# Patent

## Title
Reductase, gene thereof and method of using the same

## Abstract

The present invention relates to a protein which can asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to industrially advantageously produce an amide or ester compound of corresponding optically-active ortho-substituted mandelic acid, a DNA encoding the protein, a process for producing the protein from the DNA, and a process for asymmetrically reducing an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound.

## Background
This application is a 371 of PCT/JP2008/056258 filed Mar. 24, 2008, which claims priority to Japanese applications: 2007-074376, filed Mar. 22, 2007 and 2008-011299, filed Jan. 22, 2008, respectively.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 13, 2010, is named Q115164.txt and is 18,396 bytes in size.
TECHNICAL FIELD
The present invention relates to a novel reductase and a DNA encoding the same, a recombinant vector containing the DNA, and a transformant transformed with the recombinant vector. The present invention also relates to a process for producing an optically-active mandelic acid compound which is an optically active alcohol, using the novel reductase or the transformant.
BACKGROUND ART
Optically-active ortho-substituted mandelic acid compounds are compounds useful in production of medicines, agricultural chemicals and so on, and production processes using asymmetric reducing reagents or the like have been proposed, but in the process described in International Publication No. WO 0210101, further recrystallization or the like is performed after a reaction in order to increase an optical purity and, also in a reaction described in Organic Letters, 2005, vol. 7, No. 24, 5425-5427, it cannot be necessarily said that an optical yield of the reaction is sufficient. In addition, has not been known a method of asymmetrically reducing a keto group of a phenylglyoxylic acid compound, the group having a substituent at an ortho-position and being in the sterically-complicated environment, to an optically-active mandelic acid compound using a microorganism or the like.
DISCLOSURE OF THE INVENTION
The present invention provides a method of reducing a phenylglyoxalic acid compound having a substituent at an ortho-position to an optically active mandelic acid compound with a good optical yield.
More specifically, the present invention provides a protein which can asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to industrially advantageously produce an amide or ester compound of corresponding optically-active ortho-substituted mandelic acid, a DNA encoding the protein (hereinafter, abbreviated as invented DNA), a transformant comprising the DNA, a process for producing the protein from the DNA, and a process for asymmetrically reducing an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound.
That is, the present invention provides:
1. A DNA comprising any nucleotide sequence of the following a) to d):
a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1;
b) a nucleotide sequence of a DNA: i) wherein the DNA has at least 90% sequence homology with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotide sequence encodes an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce corresponding optically active ortho-substituted mandelic acid;
c) a nucleotide sequence of a DNA: i) wherein the DNA hybridizes under a stringent condition with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotide sequence encodes an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce corresponding optically-active ortho-substituted mandelic acid compound; and
d) the nucleotide sequence of SEQ ID NO:2;
2. A DNA in which a promoter functional in a host cell and the DNA according to the item 1 are operably linked;
3. A recombinant vector comprising the DNA according to the item 1 or 2;
4. A transformant in which the DNA of the item 2 or the recombinant vector of the item 3 has been introduced into a host cell;
5. The transformant according to the item 4, wherein the host cell is a microorganism;
6. The transformant according to the item 4, wherein the host cell is Escherichia coli; 
7. A transformant comprising the DNA of the item 1; 8. A process for producing a transformant comprising a step of introducing the recombinant vector of the item 3 into a host cell;
9. A protein comprising any amino acid sequence of the following a) to e);
a) the amino acid sequence of SEQ ID NO:1;
b) an amino acid sequence: i) which is encoded by a nucleotide sequence of a DNA having at least 90% sequence homology with a DNA comprising the nucleotide sequence of SEQ ID NO:2, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound;
c) an amino acid sequence: i) which is encoded by a nucleotide sequence of a DNA which hybridizes under a stringent condition with a DNA comprising the nucleotide sequence of SEQ ID NO:2, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound;
d) an amino acid sequence: i) in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO:1, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound; and
e) an amino acid sequence: i) which has at least 90% sequence homology with the amino acid sequence of SEQ ID NO:1, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound;
10. A recombinant vector comprising the DNA of the item 1 and a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form;
11. The recombinant vector according to the item 10, wherein the protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form is glucose dehydrogenase;
12. The recombinant vector according to the item 11, wherein the protein having glucose dehydrogenase activity is glucose dehydrogenase derived from Bacillus megaterium; 
13. A transformant, in which any of recombinant vector of the items 10 to 12 has been introduced into a host cell;
14. The transformant according to the item 13, wherein the host cell is a microorganism;
15. The transformant according to the item 13, wherein the host cell is Escherichia coli; 
16. A transformant comprising the DNA of the item 1 and a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form;
17. A process for producing an optically-active alcohol compound comprising reacting the protein of the item 9, or the transformant of any of the items 4 to 7, and 13 to 16, or a treated product thereof with a prochiral carbonyl compound;
18. The process according to the item 17, wherein the prochiral carbonyl compound is an amide or ester compound of the ortho-substituted phenylglyoxalic acid, and the corresponding optically-active alcohol compound is an amide or ester compound of the optically-active ortho-substituted mandelic acid;
19. The process according to the item 18, wherein the amide or ester compound of the ortho-substituted phenylglyoxalic acid is a compound represented by the formula (1):




(wherein R1 represents an optionally substituted amino group, or an optionally substituted alkoxy group, and R2 represents an optionally substituted C1-8 alkyl group),

and the amide or ester compound of the optically-active ortho-substituted active mandelic acid is an optically-active compound represented by the formula (2):





(wherein R1 and R2 are as defined above, and a carbon atom with a symbol is an asymmetric carbon atom); and

20. Stenotrophomonas sp. SC-1 (FERM BP-10785); and the like.
According to the present invention, amide or ester compounds of optically-active ortho-substituted mandelic acid useful as medicines or agricultural chemicals, or intermediates thereof or the like can be produced with a good optical purity.


BEST MODE FOR CARRYING OUT THE INVENTION
First, the invented DNA will be described.
The invented DNA can be obtained from a microorganism having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to a corresponding optically-active ortho-substituted mandelic acid compound, for example, a microorganism belonging to genus Stenotrophomonas such as Stenotrophomonas sp. SC-1 strain (FERM-BP 10785). The invented DNA may be such a natural DNA, or a DNA produced by introducing mutation into such a natural DNA by a method described later (site-directed mutagenesis, mutation treatment etc.).
The Stenotrophomonas sp. SC-1 strain has been deposited in International Patent Organism Depositary (IPOD) National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566 Japan, and the accession number of FERM BP-10785 was assigned (date of original deposit: Feb. 21, 2007). Its mycological properties are as follows.
1. Colony Morphology (30° C., 24 Hours)

    
    
        (1) Cellular morphology: rod-shaped, 0.7 to 0.8×1.5 to 2.0 μm
        (2) Gram staining property: negative
        (3) Presence or absence of spore: absence
        (4) Presence or absence of mobility: presence
    
    


2. Colony Morphology on Nutrient Agar

    
    
        Color of colony: pale yellow
        Shape of colony: circular
        Periphery of colony: entire periphery smooth
        Prominence of colony: lens-like
        Transparency: opaque
    
    


3. Physiological Properties

    
    
        (1) Catalase: positive
        (2) Oxidase: negative
        (3) OF test: positive/negative
    
    


4. Nucleotide Sequence of DNA Encoding 16S Ribosomal RNA
About 500 bp of a nucleotide sequence of 16S ribosomal DNA was amplified by PCR from the Stenotrophomonas sp. SC-1 strain, and the nucleotide sequence was analyzed. Using the resulting nucleotide sequence of the 16S ribosomal DNA, homology retrieval by BLAST was performed and, as a result, the homology was 99.6%, showing highest homology with 16S ribosomal DNA of the Stenotrophomonas rhizophila standard strain. Also in BLAST retrieval using International Nucleotide Sequence Database, highest homology with 16S ribosomal DNA derived from genus Stenotrophomonas was exhibited.
From the above mycological properties, the present bacterium was identified as Stenotrophomonas sp.
The “DNA which hybridizes under a stringent condition with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1” in the invented DNA refers to a DNA such that, in Southern hybridization method described, for example, in “Cloning and Sequence” (supervised by Itaru Watanabe, edited by Masahiro Sugiura, 1989, published by Nouson Bunkasha), (1) it forms a DNA-DNA hybrid with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 by hybridization at 65° C. under a high ion concentration [for example, 6×SSC (900 mM sodium chloride, 90 mM sodium citrate)], and (2) the hybrid is retained even after incubation at 65° C. for 30 minutes under a low ion concentration [for example, 0.1×SSC (15 mM sodium chloride, 1.5 mM sodium citrate)].
Specifically, examples include a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, a DNA comprising a nucleotide sequence in which, in a nucleotide sequence encoding the amino acid sequence of SEQ ID No: 1, apart of nucleotides are deleted, substituted or added, a DNA having at least 90% sequence homology, preferably at least 95% sequence homology, more preferably at least 99% sequence homology with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID No: 1, and the like.
The DNA may be a DNA cloned from naturally occurring DNAs, a DNA in which, in a nucleotide sequence of this cloned DNA, deletion, substitution or addition of a part of nucleotides is artificially introduced, or an artificially synthesized DNA. The sequence homology can be calculated using a tool for sequence analysis such as BESTFIT program supplied by UWGCG Package (Devereux et al (1984) Nucleic Acids Research 12, p 387-395), and PILEUP and BLAST algorism (Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul S. F. (1990) Mol Biol 215:403-10).
The invented DNA can be prepared, for example, as follows.
The invented DNA can be prepared by amplifying a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, a DNA comprising a nucleotide sequence encoding an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO:1 and/or a DNA comprising the nucleotide sequence of SEQ ID NO:2, by preparing a DNA library from a microorganism belonging to genus Stenotrophomonas such as Stenotrophomonas sp. according to conventional genetic engineering procedures (e.g. method described in “New Cellular Technology Experimental Protocol” (edited by the Institute of Medical Science, the University of Tokyo, Anticancer Research Section, Shujunsha, 1993)), and performing PCR employing the prepared DNA library as a template, and using appropriate primers.
In addition, the invented DNA can be prepared by amplifying a DNA comprising the nucleotide sequence of SEQ ID NO: 2 by performing PCR employing the DNA library as a template, and using an oligonucleotide comprising the nucleotide sequence of SEQ ID NO:12 and an oligonucleotide comprising the nucleotide sequence of SEQ ID NO:13 as primers.
Examples of the condition of the PCR include the condition of heating a reaction solution obtained by mixing each 20 μM of four kinds of dNTPs, each 15 pmol of two kinds of oligonucleotide primers, 1.3 U of Taqpolymerase, and a DNA library as a template at 94° C. for 2 minutes, thereafter, performing 10 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (90 seconds), then, 20 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and further retaining the solution at 72° C. for 7 minutes.
In addition, a restriction enzyme recognition sequence or the like may be added to the 5′ end and/or the 3′ end of the primer used in the PCR.
In addition, also by performing PCR employing the DNA library as a template, and using an oligonucleotide comprising a partial nucleotide sequence selected from nucleotide sequences encoding the amino acid sequence of SEQ ID NO:1 (e.g. an oligonucleotide comprising a nucleotide sequence of about 14 or more nucleotides on the 5′-terminal side encoding the amino acid sequence of SEQ ID No:1) or the like and an oligonucleotide of about 14 or more nucleotides comprising a nucleotide sequence complementary to a nucleotide sequence adjacent to a DNA-insertion site of a vector used in construction of the DNA library as primers, a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, a DNA comprising a nucleotide sequence encoding an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO:1, or the like can be amplified to prepare the invented DNA.
The thus amplified DNA is cloned into a vector according to a method described in “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Molecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X or the like, whereby the invented vector can be obtained. Specific examples of the vector used include pUC119 (manufactured by Takara Shuzou), pTV118N (manufactured by Takara Shuzou), pBluescriptII (manufactured by Toyobo), pCR2.1-TOPO (manufactured by Invitrogen), pTrc99A (manufactured by Pharmacia), pKK223-3 (manufactured by Pharmacia) and the like.
In addition, the invented DNA can also be obtained, for example, by hybridizing, as a probe, a DNA comprising a nucleotide sequence of about 15 or more nucleotides having a partial nucleotide sequence selected from nucleotide sequences encoding the amino acid sequence of SEQ ID NO:1 to a DNA library inserted in a vector derived from a microorganism or a phage under the condition described later, and detecting a DNA to which the probe specifically binds.
Examples of a method of hybridizing a probe to a chromosomal DNA or a DNA library include colony hybridization and plaque hybridization, and the method can be selected depending on a kind of a vector used in preparing a library.
When the library used is prepared using a plasmid vector, colony hybridization may be utilized. Specifically, a transformant is obtained by introducing a DNA of a library into a host microorganism, the result transformant is diluted, and then the dilution is seeded on an agar medium, and this is cultured until a colony appears.
When the library used is prepared using a phage vector, plaque hybridization may be utilized. Specifically, a host microorganism and a phage of a library are mixed under the infectable condition, the mixture is further mixed with a soft agar medium, and then the mixture is seeded on an agar medium, and this is cultured until a plaque appears.
Then, in either hybridization, a membrane is placed on an agar medium on which the culturing has been performed, and a transformant or a phage is adsorbed or transferred on the membrane. This membrane is alkali-treated, and subjected to neutralization treatment and, then, a DNA is subjected to fixation on the membrane. More specifically, in the case of plaque hybridization, a nitrocellulose membrane or a nylon membrane (e.g. Hybond-N+ (trade mark, Amersham)) is placed on the agar medium, and this is allowed to stand still for about 1 minute to adsorb or transfer phage particles onto the membrane. Then, the membrane is immersed in an alkali solution (e.g. 1.5 M sodium chloride, 0.5 M sodium hydroxide) for about 3 minutes to lyse the phage particles, whereby a phage DNA is eluted on the membrane, and this is immersed in a neutralization solution (e.g. 1.5 M sodium chloride, 0.5 M Tris-hydrochloric acid buffer pH 7.5) for about 5 minutes. Then, the membrane is washed with a washing solution (e.g. 0.3 M sodium chloride, 30 mM citric acid, 0.2 M Tris-hydrochloric acid buffer pH 7.5) for about 5 minutes, thereafter, the phage DNA is fixed on the membrane, for example, by heating at about 80° C. for about 90 minutes.
The thus prepared membrane is used to perform hybridization using the DNA as a probe. Hybridization can be performed, for example, according to the description of J. Sambrook, E. F. Frisch, T. Maniatis “Molecular Cloning: A Laboratory Manual 2nd edition (1989)” Cold Spring Harbor Laboratory Press or the like.
The DNA used in the probe may be labeled with a radioactive isotope element, or may be labeled with a fluorescent dye.
Examples of a method of labeling the DNA used in the probe with a radioactive isotope element include a method of performing PCR using the DNA used in the probe as a template, by replacing dCTP in a PCR reaction solution with (α-32P) dCTP utilizing, for example, Random Primer Labeling Kit (manufactured by Takara Shuzou).
In addition, when the DNA used in the probe is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Labeling and Detection System manufactured by Amersham can be used.
Hybridization can be performed, for example, as follows.
A pre-hybridization solution containing 450 to 900 mM sodium chloride, 45 to 90 mM sodium citrate, sodium dodecylsulfate (SDS) at a concentration of 0.1 to 1.0% by weight, a denatured non-specific DNA at a concentration of 0 to 200 μl/ml, and optionally containing albumin, Ficoll, polyvinylpyrrolidone or the like at a concentration of 0 to 0.2% by weight, respectively (preferably, a pre-hybridization solution containing 900 mM sodium chloride, 90 mM sodium citrate, 1.0% by weight of SDS and 100 μl/ml denatured Calf-thymus DNA) is prepared at a ratio of 50 to 200 μl per 1 cm2 of the membrane prepared above, and the membrane is immersed in the pre-hybridized solution, and retained at 42 to 65° C. for 1 to 4 hours.
Then, for example, a solution obtained by mixing a hybridization solution containing 450 to 900 mM sodium chloride, 45 to 90 mM sodium citrate, SDS at a concentration of 0.1 to 1.0% by weight, a denatured non-specific DNA at a concentration of 0 to 200 μg/ml, and optionally containing albumin, Ficoll, polyvinylpyrrolidone or the like at a concentration of 0 to 0.2% by weight, respectively (preferably, a hybridization solution containing 900 mM sodium chloride, 90 mM sodium citrate, 1.0% by weight of SDS and 100 μg/ml denatured Calf-thymus DNA), and the probe prepared by the aforementioned method (1.0×104 to 2.0×106 cpm equivalent amount per 1 cm2 of the membrane) is prepared at a ratio of 50 to 200 μl per 1 cm2 of the membrane, and the membrane is immersed in the hybridization solution, and retained at 42 to 65° C. for 12 to 20 hours.
After the hybridization, the membrane is removed, and washed using a washing solution containing 15 to 300 mM sodium chloride, 1.5 to 30 mM sodium citrate and 0.1 to 1.0% by weight of SDS at 42 to 65° C. (preferably, a washing solution containing 15 mM sodium chloride, 1.5 mM sodium citrate and 1.0% by weight of SDS at 65° C.) or the like. The washed membrane is mildly rinsed with 2×SSC (300 mM sodium chloride, 30 mM sodium citrate), and dried. This membrane is subjected to, for example, autoradiography, and a position of the probe on the membrane is detected, whereby a clone corresponding to a position on the membrane of a DNA which hybridizes with the probe used is specified on the original agar medium, and this is picked up, whereby a clone having the DNA is isolated.
From a cultured bacterial cell obtained by culturing the thus obtained clone, the invented DNA can be prepared.
The DNA prepared as described above is cloned into a vector according to a method described in “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Molecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X or the like, whereby the invented recombinant vector can be obtained. Examples of the vector used include, pUC119 (manufactured by Takara Shuzou), pTV118N (manufactured by Takara Shuzou), pBluescriptII (manufactured by Toyobo), pCR2.1-TOPO (manufactured by Invitrogen), pTrc99A (manufactured by Pharmacia), pKK223-3 (manufactured by Pharmacia) and the like.
In addition, a nucleotide sequence of the DNA can be analyzed by a dideoxy terminator method described in F. Sanger, S. Nicklen, A. R. Coulson, Proceeding of Natural Academy of Science U.S.A. (1977) 74: 5463-5467, or the like. For preparing a sample for nucleotide sequence analysis, a commercially available reagent such as ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit of Perkin Elmer may be used.
Confirmation that the DNA obtained as described above encodes an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound, typically, an amide or ester compound thereof (e.g. 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide) to a corresponding optically-active ortho-substituted mandelic acid compound, typically, an amide or ester compound thereof (e.g. (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide) can be performed, for example, as follows.
First, the DNA obtained as described above is inserted into a vector so that it can be linked to the downstream of a promoter functional in a host cell as described later, and this vector is introduced into a host cell to obtain a transformant. Then, a culture of the transformant is made to act on an ortho-substituted phenylglyoxalic acid compound (e.g. an amide or ester compound of that compound, specifically, for example, 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide). By analyzing an amount of a corresponding optically-active ortho-substituted mandelic acid compound (e.g. an amide or ester compound thereof, specifically, for example, (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide) in the reaction product, it can be confirmed that the resulting DNA encodes an amino acid sequence of a protein having such ability.
In order to express the invented DNA in a host cell, for example, a DNA in which a promoter functional in a host cell and the invented DNA are operably linked is introduced into a host cell.
Herein, the “operably” means that upon transformation of a host cell by introduction of the DNA into a host cell, the invented DNA is in the state where it is bound to a promoter so as to be expressed under control of the promoter. Examples of the promoter include a promoter of the lactose operon of Escherichia coli, a promoter of the tryptophan operon of Escherichia coli, and a synthetic promoter functional in Escherichia coli such as a tac promoter and a trc promoter, and a promoter which controls expression of the invented DNA in Stenotrophomonaes sp. may be utilized.
Generally, a recombinant vector obtained by inserting a DNA operably linked to a promoter functional in a host cell into the aforementioned vector is introduced into a host cell. When a vector including a selection marker gene (e.g. an antibiotic resistance imparting gene such as a kanamycin resistant gene, a neomycin resistant gene etc.) is used as a vector, a transformant into which the vector is introduced can be selected using a phenotype of the selection marker gene or the like as an index.
Examples of the host cell into which the invented DNA operably linked to a promoter functional in a host cell, or the invented recombinant vector is introduced include a microorganism belonging to genus Escherichia, genus Bacillus, genus Corynebacterium, genus Staphylococcus, genus Streptomyces, genus Saccharomyces, genus Kluyveromyces, genus Pichia, genus Rhodococcus and genus Aspergillus. 
A method of introducing the invented DNA operably linked to a promoter functional in a host cell, or the invented recombinant vector into a host cell may be a conventionally used introduction method depending on a host cell used, and examples include a calcium chloride method described in “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Molecular Biology” (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X or the like, and an electroporation method described in “Methods in Electroporation: Gene Pulser/E. coli Pulser System” Bio-Rad Laboratories, (1993) or the like.
In order to select a transformant into which the invented DNA operably linked to a promoter functional in a host cell, or the invented recombinant vector has been introduced, for example, the transformant may be selected using, as an index, a phenotype of a selection marker gene contained in the vector.
Confirmation that the transformant harbors the invented DNA can be performed by confirmation of a restriction enzyme site, analysis of a nucleotide sequence, Southern hybridization, Western hybridization or the like according to conventional methods described in “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press or the like.
Then, the invented protein will be described.
As the “amino acid in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO:1”, an amino acid sequence in which one amino acid is deleted, substituted or added in the amino acid sequence of SEQ ID NO:1 is preferable.
The invented protein can be produced, for example, by culturing a transformant comprising the invented DNA.
As a medium for culturing the transformant, for example, various media appropriately containing a carbon source or a nitrogen source, an organic salt or an inorganic salt, or the like which are normally used in culturing a host cell such as a microorganism can be used.
Examples of the carbon source include sugars such as glucose, dextrin and sucrose, sugar alcohols such as glycerol, organic acids such as fumaric acid, citric acid and pyruvic acid, animal oils, vegetable oils and beewax. The amount of these carbon sources to be added to a medium is usually around 0.1 to 30% (w/v) relative to the culturing solution.
Examples of the nitrogen source include natural organic nitrogen sources such as meat extract, peptone, yeast extract, malt extract, soybean powder, corn steep liquor, cottonseed powder, dry yeast and casamino acid, amino acids, ammonium salts of inorganic acids such as sodium nitrate, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, and urea. Among them, ammonium salts of organic acids, natural organic nitrogen sources, amino acids and the like can also be used as a carbon source in many cases. The amount of these nitrogen sources to be added to a medium is usually around 0.1 to 30% (w/v) relative to the culturing solution.
Examples of the organic salts or the inorganic salts include chlorides, sulfates, acetates, carbonates and phosphates of potassium, sodium, magnesium, iron, manganese, cobalt, zinc and the like. Specific examples thereof include sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acatate, potassium carbonate, monopotassium hydrogen phosphate and dipotassium hydrogen phosphate. The amount of these organic salts and/or inorganic salts to be added to a medium is usually around 0.0001 to 5% (w/v).
Further, in the case of a transformant into which a gene in which a type of a promoter induced by allolactose such as a tac promoter, a trc promoter and a lac promoter, and the invented DNA are linked operably, as an induction agent for inducing production of the invented protein, for example, isopropyl thio-β-D-galactoside (IPTG) may be added to a medium at a small amount.
Culturing of a transformant comprising the invented DNA can be performed according to a method which is conventionally used in culturing a host cell such as a microorganism, and examples thereof include liquid culturing and solid culturing such as tube shaking culturing, reciprocating shaking culturing, jar fermenter culturing, tank culturing and the like.
A culturing temperature can be appropriately changed in such a range that the transformant can be grown, and is usually about 15 to 40° C. A pH of a medium is preferably in a range of about 6 to 8. A culturing time is different depending on the culturing condition and, usually, about 1 day to about 5 days is preferable.
As a method of purifying the invented protein from a culture of the transformant harboring the invented DNA, a method which is normally used in purification of a protein can be applied, and, for example, the following methods can be exemplified.
First, after cells are collected from a culture of the transformant by centrifugation or the like, these cells are ground by a physical grinding method such as ultrasonic treatment, dino mill treatment and French press treatment, or a chemical grinding method using surfactants or a bacteriolytic enzyme such as lysozyme. A cell-free extract is prepared by removing impurities from the resulting grinding solution by centrifugation, membrane filter filtration or the like, and this is fractionated by appropriately using a separation purification method such as cationic exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and metal chelate chromatography, whereby the invented protein can be purified.
Examples of a carrier used in chromatography include an insoluble polymer carrier such as cellulose, dextrin and agarose into which a carboxymethyl (CM) group, a diethylaminoethyl (DEAE) group, a phenyl group or a butyl group is introduced. A commercially available carrier-filled column may be used, and examples of the commercially available carrier-filled column include Q-Sepharose FF, Phenyl-Sepharose HP (trade name, both manufactured by Amersham Pharmacia Biotech), TSK-gel G3000SW (trade name, manufactured by Tosoh) and the like.
In order to select a fraction containing the invented protein, for example, 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide is asymmetrically reduced, and the fraction may be selected using, as an index, the ability to preferentially produce (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.
The transformant of the present invention, and an treated product thereof can be used for asymmetrically reducing the ortho-substituted phenylglyoxalic acid compound represented by the formula (1) (e.g. an ester or amide compound thereof) which is a prochiral carbonyl compound to produce an optically active ortho-substituted mandelic compound represented by the formula (2) (e.g. an amide or ester compound thereof) which is an optically active alcohol compound.
In the ortho-substituted phenylglyoxalic acid compound of the formula (1), and the ortho-substituted mandelic acid compound of the formula (2), a substituent represented by R1 will be described below.
Examples of an optionally substituted amino group represented by R1 include, in addition to an amino group, a C1-6 alkylamino group such as a methlyamino group, an ethylamino group, a propylamino group, an isopropylamino group, a butylamino group, an isobutylamino group, a t-butylamino group, a pentylamino group, and a hexylamino group. In addition, examples of an optionally substituted alkoxy group include a C1-8 alkoxy group such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, a hexyloxy group, a heptyloxy group and an octyloxy group.
Then, a substituent represented by R2 will be described below.
Examples of a C1-8 alkyl group in an optionally substituted C1-8 alkyl group represented by R2 include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group and an octyl group.
Examples of a substituent of such a C1-8 alkyl group include an optionally substituted alkoxy group and an optionally substituted aryloxy group.
Examples of an optionally substituted alkoxyalkyl group represented by R2 include a C1-8 alkyl group substituted with a C1-4 alkoxy group such as a methoxymethyl group, a methoxyethyl group, a methoxypropyl group, a methoxybutyl group, a methoxypentyl group, a methoxyhexyl group, a methoxyheptyl group, a methoxyoctyl group, an ethoxymethyl group, an ethoxyethyl group, an ethoxypropyl group, an ethoxybutyl group, an ethoxypentyl group, an ethoxyhexyl group, an ethoxyheptyl group, an ethoxyoctyl group, a propoxymethyl group, a propoxyethyl group, a propoxypropyl group, a propoxybutyl group, a propoxypentyl group, a propoxyhexyl group, a propoxyheptyl group, a propoxyoctyl group, a butoxymethyl group, a butoxyethyl group, a butoxypropyl group, a butoxybutyl group, a butoxypentyl group, a butoxyhexyl group, a butoxyheptyl group and a butoxyoctyl group.
Examples of an optionally substituted aryloxy group include an aryloxy group represented by the formula (3):




(wherein A, B and C are the same or different from one another, and represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloakly group, a cycloalkenyl group, an alkoxy group, a halogenated alkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aminoalkyl group, an alkylaminoalkyl group, a halogen atom, a nitro group, a cyano group, an aralkyl group optionally substituted with an alkoxy group, an aryl group optionally substituted with a substituent selected from a halogen atom and an alkoxy group, an alkylthio group, an amino group, or an alkylamino group).

The substituent represented by A, B or C in the formula (3) will be described below.
Examples of the alkyl group include a C1-4 alkyl group such as a methyl group, an ethyl group, a propyl group, and a butyl group.
Examples of the alkenyl group include a C2-4 alkenyl group such as a vinyl group, an allyl group, and a crotyl group.
Examples of the alkynyl group include a C2-4 alkynyl group such as an ethynyl group, a propargyl group, and a butynyl group.
Examples of the cycloalkyl group include a C3-6 cycloalkyl group such as a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.
Examples of the cycloalkenyl group include a C5-6 cycloalkenyl group such as a cyclopentenyl group, and a cyclohexenyl group.
Examples of As the alkoxy group include a C1-4 alkoxy group such as a methoxy group, an ethoxy group, a propoxy group, and a butoxy group.
Examples of the halogenated alkyl group include a C1-3 haloalkyl group such as a trifluoromethyl group, a trichloromethyl group, a difluoromethyl group, a chloromethyl group, a 2-bromoethyl group, and a 1,2-dichloropropyl group.
Examples of the hydroxyalkyl group include a C1-2 alkyl group substituted with hydroxyl such as a hydroxymethyl group, and a 1-, 2-hydroxyethyl group.
Examples of the alkoxyalkyl group include a C1-2 alkyl group substituted with C1-2 alkoxy such as a methoxymethyl group, a methoxyethyl group, an ethoxymethyl group, and an ethoxyethyl group.
Examples of the aminoalkyl group include a C1-2 alkyl group substituted with monoamino, or a C1-2 alkyl group substituted with diamino, such as an aminomethyl group, and a 1-, 2-aminoethyl group.
Examples of the alkylaminoalkyl group include a C1-2 alkyl group substituted with (C1-2)alkylamino, or a C1-2 alkyl group substituted with di(C1-2)alkylamino, such as a methylaminomethyl group, a dimethylaminomethyl group, and a diethylaminomethyl group.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
Examples of the aralkyl group optionally substituted with an alkoxy group include a C7-8 aralkyl group optionally substituted with a C1-2 alkoxy group, such as a benzyl group, a phenethyl group, and a 4-methoxybenzyl group.
Examples of the aryl group optionally substituted with a group selected from a halogen atom and an alkoxy group include a C6-10 aryl group optionally substituted with a group selected from a halogen atom (fluorine atom, chlorine atom, bromine atom and iodine atom) and a C1-2 alkoxy group such as a 2-, 3-, 4-chlorophenyl group, a 2-, 3-, 4-methylphenyl group, a 2-, 3-, 4-methoxyphenyl group, a phenyl group, a 1-naphthyl group, and a 2-naphthyl group.
Examples of the alkylthio group include a C1-3 alkylthio group such as a methylthio group, an ethylthio group and a propylthio group.
Examples of the alkylamino group include a C1-6 alkylamino group such as a methylamino group, an ethylamino group, a propylamino group, an isopropylamino group, a butylamino group, an isobutylamino group, a t-butylamino group, a pentylamino group, and a hexylamino group.
From an ortho-substituted phenylglyoxalic acid compound having the optionally substituted aryloxy group and being represented by the formula (1′):




(wherein R1 represents an optionally substituted amino group, or an optionally substituted alkoxy group, and A, B and C are the same or different from one another, and represent a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloaklenyl group, an alkoxy group, a halogenated alkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aminoalkyl group, an alkylaminoalkyl group, a halogen atom, a nitro group, a cyano group, an aralkyl group optionally substituted with an alkoxy group, an aryl group optionally substituted with a substituent selected from a halogen atom and an alkoxy group, an alkylthio group, an amino group or an alkylamino group), a compound represented by the formula (2′):





(wherein R1, A, B and B are as defined above, and a hydrogen atom with a symbol is an asymmetric carbon atom) is obtained.

In the compounds of the formula (1) and (1′), as the optionally substituted amino group represented by R1, a methylamino group is preferable and, as the optionally substituted alkoxy group, for example, a methoxy group or an ethoxy group is preferable. In addition, as the optionally substituted C1-8 alkyl group represented by R2, a methyl group is preferable, as the optionally substituted alkoxyalkyl group, for example, a methoxymethyl group is preferable, as the optionally substituted aryloxy alkyl group, for example, a dimethylphenoxymethyl group is preferable and, more particularly, 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide, ethyl 2-(2-methyl-phenyl)oxoacetate, 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide, ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate, 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, or methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate is exemplified.
In the method of the present invention, when such an ortho-substituted phenylglyoxalic acid compound is a substrate, the compound of the formula (2) or the formula (2′) is obtained, and as a specific compound, an optically-active substance of 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide, ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate, 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide, ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxyacetate, 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide, or methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate is obtained, respectively.
The method is usually performed in the presence of water, and a coenzyme such as reduced nicotineamide adenine dinucleotide phosphate (hereinafter, referred to as NADPH). The water used thereupon may be an aqueous buffer solution. Examples of a buffer used in the aqueous buffer solution include an alkali metal salt of phosphoric acid such as sodium phosphate and potassium phosphate, an aqueous sodium acetate solution, an alkali metal salt of acetic acid such as potassium acetate, and a mixture thereof.
In the method, in addition to water, an organic solvent may be present together. Examples of the organic solvent which may be present together include ethers such as t-butyl methyl ether, diisopropyl ether and tetrahydrofuran, esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, ethyl propionate and butyl propionate, hydrocarbons such as toluene, hexane, cyclohexane, heptane and isooctane, alcohols such as methanol, ethanol, 2-propanol, butanol and t-butyl alcohol, organic sulfur compounds such as dimethyl sulfoxide, ketones such as acetone, nitriles such as acetonitrile, and a mixture thereof.
For example, a reaction in the method is performed by mixing through stirring, shaking or the like in the state where water, an amide or ester compound of ortho-substituted phenylglyoxalic acid (e.g. 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide), and NADPH, together with the invented protein, or a transformant producing the same, or a treated product thereof and, if necessary, further an organic solvent or the like are contained.
A pH at a reaction in the method can be appropriately selected, and is usually in a range of a pH of 3 to 10. In addition, a reaction temperature can be appropriately selected, and is usually in a range of 0 to 60° C. from the viewpoint of stability of a raw material and a product, and a reaction rate.
An endpoint of the reaction can be determined, for example, by tracing the amount of an amide or ester compound of ortho-substituted phenylglyoxalic acid (e.g. 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide) in the reaction solution by liquid chromatography or the like.
A reaction time can be appropriately selected, and is usually in a range of 0.5 hour to 10 days.
The recovery of an amide or ester compound of ortho-substituted optically active mandelic acid (e.g. (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide) from the reaction solution may be performed by a generally known arbitrary method.
Examples thereof include a purification method by performing post-treatment such as organic solvent extraction operation and concentration operation of the reaction solution, if necessary, in combination with column chromatography, distillation or the like.
The invented protein, a transformant producing the same, or a treated product thereof can be used in the method in various forms.
Examples of the specific form include a culture of a transformant comprising the invented DNA, and examples of the treated product of the transformant include a cell-free extract, a crude purified protein, a purified protein and the like, and an immobilized product thereof. Herein, examples of the treated product of the transformant include a lyophilized transformant, an organic solvent-treated transformant, a dry transformant, a transformant ground product, a self-digestion product of a transformant, an ultrasonic-treated product of a transformant, a transformant extract, and an alkali-treated product of a transformant. In addition, examples of a method of obtaining an immobilized product include a carrier binding method (a method of adsorbing the invented protein or the like onto an inorganic carrier such as silica gel and a ceramic, cellulose and an ion-exchanged resin), and an encapsulating method (a method of confining the invented protein or the like in a network structure of a polymer such as polyacrylamide, sulfur-containing polysaccharide gel (e.g. carrageenan gel), alginic acid gel and agar gel).
In addition, in view of industrial production using the transformant harboring the invented DNA, a method using a treated product obtained by killing the transformant is preferable rather than a method using a living transformant in that the restriction of a production facility is small. Examples of a method of kill treatment therefor include a physical sterilization method (heating, drying, freezing, light ray, ultrasound, filtration, powder distribution), and a sterilization method using a chemical (alkali, acid, halogen, oxidizing agent, sulfur, boron, arsenic, metal, alcohol, phenol, amine, sulfide, ether, aldehyde, ketone, cyan and antibiotic). Generally, it is desirable to select a treating method which inactivates enzyme activity of the invented protein as little as possible, and has little influence such as remaining in a reaction system, and contamination, among these sterilization methods.
In addition, a process for producing an amide or eater compound of the optically-active ortho-substituted mandelic acid of the present invention is performed in the presence of a coenzyme such as NADPH, in an amide or ester compound of the ortho-substituted phenylglyoxalic acid (e.g. 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide), as an asymmetrical reducing reaction proceeds, the NADPH is converted into oxidized β-nicotineamide adenine dinucleotide phosphate (hereinafter, referred to as NADP+). Since the NADP+ generated by conversion can be returned to original NADPH by a protein having the ability to convert NADP+ into reduced form (NADP), a protein having the ability to convert NADP+ into NADPH can be present together in the reaction system of the method.
Examples of the protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide (hereinafter, referred to as NAD+), or NADP+ into reduced β-nicotineamide adenine dinucleotide (hereinafter, referred to as NADH), or NADPH include glucose dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, amino acid dehydrogenase, organic dehydrogenase (malate dehydrogenase etc.) and the like.
In addition, when the protein having the ability to convert NAD+, or NADP+ into NADH, or NADPH is glucose dehydrogenase, by allowing glucose and the like to be present together in a reaction system, activity of the protein is enhanced in some cases, and these may be added, for example, to the reaction solution.
In addition, the protein may be an enzyme itself, or may be present together in the form of a microorganism having the enzyme, or a treated product of the microorganism in the reaction system. Further, it may be a transformant containing a gene having a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert NAD+, or NADP+ into NADH, or NADPH, or a treated product thereof. Herein, the treated product means an equivalent of the aforementioned “treated product of a transformant”.
Further, in the process for producing an amide or ester compound of the ortho-substituted optically-active mandelic acid of the present invention, the process may also be performed using a transformant simultaneously comprising a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert NAD+, or NADP+ into NADH, or NADPH such as glucose dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, amino acid dehydrogenase, and organic dehydrogenase (malate dehydrogenase etc.).
In this transformant, examples of a method of introducing both DNAs into a host cell include a method of introducing a single vector containing both DNAs into a host cell, a method of transforming a host cell with a recombinant vector in which both DNAs are separately introduced into a plurality of vectors having different replication origins, and the like. Further, one DNA or both DNAs may be introduced into a chromosome of a host cell.
In addition, as the method of introducing a single vector containing both DNAs into a host cell, for example, regions involved in expression control such as a promoter and a terminator may be connected to both DNAs to construct a recombinant vector, or a recombinant vector expressing a gene as an operon containing a plurality of cistrons such as lactose operon may be constructed.
EXAMPLES
Hereinafter, the present invention will be described in more detail by way of Examples or the like, but the present invention is not limited by those Examples at all.
Reference Example 1
Preparation of Chromosomal DNA
Each 100 ml of a medium (obtained by dissolving 2 g of glucose, 0.5 g of polypeptone, 0.3 g of yeast extract, 0.3 g of meat extract, 0.2 g of ammonium sulfate, 0.1 g of potassium dihydrogen phosphate, and 0.05 g of magnesium sulfate heptahydrate, and adjusting a pH to 6 with 2 N HCl) was placed into two 500-ml flasks, and was sterilized at 121° C. for 15 minutes. To this was added each 0.3 ml of a culture of Stenotrophomonas sp. SC-1 strain (FERM-BP 10785) cultured (30° C., 48 hours, shaking culturing) in a medium of the same composition, followed by shaking culturing at 30° C. for 24 hours. Thereafter, the resulting culture was centrifuged (8000 rpm, 4° C., 10 minutes), and the resulting precipitate was collected. The precipitate was washed with 50 ml of a 0.85% aqueous sodium chloride solution to obtain 3.5 g of wet bacterial cells.
From the bacterial cell, a chromosomal DNA (hereinafter, referred to as chromosomal DNA (A)) was obtained by using QIAprep Genomic-tip System (manufactured by Qiagen).
Example 1
Obtaining of Invented DNA, and Analysis Thereof
(1) Preparation of Invented Protein
Into 10 ml of a 20 mM potassium phosphate buffer (pH 7.0) to which PMSF (phenylmethylsulfonyl fluoride) has been added so as to be 1 mM was suspended about 7.5 g of the wet bacterial cells of the Stenotrophomonas sp. SC-1 strain (FERM-BP 10785) prepared under the same condition as that of Reference Example, and this was ground with a multi beads shocker (manufactured by Yasui Kikai), glass beads 0.1 mmΦ, 2500 rpm, 20 minutes). After 38 mg of protamine sulfate was added to the resulting ground product, this was centrifuged (8000 rpm, 4° C., 10 minutes), and the supernatant was further filtered with a filter (0.45 μm) to obtain about 8 ml of the supernatant. The same procedure was repeated to obtain about 40 ml of the supernatant.
About 10 ml of the resulting supernatant was applied on an affinity interaction chromatography column [HiTrap BlueHP (manufactured by Amersham Pharmacia Biotech)] [equilibrated with a 20 mM potassium phosphate buffer (pH 7.0)], and the column was eluted using, as a mobile phase, a potassium phosphate buffer with sodium chloride dissolved therein (concentration gradient of sodium chloride concentration 0→1.0 M) to obtain 2 ml of a fraction at a sodium chloride concentration of 0.5 to 0.8 M as a fraction having reductase activity. The same procedure was repeated to obtain about 10 ml of an active fraction.
The eluted fraction was concentrated using Amicon Ultra-4 (manufactured by MILLIPORE), and the buffer was exchanged with a 20 mM Tris-HCl buffer (pH7.5). This was applied on an ion exchange chromatography column [HiTrap DEAE Sepharose FF (manufactured by Amersham Pharmacia Biotech)] [equilibrated with a Tris-HCl buffer solution (20 mM, pH 7.5)], and the column was eluted using, as a mobile phase, a Tris-HCl buffer solution with sodium chloride dissolved therein (concentration gradient of sodium chloride concentration 0→0.5 M) to obtain 2 ml of an active fraction at a sodium chloride concentration of 0.1 to 0.2 M as a fraction having reductase activity. The same procedure was repeated to obtain about 6 ml of an active fraction.
This eluted fraction was concentrated using Amicon Ultra-4 (manufactured by MILLIPORE), and the buffer was exchanged with a 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride. This concentrated solution was passed through a gel filtration column [Superdex200 10/300GL (manufactured by Amersham Pharmacia Biotech) [mobile phase: 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride] to obtain 1 ml of an eluted fraction at a molecular weight of about 35000 dalton (hereinafter, referred to as active fraction (A)) as a fraction having reductase activity.
Regarding the fraction obtained by the chromatography and so on, reductase activity was measured by the following procedure.
Three (3) mg of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 0.2 ml of the eluted fraction obtained by the chromatography and so on, 9 mg of NADPH, 0.15 ml of butyl acetate, and 1.5 ml of a 100 mM phosphate buffer (pH 7.0) were mixed, and the mixture was stirred at 30° C. for 18 hours. Thereafter, 2 ml of ethyl acetate was added to the reaction solution, and this was centrifuged to obtain an organic layer. The organic layer was subjected to content analysis measurement by liquid chromatography under the following condition. The reductase activity was determined from the residual amount of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, and the production amount of (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.
(Content Analysis Condition)
Column: SUMICHIRAL ODS A-212
Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, B solution acetonitrile solution containing 0.1% trifluoroacetic acid













Time (min)
A solution (%):B solution (%)


















0
80:20



20
10:90



30
 1:99



30.1
80:20









Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 290 nm

(2) Analysis of Amino Acid Sequence of Partial Peptide Derived from Invented Protein
The active fraction (A) obtained by the above procedure was subjected to SDS polyacrylamide gel electrophoresis according to the method describe in Laemmli, U. K., Nature, (1970) 227, 680. The gel after electrophoresis was stained with a Coomassie brilliant blue G250 staining solution (manufactured by BIO-RAD), and a stained part of the gel was cut out. The gel was washed, and treated with trypsin, and peptides were extracted from the gel. The extracted peptides were fractionated by HPLC (column: TSK gel ODS-80Ts, 2.0 mm×250 mm (manufactured by Tosoh), mobile phase: A solution (0.1% aqueous trifluoroacetic acid), B solution (90% aqueous acetonitrile solution containing 0.09% trifluoroactic acid), concentration gradient of A/B=100/0→0/100). Two fractions from the obtained respective fractions were subjected to an amino acid sequence analysis by protein sequencer (Procise 494HT Protein Sequencing System). The determined amino acid sequence is shown in SEQ ID No: 3 or SEQ ID No: 4.
(3) Analysis of Partial Nucleotide Sequence Derived from Invented DNA (I)
Based on the amino acid sequence shown in SEQ ID NO: 3, an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 5 was synthesized. Separately, based on the amino acid sequence shown in SEQ ID NO: 4, an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 6 was synthesized.
Using the oligonucleotide primers each having the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, and employing the chromosomal DNA (A) as a template, PCR was performed under the following reaction solution composition and the reaction condition (using Expand High Fidelity PLUS PCR System manufactured by Roche Diagnostics).
[Reaction Solution Composition]
















Chromosomal DNA (A) solution
2
μl



dNTP (each 2.5 mM-mix)
1
μl



Primer (50 pmol/μl)
each 0.4
μl



5 × buffer (with MgCl)
10
μl



enz. expandHiFi (5 U/μl)
0.5
μl



Superpure water
35.7
μl









[Reaction Condition]
A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C. for 2 minutes, followed by 10 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (90 seconds) and, then, 20 times of a cycle of 94° C. (10 seconds)-65° C. (30° C. seconds)-72° C. (1 minute+5 second/cycle), and further, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquot of the PCR reaction solution was taken and was subjected to agarose gel electrophoresis resulting in detecting a band of a DNA fragment of about 600 bp.
The DNA fragment of about 600 bp was ligated to the ready-made “PCR Product insertion site” of a pCR2.1-TOPO vector (using TOPO TA cloning Kit, manufactured by Invitrogen), and E. coli TOP10F′ was transformed with the resulting ligation solution.
Thirty (30) μl of a 4% aqueous solution of 5-bromo-4-chloro-3-indolyl β-D-galactoside (hereinafter, referred to as X-gal) and 30 μl of 0.1 M IPTG were spread on an LB (1% Bacto-trypsin, 0.5% Bacto-yeast extract, 1% sodium chloride) agar medium containing 50 μg/ml of ampicillin, and the resulting transformants were inoculated thereon, followed by culturing. One white colony among formed colonies was taken, and this colony was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of ampicillin, and shaking-cultured (30° C., 24 hours) in a tube. A plasmid was taken out from the cultured bacterium cells using QIAprep Spin Miniprep Kit (manufactured by Qiagen).
A nucleotide sequence of the DNA fragment having been inserted into the resulting plasmid was analyzed, and the nucleotide sequence shown in SEQ ID NO: 7 was determined.
The analysis of the nucleotide sequence of the DNA fragment inserted in the plasmid was performed by conducting a sequencing reaction using Dye Terminator Cycle sequencing FS ready Reaction Kit (manufactured by Perkin Elmer) and employing each plasmid as a template, and analyzing the nucleotide sequence of the resulting DNA with a DNA Sequencer 373A (manufactured by Perkin Elmer).
(4) Analysis of Partial Sequence Derived from Invented DNA (II)
Based on the nucleotide sequence shown in SEQ ID NO: 7, an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 8, and an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 9 were synthesized. Separately, based on the nucleotide sequence shown in SEQ ID NO: 7, an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 10, and an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 11 were synthesized.
Using the oligonucleotide primers comprising the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 respectively, PCR was performed under the following reaction solution composition and the reaction condition using as a template a DNA obtained by treatment of the chromosomal DNA (A) with a restriction enzyme EcoRI followed by self-ligation with a 14 DNA ligase. In addition, using the oligonucleotide primers comprising the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11 respectively, PCR was performed under the following reaction solution composition and the reaction condition (using Expand High Fidelity PLUS PCR System manufactured by Roche Diagnostics) using as a template a DNA obtained by treatment of the chromosomal DNA (A) with a restriction enzyme EcoRV followed by self-ligation with a T4 DNA ligase.
[Reaction Solution Composition]
















Chromosomal DNA (A) treatment solution
2
μl



dNTP (each 2.5 mM-mix)
1
μl



Primer (50 pmol/μl)
each 0.4
μl



5 × buffer (with MgCl)
10
μl



enz. expandHiFi (5 U/μl)
0.5
μl



Superpure water
35.7
μl









[Reaction Condition]
A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C. for 2 minutes, followed by 10 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (90 seconds) and, then, 20 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and further, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquot of the PCR reaction solution was taken, and subjected to agarose gel electrophoresis. A band of a DNA fragment of about 600 bp was detected as a result of PCR performed using oligonucleotide primers comprising the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9. In addition, a band of a DNA fragment of about 1300 bp was detected as a result of PCR performed using oligonucleotide primers comprising the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11.
The DNA fragment of about 600 bp or about 1300 bp was ligated to the ready-made “PCR Product insertion site” of apCR2.1-TOPO vector (using TOPOTMTA cloning Kit, manufactured by Invitrogen), and E. coli TOP10F′ was transformed with the ligation solution.
Thirty (30) μl of an X-gal 4% aqueous solution and 30 μl of 0.1 M IPTG were spread on an LB agar medium containing 50 μg/ml of ampicillin, and the resulting transformants were inoculated thereon, followed by culturing. One white colony among formed colonies was taken, and this colony was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of ampicillin, and shaking-cultured (30° C., 24 hours) in a tube. A plasmid was taken out from each cultured bacterium cell using QIAprep Spin Miniprep Kit (manufactured by Qiagen).
A nucleotide sequence of the DNA fragment having been inserted in the resulting plasmid was analyzed. Based on the resulting nucleotide sequence, was determined the nucleotide sequence (SEQ ID NO: 2) encoding an amino acid sequence of a protein of Stenotrophomonas sp. SC-1 strain (FERM-BP 10785) having the ability to asymmetrically reduce 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide to preferentially produce (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide. Further, based on SEQ ID NO: 2, the amino acid sequence (SEQ ID NO: 1) of the protein was determined.
When SEQ ID NO: 1 was compared with SEQ ID NO: 3 or SEQ ID NO: 4, it was found that the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 is identical with a part of the amino acid sequence shown in SEQ ID NO: 1.
The analysis of the nucleotide sequence of the DNA fragment having been inserted in the plasmid was performed by conducting a sequencing reaction using Dye Terminator Cycle sequencing FS ready Reaction Kit (manufactured by Perkin Elmer) and employing each plasmid as a template, and analyzing the nucleotide sequence of the resulting DNA with a DNA sequencer 373A (manufactured by Perkin Elmer).
Example 2
Example of Production of Invented Transformant and Reducing Reaction (I)
(1) Preparation of Invented Vector
Based on the nucleotide sequence shown in SEQ ID NO: 2, an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 12, and an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 13 were synthesized.
Using the oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 12, and the oligonucleotide primer shown in SEQ ID NO: 13 as primers, and employing the chromosomal DNA (A) as a template, PCR was performed under the following reaction solution composition and the reaction condition (using Expand High Fidelity PLUS PCR System manufactured by Roche Diagnostics).
[Reaction Solution Composition]
















Chromosomal DNA (A) solution
2
μl



dNTP (each 2.5 mM-mix)
1
μl



Primer (50 pmol/μl)
each 0.4
μl



5 × buffer (with MgCl)
10
μl



enz. expandHiFi (5 U/μl)
0.5
μl



Superpure water
35.7
μl









A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C. for 2 minutes, followed by 10 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (90 seconds), and then, 20 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and further, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquot of the PCR reaction solution was taken and subjected to agarose gel electrophoresis resulting in detection of a band of a DNA fragment of about 1000 bp.
Two kinds of restriction enzymes (NcoI and XbaI) were added to the remaining PCR reaction solution to double-digest the DNA fragment of about 1000 bp, and then, the enzyme-digested DNA fragment was purified.
Separately, a plasmid vector pTrc99A (manufactured by Pharmacia) was double-digested with two kinds of restriction enzymes (NcoI and XbaI), and the enzyme-digested DNA fragment was purified.
These enzyme-digested DNA fragments were mixed, and ligated with a T4 DNA ligase, and E. coli DH5α was transformed with the resulting ligation solution.
The resulting transformants were cultured on an LB agar medium containing 50 μg/ml of amplicillin, and 8 colonies were randomly selected from the grown colonies. Each of the selected colonies was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of amplicillin, and shaking-cultured (37° C., 17 hours) in a tube. A plasmid was taken out from each cultured bacterial cell using QIAprep Spin Miniprep Kit (manufactured by Qiagen). An aliquot of each plasmid taken out was double-digested with two kinds of restriction enzymes of NcoI and XbaI, and then subjected to electrophoresis to confirm that the DNA fragment of about 1000 bp was inserted in each of the six plasmids taken out (hereinafter, this plasmid is referred to as plasmid pTrcRs).
(2) Example of Preparation of Invented Transformant and Reducing Reaction
E. coli HB101 was transformed using the plasmid pTrcRs. The resulting transformant was inoculated in a sterilized LB medium (9 ml) containing 0.2 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (37° C., 15 hours). The resulting culture was centrifuged to obtain 0.12 g of wet bacterial cells. The resulting wet bacterial cells were mixed with 1.5 mg of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 6.9 mg of NADPH, 1.5 ml of a 100 mM phosphate buffer (pH 7.0), 2.7 mg of glucose, and 0.075 ml of butyl acetate, followed by stirring at 30° C. for 23 hours. Thereafter, 2 ml of ethyl acetate was added to the reaction solution, and this was centrifuged to obtain an organic layer. The organic layer was subjected to content analysis by liquid chromatography under the following condition, and it was found that 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide was produced at 97.9% relative to the amount of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide used in the reaction. The optical purity of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide in the organic layer was measured under the following condition. The optical purity of (R) body was found to be 100% e.e.
(Content Analysis Condition)
Column: SUMICHIRAL ODS A-212
Mobile phase: A solution 0.1% aqueous trifluoroactic acid solution, B solution acetonitrile solution containing 0.1% trifluoroactic acid













Time (min)
A solution (%):B solution (%)


















0
80:20



20
10:90



30
 1:99



30.1
80:20









Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 290 nm

(Optical Purity Measuring Condition)
Column: CHIRALCEL OD-H
Mobile phase: hexane:2-propanol=9:1
Analysis time: 50 minutes
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Example 3
Example of Production of Invented Transformant and Reducing Reaction (II)
(1) Arrangement for Preparing a Gene Comprising a Nucleotide Sequence Encoding an Amino Acid Sequence of a Protein Having Ability to Convert Oxidized β-Nicotineamide Adenine Dinucleotide into Reduced Form
Bacillus megaterium IFO12108 strain was cultured in 100 ml of a sterilized LB medium to obtain 0.4 g of bacterial cells.
From the bacterial cells, a chromosomal DNA (hereinafter, referred to as chromosomal DNA (B)) was purified using Qiagen Genomic Tip (manufactured by Qiagen) according to the method described in a manual attached thereto.
(2) Preparation of a Gene Comprising a Nucleotide Sequence Encoding an Amino Acid Sequence of a Protein Having Ability to Convert Oxidized β-Nicotineamide Adenine Dinucleotide into Reduced Form
Based on the sequence of glucose dehydrogenase derived from Bacillus megaterium IWG3 described in The Journal of Biological Chemistry Vol. 264, No. 11, 6381-6385 (1989), an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 14, and an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 15 were synthesized.
Using the oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 14 and the oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 15 as primers, and employing the chromosomal DNA (B) as a template, PCR was performed under the following reaction solution composition and the reaction condition (using Expand High Fidelity PCR System manufactured by Roche Diagnostics).
[Reaction Solution Composition]
















Chromosomal DNA (B) stock solution
1
μl



dNTP (each 2.5 mM-mix)
0.4
μl



Primer (20 pmol/μl)
each 0.75
μl



10 × buffer (with MgCl)
5
μl



enz. expandHiFi (3.5 × 103 U/ml)
0.375
μl



Superpure water
41.725
μl









[PCR Reaction Condition]
A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System2400, and was heated at 97° C. for 2 minutes, followed by 10 times of a cycle of 97° C. (15 seconds) −55° C. (30 seconds) −72° C. (1.5 minutes), and then, 20 times of a cycle of 97° C. (15 seconds)-55° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and further, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquot of the PCR reaction solution was taken, and subjected to agarose gel electrophoresis resulting in detection of a band of a DNA fragment of about 950 bp.
The DNA fragment of about 950 bp obtained by PCR was ligated with the ready-made “PCR Product insertion site” of pCR2.1-TOPO vector using the resulting PCR reaction solution and TOPO TA cloning Kit Ver. E manufacturedby Invitrogen, and E. coli DH5α was transformed with the ligation solution.
Thirty (30) μg of an X-gal 4% aqueous solution and 30 μl of 0.1M IPTG were spread on an LB agar medium containing 50 μg/ml of ampicillin, and the resulting transformants were inoculated thereon, followed by culturing. One white colony among formed colonies was taken, and this colony was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of ampicillin, and this was shaking-cultured (30° C., 24 hours) in a tube. Then, a plasmid was taken out from the cultured bacterial cells using QIAprep Spin Miniprep Kit (manufactured by Qiagen). An aliquot of the plasmid taken out was digested with the restriction enzyme (EcoRI), and it was subjected to electrophoresis, whereby it was confirmed that the DNA fragment of about 950 bp was inserted in the plasmid (hereinafter, this plasmid is referred to as plasmid pSDGDH12).
A nucleotide sequence of the DNA fragment having been inserted in the Plasmid pSDGDH12 was analyzed. The result is shown in SEQ ID No: 16.
The analysis of the nucleotide sequence of the DNA fragment having been inserted in the plasmid was performed by conducting a sequencing reaction using Dye Terminator Cycle sequencing FS ready Reaction Kit (manufactured by Perkin Elmer), and employing the Plasmid pSDGDH12 as a template, and analyzing the nucleotide sequence of the resulting DNA with DNA Sequencer 373A (manufactured by Perkin Elmer).
Then, based on the nucleotide sequence shown in SEQ ID NO: 16, oligonucleotide primers comprising the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 were synthesized.
PCR was performed using the oligonucleotide primes having the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO 18, and employing the chromosomal DNA (B) under the following reaction solution composition and the reaction condition (using Expand High Fidelity PCR System manufactured by Roche Diagnostics).
[Reaction Solution Composition]
















Chromosomal DNA (B) stock solution
1
μl



dNTP (each 2.5 mM-mix)
0.4
μl



Primer (20 pmol/μl)
each 0.75
μl



10 × buffer (with MgCl)
5
μl



enz. expandHiFi (3.5 × 103 U/ml)
0.375
μl



Superpure water
41.725
μl









[PCR Reaction Condition]
A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System2400, and was heated at 97° C. for 2 minutes, followed by 10 times of a cycle of 97° C. (15 seconds)-55° C. (30 seconds)-72° C. (1.5 minutes), and then, 20 times of a cycle of 97° C. (15 seconds)-55° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and then, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquot of the PCR reaction solution was taken, and subjected to agarose gel electrophoresis resulting in detection of a band of a DNA fragment of about 800 bp.
Two kinds of restriction enzymes (NcoI and BamHI) were added to the remaining PCR reaction solution to double-digest the DNA fragment of about 800 bp, and then, the enzyme-digested DNA fragment was purified.
Separately, the plasmid vector pTrc99A (manufactured by Pharmacia) was double-digested with two kinds of restriction enzymes (NcoI and BamHI), and the enzyme-digested DNA fragment was purified.
These enzyme-digested DNA fragments were mixed, and ligated with a T4 DNA ligase, and E. coli DH5α was transformed with the resulting ligation solution.
The resulting transformants were cultured on an LB agar medium containing 50 μg/ml of ampicillin, and 10 colonies were randomly selected from the grown colonies. Each of the selected colonies was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of ampicillin, and this was shaking-cultured (37° C., 17 hours) in a tube. A plasmid was taken out from each cultured bacterial cell using QIAprep Spin Miniprep Kit (manufactured by Qiagen). An aliquote of each plasmid taken out was double-digested with two kinds of restriction enzymes of NcoI and BamHI, and then subjected to electrophoresis to confirm that the DNA fragment of about 800 bp was inserted in each of four plasmids taken out (hereinafter, this plasmid is referred to as Plasmid pTrcGDH).
(3) Preparation of Invented Vector
Using as primers an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 19 and an oligonucleotide primer shown in SEQ ID NO: 12, and employing the plasmid pTrcRs as a template, PCR was performed under the following reaction solution composition and the reaction condition (using Expand High Fidelity PCR System manufactured by Roche Diagnostics).
[Reaction Solution Composition]














Plasmid pTrcRs
2
μl


dNTP (each 2.5 mM-mix)
1
μl


Primer (50 pmol/μl)
each 0.4
μl


5 × buffer (with MgCl)
10
μl


Expand High Fidelity PLUS Taq polymerase
0.5
μl (2.5 U)


Superpure water
35.7
μl









[Reaction Condition]
A container containing a reaction solution of the above composition was set in PERKIN ELMER-GeneAmp PCR System 9700, and was heated at 94° C. for 2 minutes, followed by 10 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (90 seconds), and then, 20 times of a cycle of 94° C. (10 seconds)-65° C. (30 seconds)-72° C. (1 minute+5 seconds/cycle), and further, the container was incubated at 72° C. for 7 minutes.
Thereafter, an aliquote of the PCR reaction solution was taken, and subjected to agarose gel electrophoresis resulting in detection of a band of a DNA fragment of about 1000 bp.
Two kinds of restriction enzymes (BamH and XbaI) were added to the remaining PCR reaction solution to double-digest the DNA fragment of about 1000 bp, and then, the enzyme-digested DNA fragment was purified.
Separately, the plasmid pTrcGDH was double-digested with two kinds of restriction enzymes (BamHI and XbaI), and the enzyme-digested DNA fragment was purified.
These enzyme-digested DNA fragments were ligated with a T4 DNA ligase, and E. coli DH5α was transformed with the ligation solution. The resulting transformants were cultured on an LB agar medium containing 50 μg/ml of ampicillin, and 6 colonies were randomly selected from grown colonies. Each of the selected colonies was inoculated in a sterilized LB medium (2 ml) containing 50 μg/ml of ampicillin, and shaking-cultured (30° C., 7 hours) in a tube. A plasmid was taken out from each cultured bacterial cell using QIAprep Spin Miniprep Kit (manufactured by Qiagen). An aliquote of each plasmid taken out was double-digested with two kinds of restriction enzymes of BamHI and XbaI, and then subjected to electrophoresis to confirm that the DNA fragment of about 1000 bp of interest was inserted in all of the plasmid taken out (hereinafter, this plasmid is referred to as plasmid pTrcGSRs).
(4) Example of Preparation of Invented Transformant and Reducing Reaction
Using the plasmid pTrcGSRs, E. coli HB101 was transformed. The resulting transformant was inoculated in a sterilized LB medium (5 ml) containing 0.2 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (30° C., 24 hours). The resulting culture was centrifuged to obtain about 0.1 g of wet bacterial cells. The resulting wet bacterial cells were mixed with 3 mg of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 1.8 mg of NADP+, 1.5 ml of 100 mM phosphate buffer (pH 7.0), 2.8 mg of glucose, and 0.15 ml of tetrahydrofuran, followed by stirring at 30° C. for 24 hours. Thereafter, 2 ml of ethyl acetate was added to the reaction solution, and this was centrifuged to obtain an organic layer. The organic layer was subjected to content analysis by liquid chromatography under the following condition, and it was found that 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide is produced at 96.8% relative to the amount of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide used in the reaction. The optical purity of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-ace tamide in the organic layer was measured under the following condition. The optical purity of (R) body was found to be 100% e.e.
(Content Analysis Condition)
Column: SUMICHIRAL ODS A-212
Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, B solution acetonitrile solution containing 0.1% trifluoroacetic acid













Time (min)
A solution (%):B solution (%)


















0
80:20



20
10:90



30
 1:99



30.1
80:20









Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 290 nm

(Optical Purity Measuring Condition)
Column: CHIRALCEL OD-H
Mobile phase: hexane:2-propanol=9:1
Analysis time: 50 minutes
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Example 4
Example of Reducing Reaction by Invented Transformant (III)
Using the plasmid pTrcRs, E. coli HB101 was transformed. The resulting transformant was inculcated in a sterilized LB medium (5 ml) containing 0.1 mM of IPTG and 50 μg/ml of ampicillin, and shaking-cultured (37° C., 15 hours). The resulting culture was centrifuged to obtain about 0.1 g of wet bacterial cells. The resulting wet bacterial cells were mixed with 3 mg of methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate, 13.5 mg of NADPH, 1.5 ml of 100 mM phosphate buffer (pH 7.0), and 0.15 ml of butyl acetate, followed by stirring at 30° C. for 24 hours. Thereafter, 2 ml of ethyl acetate was added to the reaction solution, and this was centrifuged to obtain an organic layer. The organic layer was subjected to content analysis by liquid chromatography under the following condition, and it was found that methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate is produced at 28.8% relative to the amount of methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-oxoacetate used in the reaction. The optical purity of methyl 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-2-hydroxyacetate in the organic layer was measured under the following condition. The optical purity of (R) body was found to be 100% e.e.
(Content Analysis Condition)
Column: SUMICHIRAL ODS A-212
Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, B solution acetonitrile solution containing 0.1% trifluoroacetic acid













Time (min)
A solution (%):B solution (%)


















0
80:20



20
10:90



30
 1:99



30.1
80:20









Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 290 nm

(Optical Purity Measuring Condition)
Column: CHIRALCEL OD-H
Mobile phase: hexane:2-propanol=9:1
Analysis time: 50 minutes
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Example 5
Various Natures of Invented Protein
(1) Preparation of Invented Protein
Using the plasmid pTrcRs, E. coli HB101 was transformed. The resulting transformant was inoculated in a sterilized LB medium (100 ml×8) containing 0.1 mM of IPTG and 50 μg/ml of ampicillin, and was shaking-cultured (37° C., 13.5 hours). The resulting culture was centrifuged to obtain about 5.4 g of wet bacterial cells. The wet bacterial cells (about 5.4 g) were suspended in 20 ml of 20 mM potassium phosphate buffer (pH 7.0), and ground with a multibeads shocker (manufactured by Yasui Kikai, glass beads 0.1 mmΦ, 2500 rpm, 20 minutes). The resulting ground product was centrifuged (8000 rpm, 4° C., 10 minutes), and the supernatant was further filtered with a filter (0.45 μm) to obtain about 18 ml of the supernatant. The resulting supernatant was concentrated using Amicon Ultra-4 (manufactured by MILLIPORE) to obtain about 4 ml of the concentrated supernatant.
About 4 ml of the resulting concentrated supernatant was applied on an affinity interaction chromatography column [HiTrap BlueHP (manufactured by Amersham Pharmacia Biotech)] [equilibrated with a 20 mM potassium phosphate buffer (pH 7.0)], and the column was eluted using, as a mobile phase, a potassium phosphate buffer with sodium chloride dissolved therein (concentration gradient of sodium chloride concentration 0→1.2 M) to obtain about 6 ml of a fraction at a sodium chloride concentration of 0.01 to 0.34 M as a fraction having reductase activity.
The eluted fraction was concentrated using Amicon Ultra-4 (manufactured by MILLIPORE), and the buffer was exchanged with a 20 mM Tris-HCl buffer (pH 7.5). This was applied on an ion exchange chromatography column [HiTrap DEAE Sepharose FF (manufactured by Amersham Pharmacia Biotech)] [equilibrated with a Tris-HCl buffer solution (20 mM, pH 7.5)], and the column was eluted using, as a mobile phase, a Tris-HCl buffer solution with sodium chloride dissolved therein (concentration gradient of sodium chloride concentration 0→0.5 M) to obtain 2 ml of an active fraction at a sodium chloride concentration of 0.1 to 0.2 M as a fraction having reductase activity.
The eluted fraction was concentrated using Amicon Ultra-4 (manufactured by MILLIPORE), and the buffer was exchanged with a 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride. The concentrated solution was passed through a gel filtration column [Superdex200 10/300GL (manufactured by Amersham Pharmacia Biotech)] [mobile phase: 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride] to obtain 1 ml of an eluted fraction at a molecular weight of about 32200 dalton (hereinafter, referred to as active fragment (C)) as a fraction having reductase activity.
Regarding the fraction obtained by the chromatography and so on, reductase activity was measured by the following procedure.
Three (3) mg of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, 0.2 ml of the eluted fraction obtained by chromatography and so on, 9 mg of NADPH, 0.15 ml of butyl acetate, and 1.5 ml of a 100 mM phosphate buffer (pH 7.0) were mixed, and the mixture was stirred at 30° C. for 18 hours. Thereafter, 2 ml of ethyl acetate was added to the reaction solution, and this was centrifuged to obtain an organic layer. The organic layer was subjected to content analysis measurement by liquid chromatography under the following condition. The reductase activity was determined from the residual amount of 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide, and the production amount of (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide.
(Content Analysis Condition)
Column: SUMICHIRAL ODS A-212
Mobile phase: A solution 0.1% aqueous trifluoroacetic acid solution, B solution acetonitrile solution containing 0.1% trifluoroacetic acid













Time (min)
A solution (%):B solution (%)


















0
80:20



20
10:90



30
 1:99



30.1
80:20









Flow rate: 0.5 ml/min

Column temperature: 40° C.

Detection: 290 nm

(2) Enzyme Chemical Nature of Invented Protein
An enzyme chemical nature of the invented protein was investigated using the active fragment (C).
(1) Action
Using NADPH as a coenzyme, the invented protein was acted on 2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-oxo-acetamide to reduce to (R)-2-(2-(2,5-dimethylphenoxymethyl)phenyl)-N-methyl-2-hydroxy-acetamide having an optical purity of 100% e.e.
(2) Optimal pH
In order to investigate an acting optimal pH, the reduction activity of ethyl benzoylformate in a pH 5.5 to 10 was measured. As a buffer, a 100 mM phosphate buffer (pH 5.5 to 8), a 100 mM Tris-Hcl buffer (pH 7 to 9), and a 100 mM Tris-glycine buffer (pH 9 to 10) were used. The reduction activity on ethyl benzoylformate was obtained by dissolving a substrate ethyl benzoylformate so as to be a final concentration of 10 mM, and a coenzyme NADPH so as to be a final concentration of 0.5 mM in various buffer solutions, adding the purified invented protein (active fragment (C)), performing a reaction at 30° C. for 1 minute, and calculating the activity from a rate of reduction in absorbance of the reaction solution at a wavelength of 340 nm. In the present reaction condition, the activity of oxidizing 1 μmol of NADPH to NADP in one minute was defined as 1 unit. As a result, an optimal pH was 7.0.
(3) Substrate Specificity
In a 100 mM phosphate buffer solution (pH 7), a carbonyl compound as a substrate was dissolved so as to be a final concentration of 1 mM, and a coenzyme NADPH was dissolved so as to be a final concentration of 0.5 mM. An appropriate amount of the purified invented protein (active fragment (C)) was added thereto, followed by a reaction at 30° C. for 5 minutes. From a rate of reduction in absorbance of the reaction solution at a wavelength of 340 nm, the reduction activity on each carbonyl compound was calculated, and the activity was expressed as a relative value when activity on ethyl benzoylformate was made to be 100%, and the results were shown in Table 1.









TABLE 1






Substrate
Relative activity (%)


















Eethyl benzoylformate
100



Ethyl pyruvate
33.7



Ethyl 4-phenyl2-oxobutyrate
1.5



Methyl 4-bromo-3-oxobutyrate
3.9



Ethyl 4-chloro-3-oxobutyrate
0.5



3-Benzoylpropionic acid
3.3



Propionaldehyde
8.6



o-Chlorophenacyl chloride
1.0









Example 6
Example of Reducing Reaction by Invented Transformant (IV)
(1) Synthesis of ethyl 2-(2-methyl-phenyl)-2-oxoacetate
To a mixture of 50 g of tatrahydrofuran and 7.3 g of magnesium was added 4.4 g of 2-bromotoluene to activate the magnesium. A temperature was raised to 40° C., a solution of 45 g of 2-bromotoluene in tetrahydrofuran (130 g) was added dropwise until the disappearance of 2-bromotoluene was confirmed. Thereafter, the mixture was cooled to room temperature to prepare a Grignard reagent.
A solution of 83 g of diethyl oxalate in toluene (170 g) was cooled to −40° C. or lower. The Grignard reagent prepared as described above was added dropwise at −40° C. or lower, and this was retained at −40° C. for 2 hours. To the reaction mixture was added 234 g of 10% sulfuric acid, and an organic layer was recovered. The resulting organic layer was washed with 134 g of water, and dried with magnesium sulfate. After concentration with an evaporator, distillation under high vacuum, and silica gel column purification afforded 33.1 g of ethyl 2-(methyl-phenyl)-2-oxoacetate.
1H-NMR (300 MHz, CDCl3): δppm: 1.41 (t, J=7.2 Hz, 3H), 2.61 (s, 3H), 4.39-4.47 (m, 2H), 7.26-7.70 (m, 4H)
(2) Synthesis of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide
The ethyl 2-(2-methyl-phenyl)-2-oxoacetate of 18.1 g obtained in (1), 72 g of toluene, and 36 g of methanol were mixed, 23.2 g of a 40% aqueous methylamine solution was added while retaining at 25° C., and the mixture was retained at 25° C. for 1 hour. Thereafter, 36.2 g of water was added, and an organic layer was recovered. The layer was washed with 143 g of 5% hydrochloric acid, 360 g of an aqueous saturated sodium bicarbonate solution, and 36 g of water, respectively, and dried with magnesium sulfate. After concentration with an evaporator, silica gel column purification afforded 11.4 g of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide.
1H-NMR (300 MHz, CDCl3): δppm: 2.48 (s, 3H), 2.96 (d, J=5.13 Hz, 3H), 7.1 (6s, 1H), 7.25-7.93 (m, 4H)
(3) Asymmetrical reduction of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide Using Invented Transformant
Using the plasmid pTrcGSRs, E. coli HB101 was transformed. The resulting transformant was inoculated in a sterilized LB medium (100 ml) containing 0.1 mM of IPTG and 50 μg/ml of ampicillin, and was shaking-cultured (37° C., 15 hours). The resulting culture was centrifuged to obtain 0.5 g of wet bacterial cells. To a reaction tube were added 30 mg of 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide, 0.5 g of the wet bacterial cells, 30 mg of NADP+, 45 mg of glucose, and 5 ml of a 100 mM phosphate buffer solution (pH 7), and these were reacted at a stirring rate of 1000 rpm at 30° C. for 24 hours. After the completion of the reaction, 5 ml of ethyl acetate was added to the reaction solution, and this was centrifuged (1000×g, 5 minutes) to recover an organic layer. The organic layer was distilled off to obtain 22.5 mg of oily 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide. 1H-NMR analysis result of the resulting 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide is shown below.
1H-NMR (300 MHz, CDCl3) δ2.37 (S, 3H), 2.79 (d, J=4.96 Hz, 3H), 3.87 (1H), 5.15 (S, 1H), 6.21 (S, 1H), 7.15-7.26 (m, 4H)
To the resulting 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide was added hexane containing 10% 2-propanol, this was subjected to optical purity analysis by liquid chromatography under the following condition, and the optical purity was found to be 100% e.e. (elution time: 22.5 minutes).
Racemic 2-(2-methyl-phenyl)-N-methyl-2-hydroxy-acetamide was synthesized by reducing 2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide with NaBH4 in methanol.
(Optical Isomer Analysis Condition)
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane/2-propanol=90/10
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Elusion time
2-(2-methyl-phenyl)-N-methyl-2-oxo-acetamide: 16.0 minutes
2-(2-methyl-phenyl)-N-methyl-2-hydroxyacetamide: 18.5 minutes, 22.4 minutes
Example 7
Example of Reducing Reaction by Invented Transformant (V)
A reaction was performed according to the same manner as in Example 6 (3) except that ethyl2-(2-methyl-phenyl)-2-oxoacetate was used as a substrate. As a result, 26.5 mg of oily ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate was obtained. 1H-NMR analysis result of the resulting ethyl2-(2-methyl-phenyl)-2-hydroxy-acetate is shown below.
1H-NMR (300 MHz, CDCl3) δ1.21 (t, J=7.2 Hz, 3H), 2.43 (S, 3H), 3.56 (d, J=7.7 Hz, 1H), 4.13-4.29 (m, 2H), 5.35 (S, 1H), 7.15-7.30 (m, 4H)
To the resulting ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate was added hexane containing 10% 2-propanol, this was subjected to optical purity analysis by liquid chromatography under the following condition, and an optical purity was found to be 99% e.e. (elution time: 13.3.minutes).
Racemic ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate was synthesized by reducing ethyl2-(2-methyl-phenyl)-2-oxoacetate with NaBH4 in methanol.
(Optical Isomer Analysis Condition)
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane/2-propanol=90/10
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Elusion time
ethyl 2-(2-methyl-phenyl)-2-oxoacetate: 8.9 minutes
ethyl 2-(2-methyl-phenyl)-2-hydroxy-acetate: 11.6 minutes, 13.3 minutes
Example 8
Example of Reducing Reaction by Invented Transformant (VI)
(1) Synthesis of ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate
A temperature of 59.8 g of a 28% solution of sodium methoxide in methanol was raised to 60° C., a mixture solution of 70.4 g of o-bromobenzyl bromide and 70.4 g of methanol was added dropwise, and this was retained at 60° C. for 2 hours. After the mixture was cooled to room temperature, 211 g of toluene and 211 g of water were added thereto, the mixture was stirred, and layers were separated, and an organic layer was recovered. An aqueous layer was extracted with 211 g of toluene three times, the recovered organic layer and 211 g of water were added, and this was washed, and concentrated to obtain 55.3 g of 1-methoxymethyl-2-bromobenzene.
To a mixture of 46.4 g of tetrahydrofuran and 5.4 g of magnesium was added 4.6 g of 1-methoxymethyl-2-bromobenzene to activate the magnesium. A temperature was raised to 40° C., a solution of 41.7 g of 1-methoxymethyl-2-bromobenzene in tetrahydrofuran (139 g) was added dropwise until the disappearance of 1-methoxymethyl-2-bromobenzene was confirmed. Thereafter, the mixture was cooled to room temperature to prepare a Grignard agent.
A solution of 64.4 g of diethyl oxalate in toluene (177 g) was cooled to −40° C. or lower. The Grignard agent prepared as described above was added dropwise at −40° C. or lower, and this was retained at −40° C. for 4 hours. To the reaction mixture was added 211 g of 10% sulfuric acid, and an organic layer was recovered. The resulting organic layer was washed with 133 g of water, and dried with magnesium sulfate. After concentration with an evaporator, silica gel column purification and distillation under the high pressure afforded 38.5 g of ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate.
1H-NMR (300 MHz, CDCl3): δppm: 1.41 (t, J=7.08 Hz, 3H), 3.44 (s, 3H), 4.41 (q, 2H), 4.75 (s, 2H), 7.55-7.69 (m, 4H)
(2) Synthesis of 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide
28.3 g of the ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate obtained in (1), 109 g of toluene, and 54.4 g of methanol were mixed, 28.6 g of a 40% aqueous methylamine solution was added while retaining at 25° C., and the mixture was retained at 25° C. for 2 hours. Thereafter, 54.4 g of water was added, 80 g of toluene were further added, this was allowed to stand still, and an organic layer was recovered. The organic layer was washed with 178 g of 5% hydrochloric acid, 54.4 g of an aqueous saturated sodium bicarbonate solution, and 54.4 g of water, respectively, and concentrated with an evaporator to obtain 12.0 g of 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide.
1H-NMR (300 MHz, CDCl3): δppm: 2.95 (d, J=5.14 Hz, 3H), 3.28 (s, 3H), 4.66 (s, 2H), 7.04 (s, 1H), 7.36-7.72 (m, 4H)
(3) Asymmetric Reduction of 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide Using Invented Transformant
A reaction was performed according to the same manner as in Example 6(3) except that 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide was used as a substrate. As a result, 19.5 mg of oily 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide was obtained. 1H-NMR analysis result of the resulting 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide is shown below.
1H-NMR (300 MHz, CDCl3) δ2.77 (d, J=4.85 Hz, 3H), 3.47 (S, 3H), 4.38 (d, J=10.6 Hz, 1H), 4.73 (d, J=10.6 Hz, 1H), 4.81 (S, 1H), 5.23 (S, 1H), 7.16 (S, 1H), 7.26-7.43 (m, 4H)
To the resulting 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxyacetamide was added hexane containing 10% 2-propanol, this was subjected to optical purity analysis by liquid chromatography under the following condition, and the optical purity was found to be 100% e.e. (elution time: 20.3 minutes).
Racemic 2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxyacetamide was synthesized by reducing 2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide with NaBH4 in methanol.
(Optical Isomer Analysis Condition)
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane/2-propanol=90/10
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Elusion time
2-(2-methoxymethyl-phenyl)-N-methyl-2-oxo-acetamide: 18.5 minutes
2-(2-methoxymethyl-phenyl)-N-methyl-2-hydroxy-acetamide: 20.4 minutes, 21.0 minutes
Example 9
Example of Reducing Reaction by Invented Transformant (VII)
A reaction was performed according to the same manner as in Example 6(3) except that ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate was used as a substrate. As a result, 27.6 mg of oily ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate was obtained.
1H-NMR analysis result of the resulting ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate is shown below.
1H-NMR (300 MHz, CDCl3) δ1.21 (t, J=7.09 Hz, 3H), 3.39 (S, 3H), 4.16-4.26 (m, 2H), 4.59 (d, J=6.36 Hz, 2H), 5.39 (S, 1H), 7.31-7.37 (m, 4H)
To the resulting ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate was added hexane containing 10% 2-propanol, this was subjected to optical purity analysis by liquid chromatography under the condition, and the optical purity was found to be 100% e.e. (elution time: 13.6 minutes).
Racemic ethyl2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate was synthesized by reducing ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate with NaBH4 in methanol.
(Optical Isomer Analysis Condition)
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane/2-propanol=90/10
Flow rate: 0.5 ml/min
Column temperature: 40° C.
Detection: 230 nm
Elusion time
ethyl 2-(2-methoxymethyl-phenyl)-2-oxoacetate: 9.3 minutes
ethyl 2-(2-methoxymethyl-phenyl)-2-hydroxy-acetate: 12.8 minutes, 13.6 minutes
INDUSTRIAL APPLICABILITY
According to the present invention, optically-active mandelic acid compounds can be easily obtained.

## Claims

1. An isolated DNA comprising any nucleotide sequence of the following a) to d):
a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1;
b) a nucleotide sequence of a DNA: i) wherein the DNA has at least 90% sequence homology with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotide sequence encodes an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce corresponding optically active ortho-substituted mandelic acid;
c) a nucleotide sequence of a DNA: i) wherein the DNA hybridizes under a stringent condition including washing for 30 minutes at 65° C. in 0.1×SSC with a DNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, and ii) wherein the nucleotide sequence encodes an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce corresponding optically-active ortho-substituted mandelic acid compound; and
d) the nucleotide sequence of SEQ ID NO:2.

2. A DNA in which a promoter functional in a host cell and the DNA according to claim 1 are operably linked.

3. A recombinant vector comprising the DNA according to claim 1.

4. A transformant in which the DNA of claim 2 has been introduced into a host cell.

5. The transformant according to claim 4, wherein the host cell is a microorganism.

6. The transformant according to claim 4, wherein the host cell is Escherichia coli.

7. A transformant comprising the DNA of claim 1.

8. A process for producing a transformant comprising a step of introducing the recombinant vector of claim 3 into a host cell.

9. An isolated protein comprising any amino acid sequence of the following a) to e);
a) the amino acid sequence of SEQ ID NO:1;
b) an amino acid sequence: i) which is encoded by a nucleotide sequence of a DNA having at least 90% sequence homology with a DNA comprising the nucleotide sequence of SEQ ID NO:2, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound;
c) an amino acid sequence: i) which is encoded by a nucleotide sequence of a DNA which hybridizes under a stringent condition including washing for 30 minutes at 65° C. in 0.1×SSC with a DNA comprising the nucleotide sequence of SEQ ID NO:2, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound;
d) an amino acid sequence: i) in which one amino acid is deleted, substituted or added in the amino acid sequence of SEQ ID NO:1, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound; and
e) an amino acid sequence: i) which has at least 90% sequence homology with the amino acid sequence of SEQ ID NO:1, and ii) which is an amino acid sequence of a protein having the ability to asymmetrically reduce an ortho-substituted phenylglyoxalic acid compound to produce a corresponding optically-active ortho-substituted mandelic acid compound.

10. A recombinant vector comprising the DNA of claim 1 and a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form.

11. The recombinant vector according to claim 10, wherein the protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form is glucose dehydrogenase.

12. The recombinant vector according to claim 11, wherein the protein having glucose dehydrogenase activity is glucose dehydrogenase derived from Bacillus megaterium.

13. A transformant, in which the recombinant vector of claim 10 has been introduced into a host cell.

14. The transformant according to claim 13, wherein the host cell is a microorganism.

15. The transformant according to claim 13, wherein the host cell is Escherichia coli.

16. A transformant comprising the DNA of claim 1 and a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein having the ability to convert oxidized β-nicotineamide adenine dinucleotide or oxidized β-nicotineamide adenine dinucleotide phosphate into a reduced form.

17. A process for producing an optically-active alcohol compound comprising reacting the protein of claim 9, or the transformant of any of claims 4, 7, 13 and 16, or a treated product thereof with a prochiral carbonyl compound.

18. The process according to claim 17, wherein the prochiral carbonyl compound is an amide or ester compound of the ortho-substituted phenylglyoxalic acid, and the optically-active alcohol compound is an amide or ester compound of the optically-active ortho-substituted mandelic acid.

19. The process according to claim 18, wherein the amide or ester compound of the ortho-substituted phenylglyoxalic acid is a compound represented by the formula (1):



(wherein R1 represents an optionally substituted amino group, or an optionally substituted alkoxy group, and R2 represents an optionally substituted C1-8 alkyl group),
and the amide or ester compound of the optically-active ortho-substituted active mandelic acid is an optically-active compound represented by the formula (2):




(wherein R1 and R2 are as defined above, and a carbon atom with a * symbol is an asymmetric carbon atom).

20. Stenotrophomonas sp. SC-1 (FERM BP-10785).