JP2010279272A - New carbonyl reductase; gene, vector, and transformant thereof; and method for producing optically active alcohol by utilization of them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide: a new carbonyl reductase; a gene thereof; a vector containing the gene; a transformant transformed with the vector; and a method for producing an optically active alcohol by the utilization of them. <P>SOLUTION: There are provided a new polypeptide isolated from Candida tenuis; a DNA encoding the polypeptide; a transformant producing the polypeptide; and a method for producing an optically active alcohol, comprising reducing a carbonyl compound with the polypeptide or the transformant. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel carbonyl reductase, its gene, a vector containing the gene, a transformant transformed with the vector, and a method for producing an optically active alcohol using them.

  Optically active alcohols are useful compounds as synthetic raw materials and intermediates for pharmaceuticals and agricultural chemicals. As a method for producing an optically active alcohol, a method in which the carbonyl group of a carbonyl compound is asymmetrically reduced by a microorganism or an enzyme is known. Enzymes that asymmetrically reduce carbonyl compounds (hereinafter referred to as carbonyl reductases) are useful because they can be used in the production of various optically active alcohols.

  Optically active 3-quinuclidinol is a compound useful as a synthetic raw material and intermediate for pharmaceutical products. As a method for producing optically active 3-quinuclidinol, a method of asymmetric reduction of a carbonyl group of 3-quinuclidinone by a microorganism or an enzyme is known, and many microorganisms serving as an enzyme source have been reported (Patent Documents 1, 2, 3, 4). However, production by such a microbial reduction method is low in productivity, and the product isolation is often complicated due to impurities derived from bacterial cells, side reactions by other enzymes, and the like. On the other hand, if a structural gene encoding a carbonyl reductase (hereinafter referred to as quinuclidinone reductase) that produces optically active 3-quinuclidinol is obtained and quinuclidinone reductase can be mass-produced by using genetic engineering techniques, high production will be achieved. It is possible to produce optically active 3-quinuclidinol efficiently and efficiently. However, as for the structural gene encoding quinuclidinone reductase, only those derived from the following microorganisms or plants are known.

  Examples of genes encoding quinuclidinone reductase that generates R-quinuclidinol include Rhodotolura rubra JCM3782, Datura stramon, Hyoscyamus niger, Agrobacterium tumefaciens ( Agrobacterium tumefaciens) NK-1 strain is known (Patent Documents 5, 6, and 7).

Japanese Patent Laid-Open No. 10-243795 Japanese Patent No. 3891522 JP 2002-153293 A Japanese Patent No. 3858505 JP 2007-124922 A JP 2003-230398 A JP 2008-212144 A

  An object of the present invention is to provide a novel carbonyl reductase, a gene thereof, a vector containing the gene, a transformant transformed with the vector, and a method for producing an optically active alcohol using the same.

  As a result of intensive studies on the above problems, the present inventors have discovered a novel carbonyl enzyme and found that the enzyme can be used for the production of various optically active alcohols such as (R) -3-quinuclidinol. It came to be completed.

  The present invention has one or more of the following features.

One feature of the present invention is a polypeptide according to any of the following (a) to (c):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added, and acting on 3-quinuclidinone in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, A polypeptide having an activity of reducing to 3-quinuclidinol;
(C) It consists of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and has an activity of acting on 3-quinuclidinone to reduce it to R-form 3-quinuclidinol. Polypeptide.

Another feature of the present invention is a polypeptide having the physicochemical properties shown in the following (1) to (6):
(1) Action:
A carbonyl group in at least one carbonyl compound is reduced to produce an alcohol. NADPH or NADH can be used as a coenzyme;
(2) Substrate specificity:
It acts on 3-quinuclidinone and is reduced to R form 3-quinuclidinol. Alternatively, it acts on methyl 3-oxopentanoate and reduces to S-form methyl 3-hydroxypentanoate. Alternatively, it acts on ethyl cyclopentanone-2-carboxylate to reduce to 1R-2S ethyl cyclopentanol-2-carboxylate;
(3) Molecular weight:
Is a monomeric protein and exhibits a molecular weight of about 35,000 in SDS polyacrylamide electrophoresis;
(4) Optimum pH:
The optimum pH of action is in the range of pH 6-8;
(5) Optimal temperature:
The optimum temperature of action is 20 ° C to 40 ° C;
(6) Enzyme activity is inhibited by mercury chloride and p-chloromercurybenzoic acid, but not inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, o-phenanthroline and dithiothreitol.

  Another feature of the present invention is DNA encoding the polypeptide.

Another feature of the present invention is the following DNA (A), (B), (C) or (D), a vector containing the DNA, and a transformant obtained by transforming a host cell with the vector: :
(A) DNA containing the base sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) Hybridizes under stringent conditions with DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing and acts on 3-quinuclidinone to reduce to R-form 3-quinuclidinol DNA encoding a polypeptide having the activity of:
(C) Encodes a polypeptide that exhibits 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing and has an activity of acting on 3-quinuclidinone to reduce to R-quinuclidinol. DNA;
(D) In the base sequence shown in SEQ ID NO: 2 in the sequence listing, consists of a base sequence in which one or more bases are deleted, inserted, substituted and / or added, and acts on 3-quinuclidinone, A DNA encoding a polypeptide having an activity of reducing to 3-quinuclidinol.

  Another feature of the present invention is an alcohol, in particular an optically active alcohol, characterized in that the transformant into which the polypeptide of the present invention or the DNA of the present invention has been introduced and its treated product are allowed to act on a compound having a carbonyl group. It is a manufacturing method.

  The present invention provides a novel carbonyl reductase, its gene, a vector containing the gene, a transformant transformed with the vector, and a method for producing an optically active alcohol using the same.

It is a construction figure of the recombinant vector concerning Example 4 and Example 5 of the present invention.

  Hereinafter, the present invention will be described in detail using embodiments. In addition, this invention is not limited by these.

Physical properties of the polypeptide of the present invention The polypeptide isolated by the method described later in the present invention is a polypeptide having the following physicochemical properties (1) to (6).

(1) Action The polypeptide of the present invention has the ability to reduce a carbonyl compound and convert it to an alcohol compound in the presence of NADPH or NADH. The ability to reduce a carbonyl compound can be evaluated, for example, by the following method.

<Evaluation method of reducing ability for carbonyl compounds>
Reaction containing NADPH or NADH 0.25 mM, carbonyl compound 1-50 mM whose reduction activity is to be evaluated and polypeptide of the present invention in 100 mM potassium phosphate buffer (pH 6.5) containing 0.3% (v / v) dimethyl sulfoxide The progress of the reduction reaction can be easily evaluated by reacting the solution at 30 ° C. and measuring the decrease in absorbance at a wavelength of 340 nm accompanying the decrease in the amount of NADPH or NADH. When the absorbance decreases, it can be determined that the peptide of the present invention has the ability to reduce the carbonyl compound to be evaluated. In addition, it can be said that the reduction ability with respect to the evaluation carbonyl compound is so high that the rate of decrease in absorbance is high. In addition, the reducing ability of the polypeptide can be quantified, and the reducing activity 1U is the amount of enzyme that catalyzes the consumption of 1 μmol NADPH per minute.

(2) Substrate specificity The polypeptide of the present invention can use a carbonyl compound as a substrate for the reduction reaction. This can be evaluated by the method described in <Method for evaluating reducing ability with respect to carbonyl compound> described in the above (1) action. The polypeptide of the present invention has the ability to act on 3-quinuclidinone and reduce it to R-form 3-quinuclidinol. This can be confirmed, for example, by the following method.

<Evaluation method of reducing ability for 3-quinuclidinone>
0.1M 3-quinuclidinone hydrochloride, 0.1M NADPH and the polypeptide of the present invention are added to 0.5M phosphate buffer (pH 6.5) and reacted at 30 ° C. After the reaction, an extraction operation is performed with an organic solvent such as dichloromethane, and analysis under the following gas chromatography conditions can confirm the configuration and optical purity of the produced 3-quinuclidinol. It is also possible to quantify the reducing ability of the polypeptide from the following formula. The reduction activity of 1 U was defined as the amount of enzyme that produced 1 μmol of 3-quinuclidinol per minute.

  3-quinuclidinone reduction activity (U) = ((R) -3-quinuclidinol area area + (S) -3-quinuclidinol area area) ÷ (3-quinuclidinone area area + (R) -3-quinuclidinol Area area + (S) -3-Quinuclidinol area area) × 3-quinuclidinone amount used in the reaction (μmol) ÷ reaction time (min).

<Gas chromatography analysis conditions (1)>
Column: Gamma DEX (30 m, 0.25 mm ID) manufactured by SPELCO
Column temperature: 150 ° C
Inlet temperature: 220 ° C
Detector temperature: 220 ° C
Detection: FID
Carrier gas: helium, flow rate: 100 kPa
Elution time: 3-quinuclidinone (9.8 minutes), (S) -3-quinuclidinol (11.5 minutes), (R) -3-quinuclidinol (12.0 minutes).

(3) Molecular weight Both the molecular weight of the polypeptide of the present invention in SDS polyacrylamide electrophoresis and gel filtration chromatography is about 35,000. Molecular weight measurement using SDS polyacrylamide electrophoresis and gel filtration chromatography is a known method, for example, the method described in “Neo-Chemical Chemistry Experiment Course 1 Protein I Separation / Purification / Property” (published by Tokyo Chemical Dojinsha). Can be implemented. The molecular weight can be calculated from the difference in mobility from the molecular weight standard protein.

(4) About optimal pH The optimal pH range of the polypeptide of this invention is the range of pH 6.0-8.0. Measurement of the optimum pH range can be carried out as follows, for example. Using a Britton-Robinson buffer solution (J. Chem. Soc., 1456 (1931)) having a different pH, the reduction activity against 3-quinuclidinone according to the method described in <Method for evaluating reduction ability against 3-quinuclidinone> described above. Measure. The pH at which the highest activity was 100% and the activity value at each pH was expressed as relative activity, and the pH range where the relative activity value was 70% or more was defined as the optimum pH for action.

(5) About optimal temperature The optimal temperature of action of the enzyme activity of the polypeptide of the present invention is 20 ° C to 40 ° C. The measurement of the optimum temperature for this action can be performed, for example, as follows. In the method described in <Method for evaluating reducing ability with respect to 3-quinuclidinone>, the reducing activity with respect to 3-quinuclidinone is measured by changing the measurement temperature. When the reduction activity value at the temperature having the highest activity is 100%, and the activity value at each temperature is expressed as relative activity, the temperature range in which the relative activity value is 70% or more is defined as the optimum temperature for action. did.

(6) Inhibitor The enzyme activity of the polypeptide of the present invention is inhibited by mercuric chloride and p-chloromercurybenzoic acid, but not by iodoacetic acid, ethylenediaminetetraacetic acid, o-phenanthroline and dithiothreitol. Whether a compound inhibits the enzyme activity of a polypeptide can be evaluated by, for example, the following method. A 0.5M phosphate buffer solution (pH 6.5) containing various compounds at a concentration of 0.01 to 1 mM is prepared, and this buffer solution is used as described in <Method for evaluating reducing ability for carbonyl compounds>. The reduction activity against ethyl 4-chloroacetoacetate is measured by the method. When the activity value in the presence of various compounds showed a value of 30% or less of the activity value in the absence of various compounds, it was evaluated that the added compound inhibited the enzyme activity of the polypeptide. In addition, when the activity value in the presence of various compounds shows 90% or more of the activity value in the absence of various compounds, the added compound does not inhibit the enzymatic activity of the polypeptide. It was evaluated.

<About isolation of the polypeptide of the present invention>
The polypeptide of the present invention is a polypeptide having the activity of reducing a compound having a carbonyl group to produce an alcohol, preferably a polypeptide having the activity of asymmetrically reducing an asymmetric ketone to produce an optically active alcohol, Most preferably, it can be selected from polypeptides having the activity of asymmetrically reducing 3-quinuclidinone to produce (R) -3-quinuclidinol.

  Such a polypeptide can be isolated from organisms such as microorganisms having the activity. The enzyme can be found from microorganisms by the following method, for example. The microorganism is cultured in an appropriate medium, and after collection, 3-quinuclidinone is reacted in a buffer solution in the presence of nutrients such as glucose. After the reaction, extraction with a solvent or the like is performed, and the generation of (R) -3-quinuclidinol may be confirmed by analysis under the conditions described in <Gas chromatography analysis conditions (1)>.

  As a medium for culturing a microorganism, a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like can be used as long as the microorganism grows. The culture can be performed, for example, by shaking or aeration at a temperature of 25 ° C. to 37 ° C. and a pH of 4 to 8.

  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 cell residue is removed by centrifugation to obtain a cell-free extract. And heat treatment, 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, limiting The polypeptide of the present invention is isolated from the cell-free extract by using a technique such as external filtration alone or in combination.

  The origin of the polypeptide of the present invention is not limited, but is preferably a microorganism belonging to the genus Candida. Preferably, Candida tenuis (Candida tenuis) NBRC0716 strain is more preferable. Such microorganisms can be obtained from the Biological Genetic Resource Department (NBRC: 2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture 292-0818).

<About the amino acid sequence of the polypeptide of the present invention>
Another feature of the present invention is a polypeptide according to any of the following (a) to (c):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added, and acting on 3-quinuclidinone in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, A polypeptide having an activity of reducing to 3-quinuclidinol;
(C) It consists of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and has an activity of acting on 3-quinuclidinone to reduce it to R-form 3-quinuclidinol. Polypeptide.

  Each of (a) to (c) will be described in detail below.

(A) Polypeptide As the amino acid sequence of the polypeptide of the present invention, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing encoded by the base sequence shown in SEQ ID NO: 2 in the sequence listing can be mentioned.

Polypeptide of (b) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing is a Current Protocols in Molecular It can be prepared according to a known method described in Biology (John Wiley and Sons, Inc., 1989) and the like, and is included in the polypeptide as long as it has the physicochemical properties of the polypeptide of the present invention.

  In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the place where amino acids are substituted, inserted, deleted and / or added is not particularly limited. Here, the highly conserved region represents a position where amino acids are matched between a plurality of sequences when amino acid sequences are optimally aligned and compared for a plurality of enzymes having different origins. The highly conserved region can be confirmed by comparing the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of a known microorganism-derived carbonyl reductase using a tool such as GENETYX.

  The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one type (for example, substitution) of modification, or two or more types of modification (for example, substitution and substitution). Insertion).

In the case of substitution, the amino acid to be substituted is preferably an amino acid having a property similar to that of the amino acid before substitution (cognate amino acid). Here, amino acids within the same group of the following groups are considered homologous amino acids:
(Group 1: neutral nonpolar amino acids) Gly, Ala, Val, Leu, Ile, Met, Cys, Pro, Phe;
(Group 2: neutral polar amino acids) Ser, Thr, Gln, Asn, Trp, Tyr;
(Group 3: acidic amino acids) Glu, Asp;
(Group 4: basic amino acids) His, Lys, Arg.

  The “plural amino acids” described above mean, for example, 20 amino acids, preferably 15 amino acids, more preferably 10, more preferably 5, 4, 3, or 2 amino acids. To do.

(C) Polypeptide When the polypeptide having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has physicochemical properties of the polypeptide of the present invention, this is also the case of the present invention. Included in the polypeptide. A polypeptide having 85% or more homology with the amino acid sequence of SEQ ID NO: 1 in the sequence listing is included in the polypeptide of the present invention, and the homology thereof is preferably 90% or more, more preferably 95% or more, and 98% The above is more preferable, and 99% or more is most preferable.

  The homology of the amino acid sequence is determined by comparing the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing with the amino acid sequence to be evaluated, dividing the number of amino acid positions in both sequences by the total number of amino acids compared, It is expressed as a value multiplied by 100.

  As long as it has the physicochemical properties of the polypeptide of the present invention, an additional amino acid sequence can be bound to the amino acid sequence shown in SEQ ID NO: 1. For example, a tag sequence such as a histidine tag or an HA tag can be added. Alternatively, it can be a fusion protein with another protein. Moreover, as long as it has the physicochemical properties of the polypeptide of the present invention, it may be a peptide fragment.

<Cloning of DNA encoding the polypeptide of the present invention>
The DNA encoding the polypeptide of the present invention may be any DNA as long as it can express the enzyme in a host cell introduced according to the method described below, and may contain any untranslated region. If the enzyme can be obtained, such DNA can be obtained from a living organism that is the source of the enzyme by a method known to those skilled in the art. For example, it can be acquired by the method shown below.

  Unless otherwise specified, genetic manipulations such as DNA cloning, vector preparation and transformation described later in this specification are described in documents such as Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989). Can be implemented by any method. Further,% used in the description of the present specification means% (w / v) unless otherwise specified.

  First, the polypeptide of the present invention isolated by the method described in “Isolation of the polypeptide of the present invention” is digested with an appropriate endopeptidase, and the resulting peptide fragment is subjected to reverse phase HPLC. Sort. Then, for example, part or all of the amino acid sequences of these peptide fragments are determined by a PPSQ-33A type protein sequencer (manufactured by Shimadzu Corporation).

  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, Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems Japan Co., Ltd.).

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

  Examples of the DNA encoding the polypeptide of the present invention thus obtained include DNA containing the base sequence shown in SEQ ID NO: 2 in the sequence listing.

  Hereinafter, the base sequence shown in SEQ ID NO: 2 will be described.

<Regarding the DNA sequence encoding the polypeptide of the present invention>
As DNA encoding the polypeptide of the present invention, for example, DNA comprising the base sequence shown in SEQ ID NO: 2 in the sequence listing, or DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing And DNA that hybridizes under stringent conditions.

  Here, “DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing” means the base sequence shown in SEQ ID NO: 2 in the sequence listing DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like under stringent conditions using a DNA consisting of a base sequence complementary to DNA as a probe.

  Hybridization can be performed according to the method described in Molecular Cloning, A laboratory manual, second edition (Cold Spring Harbor Laboratory Press, 1989). Here, “DNA that hybridizes under stringent conditions” means, for example, a high-temperature DNA at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After hybridization, the filter is washed at 65 ° C. using a 2 × concentration SSC solution (the composition of the 1 × concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). The DNA which can be raised can be mention | raise | lifted. It is preferably washed with an SSC solution of 0.5 times concentration at 65 ° C., more preferably washed with an SSC solution of 0.2 times concentration at 65 ° C., and more preferably washed with an SSC solution of 0.1 times concentration at 65 ° C. DNA that can be obtained by

  Although the hybridization conditions have been described as described above, the conditions are not particularly limited. A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  The DNA that can hybridize under the above conditions is 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, with the DNA shown in SEQ ID NO: 2. Most preferably, 98% or more of the DNA can be mentioned, and the encoded polypeptide is included in the DNA as long as it has the physicochemical properties of the polypeptide of the present invention.

  Here, “sequence identity (%)” means that the two DNAs to be compared are optimally aligned, and the nucleobases (eg, A, T, C, G, U, or I) match in both sequences. The number of positions is divided by the total number of comparison bases, and the result is expressed by a value obtained by multiplying by 100.

  Sequence identity can be calculated, for example, using the following sequence analysis tool: GCG Wisconsin Package (Program Manual for The Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Medison, Wisconsin, USA) 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1RQ, England) and the ExPASy World Wide Web Molecular Biology Server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

<About host-vector system and transformant>
A polypeptide expression vector can be prepared by inserting a DNA encoding the polypeptide of the present invention into an expression vector. The polypeptide of the present invention can be expressed by culturing a transformant obtained by transforming a host organism with this polypeptide expression vector. Furthermore, a method of introducing a polynucleotide encoding the polypeptide of the present invention into a chromosome can also be used.

  The expression vector used above is not particularly limited as long as it can express the polypeptide encoded by the DNA in a suitable host organism. 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.

  For example, in the case of Escherichia coli, such a vector usually contains regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc., and operates with the DNA of the present invention. It can be suitably used as an expression vector comprising expression units that are ligated together. Examples thereof include pUCN18 (see Example 4), pSTV28 (manufactured by Takara Bio Inc.), pUCNT (WO94 / 03613) and the like.

  As used herein, the term “regulatory element” refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  As used herein, 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.

  Vectors and promoters that can be used in various organisms are described in detail in "Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan".

  As long as the host organism used for expressing each polypeptide is an organism that is transformed with a polypeptide expression vector containing DNA encoding each polypeptide and can express the polypeptide encoded by the introduced DNA, There is no particular limitation. Examples of usable microorganisms include, for example, the genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, Bacteria whose host vector systems are developed such as Streptococcus genus and Lactobacillus genus, Actinomycetes and Saccharomyces whose host vector systems are developed such as Rhodococcus genus and Streptomyces genus Genus (Saccharomyces), genus Kluyveromyces, genus Schizosaccharomyces, genus Zygosaccharomyces, genus Yarrowia, genus Trichosporon, genus Rhodosporidium (Rhosporidium) Host vector systems such as (Pichia) and Candida Yeast being developed, Neurospora (Neurospora) genus Aspergillus (Aspergillus) genus Cephalosporium (Cephalosporium) genus, and Trichoderma (Trichoderma) sp host-vector systems molds being developed, such as, and the like. In addition to microorganisms, various host / vector systems have been developed for plants and animals, especially in insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system for expressing a large amount of heterologous protein has been developed and can be suitably used. Among these, bacteria are preferable from the introduction and expression efficiency, and Escherichia coli is particularly preferable.

  A polypeptide expression vector containing a DNA encoding the polypeptide of the present invention can be introduced into a host microorganism by a known method. For example, when using the plasmid pNCT (see Example 4), which is a vector of the present invention in which the DNA shown in SEQ ID NO: 2 is introduced into the above-described expression vector pUCN18 as a polypeptide expression vector and Escherichia coli is used as a host microorganism, a commercially available E . E. coli HB101 competent cell (manufactured by Takara Bio Inc.) and the like, and a transformant in which the vector is introduced into a host cell by operating according to the protocol; E. coli HB101 (pNCT) (see Example 6).

  Moreover, the transformant which expressed both the polypeptide of this invention and the polypeptide which has the reductive coenzyme reproduction | regeneration ability mentioned later in the same microbial cell can also be bred. That is, the DNA encoding the polypeptide of the present invention and the DNA encoding the polypeptide having the ability to regenerate reduced coenzyme are incorporated into the same vector and introduced into a host cell. Can be obtained by incorporating each of these DNAs into two different vectors of different incompatibility groups and introducing them into the same host cell. As the transformant thus obtained, for example, both the DNA shown in SEQ ID NO: 2 and the DNA encoding glucose dehydrogenase, which is a polypeptide having the ability to regenerate reduced coenzyme, are expressed as described above. PNCTG (see Example 5), a recombinant vector introduced into the vector pUCN18, was transformed into E. coli. E. coli which is a transformant introduced into E. coli HB101 competent cell (Takara Bio Inc.). E. coli HB101 (pNCTG) (see Example 6).

<Method for producing alcohol using polypeptide or transformant>
(Reaction conditions)
When an alcohol is produced by reducing a compound having a carbonyl group using the polypeptide of the present invention or a transformant expressing the polypeptide of the present invention, the method can be carried out as follows. However, it is not necessarily limited to the following method.

An appropriate solvent such as 100 mM phosphate buffer (pH 6.5) is added with a substrate that is a carbonyl compound, such as 3-quinuclidinone, a coenzyme such as NADPH or NADP ⁺ , and a culture of the transformant. And / or its treated product is added, and the mixture is reacted with stirring under pH adjustment.

  Here, the treated product is, for example, a crude extract, cultured cells, freeze-dried organisms, acetone-dried organisms, disrupted cells, or immobilized products thereof, and the enzyme catalytic activity of the polypeptide remains. Means what you are doing.

  This reaction is performed at a temperature of 5 to 80 ° C., preferably 10 to 60 ° C., more preferably 20 to 40 ° C., and the pH of the reaction solution during the reaction is 3 to 10, preferably 4 to 9, more preferably 5 to 5. 8 is maintained. The reaction can be carried out batchwise or continuously. In the case of a batch system, the reaction substrate may be added at a charge concentration of 0.01 to 100% (w / v), preferably 0.1 to 70%, more preferably 0.5 to 50%. Further, a substrate may be newly added during the reaction.

  In the reaction, an aqueous solvent may be used, or an aqueous solvent and an organic solvent may be mixed and used. Examples of the organic solvent include toluene, ethyl acetate, n-butyl acetate, hexane, isopropanol, diisopropyl ether, methanol, acetone, dimethyl sulfoxide and the like.

  Here, the treated product of the transformant means, for example, a crude enzyme solution, cultured cells, freeze-dried cells, acetone-dried cells, a ground product thereof, a mixture thereof, or the like. Furthermore, they can be used by immobilizing them with a known means in the form of polypeptides or cells. In addition, when this reaction is performed, a transformant that produces both the polypeptide of the present invention and the polypeptide having the ability to regenerate reduced coenzyme, for example, E. coli. If E. coli HB101 (pNCTG) (see Example 6) is used, the amount of coenzyme used can be greatly reduced. Next, the polypeptide having the ability to regenerate reduced coenzyme will be described in detail.

<Polypeptide having reductive coenzyme regeneration ability>
When a carbonyl compound is reduced to synthesize an alcohol compound using a transformant capable of producing the polypeptide of the present invention, NADPH or NADH is required as a coenzyme. As described above, the reaction can be carried out even if NADPH or NADH is added to the reaction system in a necessary amount. However, an enzyme having the ability to convert the oxidized coenzyme (NADP ⁺ or NAD ⁺ ) to reduced NADPH or NADH (hereinafter referred to as reduced coenzyme regeneration ability) together with its substrate, that is, a coenzyme regeneration system By performing the reaction in combination with the polypeptide of the present invention, the amount of expensive coenzyme used can be greatly reduced. Examples of the enzyme having the ability to regenerate reduced coenzyme include hydrogenase, formate dehydrogenase, carbonyl reductase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase. Preferably, glucose dehydrogenase is used.

  Such a reaction can also be carried out by adding a coenzyme regeneration system into the asymmetric reduction reaction system, but it encodes a DNA encoding the enzyme of the present invention and a polypeptide having a reduced coenzyme regeneration ability. When a transformant transformed with both DNAs is used as a catalyst, the reaction can be carried out efficiently without separately preparing an enzyme capable of regenerating reduced coenzyme and adding it to the reaction system. . Such a transformant can be obtained by the method described in the above-mentioned “Regarding Host-Vector System and Transformant”. For example, pNCTG (a recombinant vector in which both the DNA shown in SEQ ID NO: 2 and the DNA encoding glucose dehydrogenase, which is a polypeptide having the ability to regenerate reduced coenzyme, are introduced into the expression vector pUCN18 described above. See Example 5). E. coli which is a transformant introduced into E. coli HB101 competent cell (Takara Bio Inc.). E. coli HB101 (pNCTG) (see Example 6).

<Compounds having a carbonyl group and the resulting alcohol>
When an alcohol is produced by reducing a compound having a carbonyl group using the polypeptide of the present invention or a transformant in which the polypeptide of the present invention is expressed, there is no limitation on the carbonyl compound serving as the substrate. When the compound having a carbonyl group is an asymmetric ketone, the product becomes a useful optically active alcohol, which is a very beneficial reaction.

  Examples of the compound having a carbonyl group include the following formula (1):

(Wherein R ¹ and R ² are a hydrogen atom, a halogen atom, an optionally substituted alkyl group, an optionally substituted aralkyl group, an optionally substituted aryl group, or an optionally substituted alkoxy; Group, a hydroxyl group, an amino group, or a nitro group, or R ¹ and R ² may be bonded to each other to form a ring (provided that R ¹ and R ² have different structures). An asymmetric ketone, the product of which is represented by the following formula (2):

The method for producing an alcohol according to claim 13, wherein R ¹ and R ² are the same as those described above, and * represents an asymmetric carbon.

  The substituents mentioned above include a halogen atom, hydroxyl group, carboxyl group, amino group, cyano group, nitro group, alkyl group, aryl group, aralkyl group, alkoxy group and the like. Moreover, the halogen atom said above is a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.

R ¹ and R ² are alkyl groups having 1 to 14 carbon atoms, aryl groups having 6 to 14 carbon atoms, heteroaryl groups having 4 to 14 carbon atoms, alkoxy groups having 1 to 5 carbon atoms, and 2 to 5 carbon atoms. Alkoxy carboxyl group, straight or branched alkyl group having 1 to 5 carbon atoms, alkenyl group having 2 to 5 carbon atoms, cycloalkyl group having 5 to 10 carbon atoms, heterocycloalkyl group having 4 to 9 carbon atoms, carboxyl group , A hydrogen atom, a halogen atom, a hydroxyl group, an amino group, or a nitro group is preferable. As the asymmetric ketone as the substrate, 3-quinuclidinone, ethyl 4-chloroacetoacetate, methyl 3-oxopentanoate, and ethyl cyclopentanone-2-carboxylate are particularly preferable.

<Alcohol isolation and purification method>
The method for collecting alcohol from the reaction solution after the reaction is not particularly limited, but it is extracted directly from the reaction solution or after separation of the cells, etc., with a solvent such as ethyl acetate, toluene, t-butyl methyl ether, hexane, methylene chloride or the like. Then, after dehydration, if purified by distillation, recrystallization, silica gel column chromatography or the like, a high-purity alcohol compound can be easily obtained.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these. Detailed operation methods relating to the recombinant DNA technology used in the following examples are described in the following documents:
Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989),
Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).

(Example 1) Purification of polypeptide According to the following method, 3-quinuclidinone is asymmetrically reduced from Candida tenuis NBRC0716 strain and has the activity of producing (R) -3-quinuclidinol. Polypeptides were isolated and purified to single. Unless otherwise noted, purification operations were performed at 4 ° C. Moreover, the reduction activity with respect to 3-quinuclidinone was carried out by the method described in <Method for evaluating reduction ability with respect to 3-quinuclidinone>.

(Microbial culture)
A liquid medium (composition of 10 g of meat extract, 10 g of peptone, 5 g of yeast extract, 3 g of sodium chloride, 0.1 g of Adecanol LG-109 (manufactured by NOF Corporation) (all per 1 L) in a 5 L mini jar fermenter ( pH 7) 3000 ml was prepared and steam sterilized at 120 ° C. for 20 minutes. This medium was inoculated with 30 ml of the Candida tenuis NBRC0716 strain pre-cultured in the same medium, and cultured for 72 hours with stirring at 28 ° C., 0.3 vvM, 350 rpm. It was.

(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 1 mM dithiothreitol (hereinafter referred to as DTT) and 10% glycerol, and multi-bead shocker MB501P (S) type (manufactured by Yasui Instruments Co., Ltd.). ), And the cell residue was removed by centrifugation to obtain a cell-free extract.

(Ammonium sulfate fraction)
Ammonium sulfate was added to the cell-free extract obtained above to a final concentration of 1.6 M and stirred for 1 hour, and then the precipitate was removed by centrifugation. Ammonium sulfate was added to the supernatant to a final concentration of 3.2 M, and after stirring for 1 hour, a precipitate was obtained by centrifugation. This precipitate was dissolved in 10 mM phosphate buffer (pH 7.0) containing 1 mM DTT and 10% glycerol, and dialyzed against the same buffer overnight.

(DEAE-TOYOPEARL column chromatography)
DEAE-TOYOPEARL 650M (manufactured by Tosoh Corporation) column (400 ml) in which the active fraction of the dialyzed ammonium sulfate fraction was pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1 mM DTT and 10% glycerol. After washing the column with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 0.1 M).

(Blue-Sepharose column chromatography)
The active fraction of the dialyzed ammonium sulfate fraction was applied to a Blue-Sepharose 6 Fast Flow column (30 ml) pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1 mM DTT and 10% glycerol. After the column was washed with the same buffer, the active fraction was eluted with a linear gradient of phosphate buffer (from 10 mM to 1 M) to obtain a purified preparation of a single polypeptide electrophoretically. This enzyme had the activity of reducing 3-quinuclidinone to produce (R) -quinuclidinol. The activity when NADPH was used as a coenzyme was 4.1 times that when NADH was used.

(Example 2) Physicochemical properties of Candida tenuis-derived quinuclidinone reductase The physicochemical properties of Candida tenuis-derived quinuclidinone reductase obtained as described above were examined. Each activity was measured by the method described in <Method for evaluating reduction ability for 3-quinuclidinone> or <Method for evaluating reduction ability for carbonyl compound>.

(Substrate specificity)
Dissolve the substrate carbonyl compound to a final concentration of 2 mM and coenzyme NADPH to a final concentration of 0.25 mM in 100 mM phosphate buffer (pH 6.5) containing 0.3% (v / v) dimethyl sulfoxide. did. An appropriate amount of the purified Candida tenuis-derived quinuclidinone reductase 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 ethyl 4-chloroacetoacetate was taken as 100%. As is apparent from Table 1, Candida tenuis-derived quinuclidinone reductase showed reducing activity against a wide range of carbonyl compounds.

  Table 1: Substrate specificity of reductive activity of quinuclidinone reductase from Candida tenuis

(Optimum pH)
Using a Britton-Robinson buffer having a different pH, the reducing activity against 3-quinuclidinone is measured by the method described in <Method for evaluating reducing ability against 3-quinuclidinone>. When the reduction activity at pH 7, which had the highest activity, was 100%, the reduction activity at each pH was calculated as a relative activity and summarized in Table 2. The pH range in which the relative activity value was 70% or more was pH 6 to pH 8.

  Table 2: Optimum pH for reduction activity of 3-quinuclidinone by Candida tenuis-derived quinuclidinone reductase

(Optimum temperature)
Of the standard reaction conditions, only the temperature was changed and the reduction activity of 3-quinuclidinone was measured. The reduction activity at each temperature when the reduction activity at 35 ° C., which had the highest activity, was taken as 100%, was calculated as a relative activity and summarized in Table 3. The temperature range in which the relative activity value was 70% or more was 20 ° C to 40 ° C.

  Table 3: Optimum temperature of reduction activity of 3-quinuclidinone from Candida tenuis-derived quinuclidinone reductase

(Inhibitor)
Candida tenuis-derived quinuclidinone reductase was measured for reducing activity against ethyl 4-chloroacetoacetate by the method described in (Substrate specificity) in the presence of various reagents shown in Table 4 and 0.5% dimethyl sulfoxide. The reduction activity in the presence of each reagent when the activity value to which no reagent was added was 100% was calculated as relative activity, and is summarized in Table 4. The enzyme activity of Candida tenuis-derived quinuclidinone reductase was inhibited by mercury chloride and p-chloromercurybenzoic acid, but not by iodoacetic acid, ethylenediaminetetraacetic acid, o-phenanthroline, or dithiothreitol.

  Table 4: Inhibitors of reductive activity of Candida tenuis-derived quinuclidinone reductase against ethyl 4-chloroacetoacetate

(Molecular weight)
The molecular weight of Candida tenuis-derived quinuclidinone reductase in SDS polyacrylamide electrophoresis and gel filtration chromatography was calculated to be about 35,000 from the difference in elution time of the molecular weight standard protein.

(Example 3) Acquisition of DNA encoding quinuclidinone reductase derived from Candida tenuis (Preparation of PCR primers)
The purified Candida tenuis-derived quinuclidinone reductase 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 peptide fragment was determined by a PPSQ-33A type protein sequencer (manufactured by Shimadzu Corporation). Primer 1: 5′-CAACAYTACGCHGACGACATYAAC-3 ′ (SEQ ID NO: 3 in the sequence listing) for amplifying a part of the gene encoding quinuclidinone reductase by PCR based on the DNA sequence predicted from this amino acid sequence, and Primer 2: 5′-CATRAGTTGGGTGTGTCRTGRAC-3 ′ (SEQ ID NO: 4 in the sequence listing) was synthesized.

(Amplification of genes by PCR)
Using Example 1 Candida-Tenuisu cultured in the same manner as (Candida tenuis) NBRC0716 strain Gen GenTLE ^TM from cells of (Takara Bio Inc.), chromosomal DNA was extracted according to the manufacturer's instructions. 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 0.2 kbp that was 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 directly sequenced using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems Japan Co., Ltd.) and Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems Japan Co., Ltd.), and the base sequence was analyzed. The resulting base sequence is shown in SEQ ID NO: 5 in the sequence listing.

(Determining the full-length sequence of the target gene by inverse PCR method)
The chromosomal DNA of Candida tenuis NBRC0716 strain prepared above was completely digested with restriction enzymes XhoI or NspV, and the resulting DNA fragment mixture was intramolecularly cyclized with T4 ligase. Using this as a template, the entire base sequence of the Candida tenuis-derived quinuclidinone reductase gene containing the base sequence shown in SEQ ID NO: 5 was obtained by inverse PCR (Nucl. Acids Res., 16, 8186 (1988)). Were determined. The results are shown in SEQ ID NO: 2 in the sequence listing. Inverse PCR was performed using Prime Star GXL (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: 2 is shown in SEQ ID NO: 1.

Example 4 Construction of Recombinant Vector pNCT Using Primer 3: 5′-GGAGTCCATATGTCTACTCCCGTCGTATTT-3 ′ (SEQ ID NO: 6 in the Sequence Listing), Primer 4: 5′-ATATACGCAGCTAGTACACACCCGGTTTCT-3 ′ (SEQ ID NO: 7 in the Sequence Listing) PCR was performed using the chromosomal DNA of Candida tenuis NBRC0716 strain 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: 2 in the sequence listing and a PstI recognition site was added immediately after the termination codon was obtained. PCR was performed using Prime Star (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual.

The DNA fragment obtained by the above PCR was digested with NdeI and PstI, and the 185th T of the plasmid pUCN18 (pUC18 (manufactured by Takara Bio Inc., GenBank Accession No. L09136) was modified to A by the PCR method to the NdeI site. And the 471-472th GC is modified with TG to insert the NdeI site downstream of the lac promoter into a recombinant vector pNCT. Built. The preparation method and structure of pNCT are shown in FIG.

(Example 5) Construction of recombinant vector pNCTG further containing glucose dehydrogenase gene Primer 4: 5'-AAACTACTGCAGAGGAAACAAACAATGTATAAAAG-3 '(SEQ ID NO: 8 in the sequence listing) and primer 5: 5'-ACGCAAGCTTTTATCCGCGTCCTGCTTGG-3' PCR was performed using plasmid pGDK1 (obtained and prepared by those skilled in the art according to the method described in Eur. J. Biochem., 186, 389 (1989)) as a template using Bacillus megaterium (SEQ ID NO: 9) of the Sequence Listing. Bacillus megaterium) IAM1030-derived glucose dehydrogenase (hereinafter referred to as GDH) gene, an Escherichia coli ribosome-binding sequence is added 5 bases upstream from the start codon, and a PstI recognition site is added immediately before it. Immediately after HindIII recognition Double-stranded DNA with a site added was obtained.

  The obtained DNA fragment was digested with PstI and HindIII and inserted between the PstI recognition site and HindIII recognition site downstream of the Candida tenuis-derived quinuclidinone reductase gene of the plasmid pNCT described in Example 4, and the recombinant vector pNCTG Built. The preparation method and structure of pNCTG are shown in FIG.

Example 6 Production of Transformant Using the recombinant vector pNCT constructed in Example 4, E. coli was transformed. E. coli HB101 competent cells (manufactured by Takara Bio Inc.) E. coli HB101 (pNCT) was obtained.

  Similarly, using the recombinant vector pNCTG constructed in Example 5, E. coli HB101 competent cells (manufactured by Takara Bio Inc.) E. coli HB101 (pNCTG) was obtained.

  Further, as a comparative example, the plasmid pUCN18 used for the construction of the recombinant vector was used to construct E. coli. E. coli HB101 competent cells (manufactured by Takara Bio Inc.) coli HB101 (pUCN18) was obtained.

(Example 7) Expression of DNA in transformant Each of the three types of transformants obtained in Example 6 was treated with 2 x YT medium (1.6% tryptone, 1% yeast extract) containing 200 µg / ml ampicillin. 0%, 0.5% NaCl, pH 7.0) was inoculated and cultured at 37 ° C. for 24 hours with shaking. The cells were collected by centrifugation and suspended in 5 ml of 100 mM phosphate buffer (pH 6.5). This was crushed using a UH-50 type ultrasonic dispersing machine (manufactured by SMT Co., Ltd.), and then the cell residue was removed by centrifugation to obtain a cell-free extract. The cell-free extract was measured for 3-quinuclidinone reduction activity, GDH activity, and FDH activity by Candida tenuis-derived quinuclidinone reductase.

  The reduction activity for 3-quinuclidinone was carried out by the method described in <Method for evaluating reduction ability for 3-quinuclidinone> described above. GDH activity was determined by adding 0.1 M glucose, 2 mM coenzyme NADP, and crude enzyme solution to 1 M Tris-HCl buffer (pH 8.0), reacting at 25 ° C. for 1 minute, and increasing absorbance at a wavelength of 340 nm. Calculated. Under these reaction conditions, the enzyme activity for reducing 1 μmol of NADP to NADPH per minute was defined as 1 U.

  Candida tenuis-derived quinuclidinone reductase and GDH activity are summarized in Table 5 as specific activities. As shown in Table 5, in any of the two types of transformants obtained in Example 6 except for the comparative example, it has 3-quinuclidinone reducing activity and is a Candida tenuis-derived quinuclidinone reductase. Expression was observed. In addition, E. coli containing the GDH gene. In E. coli HB101 (pNCTG), GDH expression was observed.

(Example 8) Transformant Production of (R) -3-quinuclidinol using E. coli HB101 (pNCTG) A culture solution was obtained by culturing E. coli HB101 (pNCTG) in the same manner as in Example 7. Glucose 1.6 g, NADP ⁺ 2 mg, 3-quinuclidinone hydrochloride 1.28 g was added to 100 ml of the culture solution, and the mixture was stirred at 30 ° C. for 24 hours while adjusting to pH 6.5 by dropwise addition of 5N aqueous sodium hydroxide solution. did. After completion of the reaction, the reaction solution was extracted with n-butanol, and the obtained organic layer was dried over anhydrous sodium sulfate. After removing sodium sulfate, the organic solvent was distilled off under reduced pressure to obtain 0.8 g of (R) -3-quinuclidinol. The optical purity was 98.9% e.e. when measured under <Gas Chromatography Analysis Conditions (1)> described above. e. Met.

(Example 9) Transformant Production of methyl (S) -3-hydroxypentanoate using E. coli HB101 (pNCT) After culturing E. coli HB101 (pNCT) in the same manner as in Example 7, cell disruption with an ultrasonic homogenizer was performed to obtain 100 ml of a cell-free extract. To 100 ml of this cell-free extract, 100 U of glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Inc.), 1.7 g of glucose, and 10 mg of NADP ⁺ were added and stirred at 30 ° C. To this was added 1.0 g of methyl 3-oxopentanoate, and 5N aqueous sodium hydroxide solution was added dropwise to adjust the pH to 6.5, and stirring was continued at 30 ° C. After reaction for 20 hours, the reaction solution was extracted with ethyl acetate, and the obtained organic layers were combined and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the organic solvent was distilled off under reduced pressure to obtain 0.9 g of methyl (S) -3-hydroxypentanoate. The optical purity of this product was 92.0% e.e. e. Met. In addition, the production amount of (S) -3-hydroxypentanoic acid methyl was determined by analyzing under the following gas chromatography conditions.

<Gas chromatography analysis conditions (2)>
Column: InertCap5 (30 m × 0.25 mm) manufactured by GL Science Co., Ltd.
Detection: FID
Carrier gas: helium, flow rate: 150 kPa
Column temperature: 70 ° C
Elution time: methyl 3-oxopentanoate 9.4 minutes, methyl 3-hydroxypentanoate 8.4 minutes.

  The optical purity of the produced methyl (S) -3-hydroxypentanoate was measured by HPLC analysis after derivatization with phenyl isocyanate, and the derivatization of methyl (S) -3-hydroxypentanoate is a reaction. Methyl (S) -3-hydroxypentanoate was extracted from the solution with ethyl acetate, pyridine, phenyl isocyanate and 4-dimethylaminopyridine were added, and the mixture was stirred at 60 ° C. for 1 hour. After washing with 1N hydrochloric acid, the product was purified by preparative thin layer chromatography, dissolved in ethanol, and analyzed under the following high performance liquid chromatography conditions.

<High-performance liquid chromatography analysis conditions (1)>
Column: Daicel Chemical Industries, Ltd. Chiralpak OD-H (250 mm × 4.6 mm
Eluent: n-hexane / 2-propanol = 97.5 / 2.5
Flow rate: 1.0 ml / min
Detection: 245nm
Elution time: R-form 18.5 minutes, S-form 26.4 minutes.

(Example 10) Transformant Production of ethyl (S) -4-chloro-3-hydroxybutanoate using E. coli HB101 (pNCT) After culturing E. coli HB101 (pNCT) in the same manner as in Example 7, cell disruption with an ultrasonic homogenizer was performed to obtain 100 ml of a cell-free extract. To 100 ml of this cell-free extract, 100 U of glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Co., Ltd.), 2.0 g of glucose, and 10 mg of NADP ⁺ were added and stirred at 30 ° C. To this was added 1.0 g of ethyl 4-chloroacetoacetate, and stirring was continued at 30 ° C. while adjusting the pH to 6.5 by dropwise addition of 5N aqueous sodium hydroxide solution. After reaction for 20 hours, the reaction solution was extracted with ethyl acetate, and the obtained organic layers were combined and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the organic solvent was distilled off under reduced pressure to obtain 0.6 g of ethyl (S) -4-chloro-3-hydroxybutanoate. The optical purity of this product is 76.5% e.e. e. Met. In addition, the production amount of ethyl (S) -4-chloro-3-hydroxybutanoate was determined by analyzing under the following gas chromatography conditions.

<Gas chromatography analysis conditions (3)>
Column: InertCap5 (30 m × 0.25 mm) manufactured by GL Science Co., Ltd.
Detection: FID
Carrier gas: helium, flow rate: 150 kPa
Column temperature: 100 ° C
Elution time: ethyl 4-chloroacetoacetate 9.8 minutes, ethyl 4-chloro-3-hydroxybutanoate 11.4 minutes.

  Further, the optical purity of the produced ethyl (S) -4-chloro-3-hydroxybutanoate was measured by HPLC analysis after derivatization with 3,5-dinitrobenzoyl chloride. Derivatization of methyl acid is carried out by extracting methyl (S) -3-hydroxypentanoate from the reaction solution with ethyl acetate, adding pyridine, acetone, 3,5-dinitrobenzoyl chloride and 4-dimethylaminopyridine, and then at room temperature. This was carried out by stirring for 1 hour. After washing with 1N hydrochloric acid, the product was purified by preparative thin layer chromatography, dissolved in ethanol, and analyzed under the following high performance liquid chromatography conditions.

<High-performance liquid chromatography analysis conditions (2)>
Column: Daicel Chemical Industries, Ltd. Chiralpak AD-H (250 mm × 4.6 mm
Eluent: n-hexane / ethanol = 3/7
Flow rate: 1.0 ml / min
Detection: 245nm
Elution time: S form 10.5 minutes, R form 20.5 minutes.

(Example 11) Transformant Preparation of ethyl (1R, 2S) -cyclopentanol-2-carboxylate using E. coli HB101 (pNCTG) After culturing E. coli HB101 (pNCT) in the same manner as in Example 7, cell disruption with an ultrasonic homogenizer was performed to obtain 100 ml of a cell-free extract. To 100 ml of this cell-free extract, 100 U of glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Co., Ltd.), 2.0 g of glucose, and 10 mg of NADP ⁺ were added and stirred at 30 ° C. To this, 1.0 g of ethyl cyclopentanone-2-carboxylate was added, and stirring was continued at 30 ° C. while adjusting the pH to 6.5 by dropwise addition of 5N aqueous sodium hydroxide solution. After reaction for 20 hours, the reaction solution was extracted with ethyl acetate, and the obtained organic layers were combined and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the organic solvent was distilled off under reduced pressure to obtain 0.2 g of ethyl (1R, 2S) -cyclopentanol-2-carboxylate. This product has a cis / trans ratio of 99.5% and an optical purity of 99.3% e.e. e. Met. In addition, the production amount and cis / trans ratio of ethyl (1R, 2S) -cyclopentanol-2-carboxylate were determined by analyzing under the following gas chromatography conditions. In addition, cis / trans ratio was calculated | required from the following formula.

<Gas chromatography analysis conditions (4)>
Column: ULBON HR20-M (25 m × 0.25 mm) manufactured by Shinwa Chemical
Detection: FID
Carrier gas: helium, flow rate: 150 kPa
Column temperature: 130 ° C
Elution time: ethyl cyclopentanone-2-carboxylate 9.5 minutes, ethyl cyclopentanol-2-carboxylate cis form 8.7 minutes, trans form 17.0 minutes,
cis / trans ratio (%) = cis body area area ÷ (cis body area area + trans body area area) × 100.

  Moreover, the optical purity of the produced ethyl (1R, 2S) -cyclopentanol-2-carboxylate was purified by preparative thin layer chromatography, dissolved in ethanol, and then subjected to the following high performance liquid chromatography conditions. analyzed.

<High-performance liquid chromatography analysis conditions (3)>
Column: Daicel Chemical Industries, Ltd. Chiralcel OD-H (250 mm × 4.6 mm
Eluent: n-hexane / 2-propanol = 98/2
Flow rate: 1.0 ml / min
Detection: 220nm
Elution time: (1R, 2S) body 8.4 minutes, (1S, 2R) body 11.5 minutes, (1R, 2R) body 12.8 minutes, (1S, 2S) body 13.9 minutes.

Claims (15)

The polypeptide according to any one of the following (a) to (c):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added, and acting on 3-quinuclidinone in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, A polypeptide having an activity of reducing to 3-quinuclidinol;
(C) It consists of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and has an activity of acting on 3-quinuclidinone to reduce it to R-form 3-quinuclidinol. Polypeptide. Polypeptides having the physicochemical properties shown in the following (1) to (6):
(1) Action:
A carbonyl group in at least one carbonyl compound is reduced to produce an alcohol. NADPH or NADH can be used as a coenzyme;
(2) Substrate specificity:
It acts on 3-quinuclidinone and is reduced to R form 3-quinuclidinol. Alternatively, it acts on methyl 3-oxopentanoate and reduces to S-form methyl 3-hydroxypentanoate. Alternatively, it acts on methyl cyclopentanone-2-carboxylate to reduce it to 1R-2S ethyl cyclopentanol-2-carboxylate;
(3) Molecular weight:
Is a monomeric protein and exhibits a molecular weight of about 35,000 in SDS polyacrylamide electrophoresis;
(4) Optimum pH:
The optimum pH of action is in the range of pH 6-8;
(5) Optimal temperature:
The optimum temperature of action is 20 ° C to 40 ° C;
(6) Enzyme activity is inhibited by mercury chloride and p-chloromercurybenzoic acid, but not inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, o-phenanthroline and dithiothreitol. The polypeptide according to claim 2, which is derived from a microorganism belonging to the genus Candida. The polypeptide according to claim 3, wherein the microorganism is Candida tenuis. DNA encoding the polypeptide according to any one of claims 1 to 4. The following DNA (A), (B), (C) or (D):
(A) DNA containing the base sequence shown in SEQ ID NO: 2 in the sequence listing;
(B) Hybridizes under stringent conditions with DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing and acts on 3-quinuclidinone to reduce to R-form 3-quinuclidinol DNA encoding a polypeptide having the activity of:
(C) Encodes a polypeptide that exhibits 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing and has an activity of acting on 3-quinuclidinone to reduce to R-quinuclidinol. DNA;
(D) In the base sequence shown in SEQ ID NO: 2 in the sequence listing, consists of a base sequence in which one or more bases are deleted, inserted, substituted and / or added, and acts on 3-quinuclidinone, A DNA encoding a polypeptide having an activity of reducing to 3-quinuclidinol. A vector comprising the DNA according to any one of claims 5 and 6. The vector according to claim 7, further comprising a DNA encoding a polypeptide having the ability to regenerate reduced coenzyme. The vector according to claim 8, wherein the polypeptide having reducible coenzyme regeneration ability is glucose dehydrogenase. A transformant obtained by transforming a host cell with the vector according to claim 7. The transformant according to claim 10, wherein the host cell is Escherichia coli. A polypeptide according to any one of claims 1 to 4, or a transformant according to claim 10 or claim 11 and / or a processed product thereof, which acts on a carbonyl compound. Production method. The production method according to claim 12, wherein the carbonyl compound is an asymmetric ketone and the product is an optically active alcohol. The compound having a carbonyl group is represented by the following formula (1):

(Wherein R ¹ and R ² are a hydrogen atom, a halogen atom, an optionally substituted alkyl group, an optionally substituted aralkyl group, an optionally substituted aryl group, or an optionally substituted alkoxy; A group, an amino group, or a nitro group, or R ¹ and R ² may be bonded to each other to form a ring, provided that R ¹ and R ² have different structures). The product is represented by the following formula (2):

The manufacturing method of Claim 12 which is an optically active alcohol represented by (In formula, R < ¹ >, R < ² > is the same as the above, * represents an asymmetric carbon.). The production method according to claim 14, wherein the compound having a carbonyl group is methyl 3-oxopentanoate, ethyl 4-chloroacetoacetate, ethyl cyclopentanone-2-carboxylate, 3-quinuclidinone or a salt thereof.

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