM substrate acetoacetanilide, 0.25 mM coenzyme NADPH, and crude enzyme in 100 mM phosphate buffer (pH 6.5) containing 1.3% (v / v) dimethyl sulfoxide. Was calculated from the rate of decrease in absorbance at a wavelength of 340 nm when added at 30 ° C. for 1 minute. Under these reaction conditions, the activity of oxidizing 1 μmol of NADPH to NADP per minute was defined as 1 unit.

(Microbial culture)
From 5L jar fermenter (manufactured by Maruhishi Bioengineer), meat extract 10g, peptone 10g, yeast extract 5g, sodium chloride 3g, Adecanol LG-109 (manufactured by NOF Corporation) 0.1g (all per liter) 3 L of a liquid medium (pH 7) to be prepared was prepared and steam sterilized at 120 ° C. for 20 minutes. This medium was inoculated with 50 ml of a culture solution of Paenibacillus albei NBRC3343 strain that had been pre-cultured in the same medium, and the stirring speed was 450 rpm, the aeration rate was 0.9 NL / min, and the temperature was 30 ° C. for 40 hours. Culture was performed.

(Preparation of cell-free extract)
Bacteria were collected from the culture broth by centrifugation, and washed with 0.8% aqueous sodium chloride solution. This microbial cell was suspended in 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin, crushed using a SONIFIER 250 ultrasonic crusher (manufactured by BRANSON), and then centrifuged. The cell residue was removed by separation to obtain a cell-free extract.

(Nucleic acid removal)
To the cell-free extract obtained above, a 3.5% aqueous protamine sulfate solution was added, stirred for 30 minutes, and then the precipitate was removed by centrifugation.

(DEAE-TOYOPEARL column chromatography)
The DEAE-TOYOPEARL 650M column (manufactured by Tosoh Corporation) was prepared by equilibrating the cell-free extract subjected to the nucleic acid removal treatment in advance with a 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin. (400 ml) to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 0.5 M).

(Phenyl-TOYOPEARL column chromatography)
Ammonium sulfate is dissolved in the active fraction obtained by DEAE-TOYOPEARL column chromatography to a final concentration of 1.2 M, and 10 mM phosphate buffer containing 1.2 M ammonium sulfate, 5 mM β-mercaptoethanol and 10% glycerin. It was applied to a Phenyl-TOYOPEARL 650M (manufactured by Tosoh Corporation) column (90 ml) equilibrated in advance (pH 7.0) to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a linear ammonium sulfate gradient (1.2 M to 0.2 M). The active fractions were collected and dialyzed overnight against 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin.

(Blue Sepharose column chromatography)
The active fraction obtained by Phenyl-TOYOPEARL column chromatography was pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing β-mercaptoethanol and 10% glycerin. Blue Sepharose 6 Fast Flow (Amersham Bioscience Stock) The product was applied to a column (40 ml) to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 1 M).

(Butyl-TOYOPEARL column chromatography)
Ammonium sulfate was dissolved in the active fraction obtained by Blue Sepharose column chromatography to a final concentration of 1.2 M, and 10 mM phosphate buffer containing 1.2 M ammonium sulfate, 5 mM β-mercaptoethanol and 10% glycerin ( The column was applied to a Butyl-TOYOPEARL 650M (manufactured by Tosoh Corporation) column (10 ml) previously equilibrated with pH 7.0) to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a linear ammonium sulfate gradient (1.2 M to 0.2 M). The active fractions were collected and dialyzed overnight against 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin.

(2 ′, 5′-ADP Sepharose column chromatography)
The active fraction obtained by Butyl-TOYOPEARL column chromatography was pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol and 10% glycerin 2 ′, 5′− The product was applied to an ADP Sepharose (Amersham Biosciences) column (35 ml) to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 0.5 M) to obtain a purified preparation of a single polypeptide electrophoretically.

Example 2 Gene Cloning (Preparation of PCR Primer)
The purified polypeptide obtained in Example 1 was denatured in the presence of 8M urea, and then digested with Achromobacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The amino acid sequence of the obtained peptide fragment was It was determined with an ABI492 type protein sequencer (manufactured by PerkinElmer). Primer 1: 5′-CARGAYCTNACNAAYAARAC-3 ′ (SEQ ID NO: 3 in the sequence listing) for amplifying a part of the gene encoding the polypeptide by PCR based on the DNA sequence predicted from this amino acid sequence, and Primer 2: 5′-ARNGCYTCNGTCATNCCCAT-3 ′ (SEQ ID NO: 4 in the sequence listing) was synthesized.

(Amplification of genes by PCR)
Chromosomal DNA was extracted from Paenibacillus albei NBRC3343 cells cultured in the same manner as in Example 1 according to the method of Visser et al. (Appl. Microbiol. Biotechnol., 53 , 415 (2000)). Next, PCR was performed using the DNA primers 1 and 2 prepared above and the obtained chromosomal DNA as a template. As a result, a DNA fragment of about 0.5 kbp, which is considered to be a part of the target gene, was amplified. PCR was performed using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual. This DNA fragment was cloned into the plasmid pT7Blue T-Vector (Novagen), and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and ABI 373A inc. The sequence was analyzed. The resulting base sequence is shown in SEQ ID NO: 5 in the sequence listing.

(Determination of full-length sequence of target gene by i-PCR method)
The chromosomal DNA of Paenibacillus albei NBRC3343 prepared above was completely digested with the restriction enzyme EcoRI, and the resulting mixture of DNA fragments was intramolecularly cyclized with T4 ligase. Using this as a template, the entire base sequence of the gene containing the base sequence shown in SEQ ID NO: 5 was determined by i-PCR (Nucl. Acids Res., 16 , 8186 (1988)). The result is shown in SEQ ID NO: 1 in the sequence listing. i-PCR was performed using TaKaRa LA Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual. The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

(Example 3) Expression vector construction Primer 3: 5′-ATTAAACATATGAAATTGCAAGATCTTACG-3 ′ (SEQ ID NO: 6 in the sequence listing) and primer 4: 5′-AAAGAATTCTTACTGTGGATTGGTTGTCC-3 ′ (SEQ ID NO: 7 in the sequence listing) PCR was performed using the chromosomal DNA of Paenibacillus albei NBRC3343 obtained in Example 2 as a template. As a result, a double-stranded DNA in which an NdeI recognition site was added to the start codon portion of the gene consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing and an EcoRI recognition site was added immediately after the termination codon was obtained. PCR was performed using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual. This DNA was digested with NdeI and EcoRI and inserted between the NdeI recognition site and EcoRI recognition site downstream of the lac promoter of plasmid pUCNT (International Publication No. WO94 / 03613) to construct a recombinant vector pNTBA.

(Example 4) Preparation of transformant Using the recombinant vector pNTBA constructed in Example 3, E. coli HB101 competent cells (Takara Bio Inc.) were transformed. coli HB101 (pNTBA) was obtained. This was inoculated into 50 ml of 2 × YT medium (tripton 1.6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0) containing 200 μg / ml ampicillin and cultured with shaking at 37 ° C. for 24 hours. . Further, as a comparative example, an E. coli containing plasmid vector pUCNT. E. coli HB101 (pUCNT) was also cultured in the same manner. The cells were collected by centrifugation and suspended in 50 ml of 100 mM phosphate buffer (pH 6.5). These were crushed using a UH-50 type ultrasonic homogenizer (manufactured by SMT), and then the cell residue was removed by centrifugation to obtain a cell-free extract. The cell-free extracts were measured for acetoacetanilide reduction activity. E. coli HB101 (pNTBA) 31.7 U / mg, E. coli. No activity was detected with E. coli HB101 (pUCNT). The protein concentration in the cell-free extract was measured using a protein assay kit (manufactured by BIO-RAD).

(Example 5) Production of ethyl (R) -4-chloro-3-hydroxybutyrate E. coli prepared in the same manner as in Example 4. 1 ml of cell-free extract of E. coli HB101 (pNTBA) cells, 10 mg of ethyl 4-chloroacetoacetate (contained in β-ketoesters), 25 mg of glucose, 0.5 mg of NADP, glucose dehydrogenase “GLUCDH“ Amano2 ”” (Amano) Enzyme Co., Ltd.) 1 mg, 1.0 M phosphate buffer solution (pH 6.5) 0.1 ml was added and shaken at 30 ° C. for 16 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate and derivatized with phenyl isocyanate. This was purified by thin layer chromatography. When optical purity was measured using this derivatized ethyl 4-chloro-3-hydroxybutyrate, it was 99% ee or more. The measurement of the optical purity of ethyl 4-chloro-3-hydroxybutyrate (included in β-hydroxyesters) was carried out by high performance liquid chromatography (column: CHIRALCEL OJ (ID 4.6 mm × 250 mm) manufactured by Daicel Chemical Industries, Ltd.). Performed using a fitted HPLC.

(Example 6) Substrate specificity of polypeptide A 100 mM phosphate buffer (pH 6.5) containing 0.2% (v / v) dimethyl sulfoxide was added with a carbonyl compound serving as a substrate at a final concentration of 1 mM. The enzyme NADPH was dissolved to a final concentration of 0.25 mM. An appropriate amount of the purified polypeptide prepared in Example 1 was added thereto, and the reaction was performed at 30 ° C. for 3 minutes. The reduction activity for each carbonyl compound was calculated from the rate of decrease in absorbance at a wavelength of 340 nm of the reaction solution, and this was expressed as a relative value when the activity for acetoacetanilide was taken as 100%. As is clear from Table 1, the polypeptide of the embodiment of the present invention showed a reducing activity against compounds having a wide range of carbonyl groups.

Claims (11)

The following DNA (a) or (b):
(A) DNA containing the base sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, 75 mM trisodium citrate, 750 mM sodium chloride, 0.5% sodium dodecyl sulfate, 0.1% bovine serum albumin, After hybridization at 65 ° C. in an aqueous solution consisting of 0.1% polyvinylpyrrolidone and 0.1% Ficoll 400, 1.5 mM trisodium citrate, 15 mM sodium chloride, and 0.1% dodecyl sulfate DNA that encodes a polypeptide that has an activity of producing an optically active alcohol by hybridizing under conditions where washing is performed at 60 ° C. using an aqueous solution of a sodium composition and reducing a compound having a carbonyl group . A DNA encoding the polypeptide according to claim 1 and having an activity of asymmetrically reducing β-ketoesters. A polypeptide comprising an amino acid sequence encoded by the DNA according to claim 1. The following polypeptide (a) or (b):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) a polypeptide comprising an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having an activity of reducing a compound having a carbonyl group to produce an optically active alcohol Having a polypeptide. DNA encoding the polypeptide of claim 4. A vector comprising the DNA according to any one of claims 1, 2, and 5. The vector according to claim 6, further comprising DNA encoding a polypeptide having reduced coenzyme regeneration activity. A transformant obtained by transforming a host cell with the vector according to claim 6 or 7 . The transformant according to claim 8 , wherein the host cell is Escherichia coli. A method for producing an optically active alcohol, comprising reacting the polypeptide according to claim 3 or 4 or the transformant according to claim 8 or 9 with a compound having a carbonyl group. A method for producing optically active β-hydroxyesters, comprising reacting the polypeptide according to claim 3 or 4 or the transformant according to claim 8 or 9 with β-ketoesters.