JP3496640B2 - Oxidoreductase, gene encoding the enzyme, transformant containing the enzyme gene, and method for producing the enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to oxidoreductases derived from Bacillus subtilis and Saccharomyces cerevisiae, genes encoding the same, vectors containing the same, and vectors transformed with the same. The present invention relates to a transformant and a method for producing the same enzyme.

[0002]

2. Description of the Related Art Saccharomyces cerevisiae has been known as a redox enzyme-producing microorganism having a reducing ability for diketone compounds. However, in the Saccharomyces cerevisiae, for example, one of aromatic diketone compounds
When the seed benzil is reduced, racemic benzoin is produced. Therefore, the enzyme produced by the microorganism has a low stereoselectivity (Chemistr.
y Letters, pp. 1191-1192, 1988). Xanthomonas oryzae is also known as a microorganism that produces an oxidoreductase having an asymmetric reduction ability for benzyl which is an aromatic diketone compound. When benzyl is asymmetrically reduced by the same enzyme-producing microorganism, (R) -benzoin is stereoselectively produced as a reduction product (Chemistry Letters, 1111-111).
2 1985). Saccharomyces cerevisiae is known to produce 7 kinds of enzymes that reduce 4-chloroacetoacetic acid ester, which is an aliphatic diketone compound, to 4-hydroxybutanoic acid ester,
All the genes of these seven enzymes have not yet been isolated (Chemistry and Biology, 35, 590-598, 19).
1997), their amino acid sequences were also only deduced from the genomic sequences of Saccharomyces cerevisiae for the two enzymes (Biosci. Biotech. Biochem.,
60, 1538-1539, 1996).

Regarding Bacillus subtilis, the nucleotide sequence of DNA has been clarified by the Genome Project. However, the function of many genes remains unknown.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides an oxidoreductase derived from Bacillus subtilis having an ability to reduce an aromatic diketone compound, and (S) -benzoin by asymmetrically reducing benzyl, for example. It is an object of the present invention to provide an oxidoreductase derived from Saccharomyces cerevisiae, a gene encoding these enzymes, a vector containing the gene, a transformant transformed with the vector, and a method for producing the enzyme.

[0005]

[Means for Solving the Problems] In the Japanese Patent Application No. 2000-257829 of the prior application, the present inventors have developed a novel oxidoreductase derived from Bacillus cereus having a reducing ability to an aromatic diketone compound, and a novel enzyme encoding the same. There is provided a gene, a vector containing the gene, a transformant transformed with the vector, and a method for producing an oxidoreductase characterized by culturing the transformant. Among them, the amino acid sequence of Bacillus cereus-derived oxidoreductase was the amino acid sequence encoded by the yueD gene of Bacillus subtilis, which has a similar function to sepiapterin reductase, and Saccharomyces. It was found that they have 40% and 30% similarity with the amino acid sequence encoded by YIR036C of S. cerevisiae open reading frame (hereinafter abbreviated as ORF), respectively.

[0006] As a result of further research based on this finding, a protein defined by the yueD gene of Bacillus subtilis and YI of Saccharomyces cerevisiae
It was found that the protein defined by R036C has a reducing ability for aromatic diketone compounds. Also,
It has also been found that the protein defined by YIR036C asymmetrically reduces, for example, benzyl to produce (S) -benzoin.

Therefore, the present inventors solve the above problems by providing the following inventions. That is, the present invention provides (1) oxidoreductase comprising an amino acid sequence shown in SEQ ID NO 3, the redox enzyme encoded by the nucleotide sequence shown in (2) SEQ ID NO 4, (3) SEQ ID NO 4 base oxidoreductase gene containing sequence, (4) oxidoreductase gene encoding the amino acid sequence shown in SEQ ID NO 3 shown in, (5) obtained by expressing a DNA hybridizing to a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO 4, and a protein or polypeptide having a redox potential, (6) the ( 3) or (4) a recombinant vector comprising the gene according, (7) the vector is a pUC19 (6) a recombinant vector according, (8) the (6) or (7), wherein the recombinant A transformant transformed with the vector, ( 9 ) the transformant according to ( 8 ) above, wherein the host cell is a microorganism, ( 10 ) the transformant according to ( 9 ) above, wherein the microorganism is Escherichia coli, and ( 11) the (8), (9) or (10) culturing the transformant according to the production method of the oxidoreductase, and collecting the oxidoreductase from the culture To.

The oxidoreductase, the gene encoding the enzyme and the vector containing the gene according to the present invention can be obtained based on the embodiments described below. The method for producing a reductase can also be carried out based on the embodiments described below. Hereinafter, the present invention will be described in detail.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The oxidoreductase gene derived from Bacillus subtilis and Saccharomyces cerevisiae according to the present invention may be any gene as long as it imparts the oxidoreduction activity of the enzyme. For example, the Bacillus subtilis-derived oxidoreductase gene shown in SEQ ID NO: 2 or the Saccharomyces shown in SEQ ID NO: 4.
It would be possible for a person skilled in the art to prepare a sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence of oxidoreductase gene derived from S. cerevisiae, according to the state of the art in the current scientific field. It's extremely easy. It is also extremely easy for a person skilled in the art to add a base sequence such as a promoter or an enhancer to the 5'end side of the gene and / or add a poly A addition signal base sequence to the 3'end side of the gene. Therefore, a book consisting of a nucleotide sequence having one or more base deletions, substitutions, insertions or additions at the 5'end side and the 3'end side of the base sequence shown in SEQ ID NO: 2 or 4 and / or between them. The oxidoreductase gene of the invention is included in the present invention. Furthermore, the present invention hybridizes to a DNA complementary to the base sequence shown in SEQ ID NO: 2 or 4 or a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 or 4 under stringent conditions, It also includes DNA or oligonucleotide having redox ability.
Hybridization may be performed using procedures well known to those skilled in the art (eg, Meinkoth, J and G. Wahl, Anal. Biochem., 138).
Vol., Pp. 267-284, 1984). Specific hybridization conditions include, for example, 50% formamide, 5 × SSCP.
(150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA),
(PH 7.2)), 5 × Denhardt's solution,
0.1% SDS, 10% dextran sulfate and 100
After incubation with μg / ml denatured salmon sperm DNA, the filters are then placed in 0.2 × SSCP at 42 ° C.
For example, washing with can be performed. The stringent conditions are 0.1 × SSCP and 50 ° C. in the filter washing step, and more stringent conditions are 0.1 × S in the same step.
The conditions of SCP and 65 ° C. can be mentioned. Further, the present invention provides a polymerase chain reaction (hereinafter referred to as PCR) using a primer prepared based on the nucleotide sequence of Sequence Listing 2 or 4.
And a DNA or an oligonucleotide which defines a protein having redox ability.

The oxidoreductase derived from Bacillus subtilis or Saccharomyces cerevisiae according to the present invention is
Any substance may be used as long as it has redox activity of the enzyme. The oxidoreductase of the present invention is, for example,
In order to facilitate detection or purification of the oxidoreductase in the amino acid sequence of the Bacillus subtilis-derived oxidoreductase shown in SEQ ID NO: 1 or the Saccharomyces cerevisiae-derived oxidoreductase shown in SEQ ID NO: 3, It also includes a protein to which another protein such as E. coli β-galactosidase or histidine tag is added to the amino-terminal side or the carboxyl-terminal side of E. coli using well-known genetic engineering techniques. Further, in order to analyze the function of the present oxidoreductase more deeply, substitution, deletion, insertion, and / or addition of a sufficient number of amino acids by known methods such as site-directed mutagenesis, It also includes inserted and / or added amino acid sequences.
The present invention also includes such a mutant of the oxidoreductase. Furthermore, the present invention provides SEQ ID NO: 2 or 4.
It also includes a protein or polypeptide having an amino acid sequence encoded by a DNA that hybridizes with a DNA having a base sequence complementary to the base sequence shown in 1 and having redox ability.

The oxidoreductase gene of the present invention has the desired D
Methods known per se for obtaining NA sequences, eg P
It can be obtained by CR.

When the gene is obtained by the PCR method, a sample containing the gene is used as a primer, thermostable polymerase, dNTP (deoxy ATP, deoxy T).
A reaction solution added to a solution containing TP, deoxy GTP and deoxy CTP) and a buffer solution is prepared. PC concerned
The primer used for R can be appropriately set based on the publicly available Bacillus subtilis yueD gene sequence (SEQ ID NO: 2) or Saccharomyces cerevisiae YIR036C (SEQ ID NO: 4). In addition, in order to insert the gene amplified by PCR into the vector, it is preferable to include a recognition sequence for a restriction enzyme on the 5′-terminal side of the primer. The restriction enzyme is a type II restriction enzyme, such as restriction enzyme B, in that it can correctly digest the DNA chain at the position where the recognition sequence appears.
amHI, EcoRI, HindIII, KpnI, N
coI, NdeI, PstI, SacI, SalI, S
It is preferred to choose a recognition sequence such as apl, SmaI, SphI or XbaI. In addition to the recognition sequence of the restriction enzyme, a clamp (clam
You may add several bases called p). As a sample used for PCR, any sample may be used as long as it contains a desired gene, for example, chromosome DN of Bacillus subtilis or Saccharomyces cerevisiae.
A, a DNA library of the same chromosome, a cDNA library or a plasmid containing the gene of the present invention can be used. The thermostable polymerase used in PCR is a DNA-dependent 5 ′ → 3 ′ DNA synthase having an optimum temperature of 70 ° C. or higher and hardly inactivating even at 90 ° C. Examples of the thermostable polymerase include TaKaRa
ExTaq (trademark, manufactured by Takara Shuzo Co., Ltd.) can be used.

The PCR conditions for the gene can be appropriately set by those skilled in the art. For example, after incubation at 94 ° C. for 1 minute, 94 ° C. for 1 minute and 72
10 cycles of 3 minutes at ℃ for 10 minutes, 1 minute at 94 ℃, 2 minutes at 65 ℃ and 1 cycle at 72 ℃
This is 10 cycles with 1 minute as one cycle, and further 1 minute at 94 ° C, 2 minutes at 60 ° C and 1 minute at 72 ° C.
It can be carried out by performing 15 cycles with one minute as one cycle (Genes to Cells, Volume 5, 11
1-125, 2000). After the gene amplification reaction, the PCR product is developed by electrophoresis using, for example, 1.5% agarose gel, and a fraction containing a DNA band of about 800 bp is cut out. Then, for example, Prep-A-G
A desired gene DNA can be purified by using eneDNA Purification Kit (trade name, manufactured by Bio-Rad (BIO-RAD)). Then, when a primer containing a recognition sequence for a restriction enzyme is used, a restriction enzyme that recognizes the recognition sequence for the restriction enzyme contained in the primer is allowed to act on the gene amplified by PCR.

The recombinant vector containing the oxidoreductase gene has the above-mentioned gene treated with the restriction enzyme and the vector 2 to
It can be prepared by mixing and ligating at a molar ratio of 5: 1. That is, a vector for inserting the gene is obtained by digesting the vector with a restriction enzyme. Reaction conditions such as reaction temperature and reaction time for digestion reaction with a restriction enzyme can be appropriately set according to the selected enzyme. The vector used as the recombinant vector is not limited, and for example, a plasmid vector, a bacteriophage vector, a retrovirus vector, a baculovirus vector, a papillomavirus vector or the like can be used. In particular,
For example, pUC19DNA (Takara Shuzo Co., Ltd.), pT
YB-1 or pTYB-11 (New England B
ioLabs (registered trademark)) can be used. If necessary, these vectors can be purified by a purification means such as phenol extraction, and further concentrated by a concentration means such as an ethanol precipitation method.
In order to prevent self-ligation of the vector fragment, for example, phosphatase treatment with alkaline phosphatase derived from calf small intestine may be performed. Then, the gene and the vector are mixed, and a ligase such as T
A recombinant vector can be obtained by allowing 4DNA ligase to act. Specifically, a commercially available ligation kit such as DNA Ligation Kit Ve
r. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.) or Ligat
Ionhigh (trade name, manufactured by Toyobo Co., Ltd.) is used to perform ligation under prescribed conditions,
A recombinant vector can be obtained.

The above recombinant vector is introduced into a host by a known transformation method. This transformation is carried out by a conventional method (for example,
Methods Enzymol., 204, 63-113, 19
1991). As the host cell in the present invention, any cell can be used without limitation as long as it can replicate the recombinant vector. Specific examples of the host cell include microorganisms such as Escherichia coli and yeast, insect cells such as sf9 strain, or CO.
Animal cells such as S cells can be mentioned. Specifically, for example, Escherichia coli DH5α, JM109, ER
2566 or Epicurian Coli® BL21-CodonPlus ™ Compe.
tent Cells (trade name, manufactured by Toyobo Co., Ltd.)
Can be used.

According to a further embodiment of the present invention, the oxidoreductase according to the present invention is industrially produced by culturing a transformant transformed with a recombinant vector containing the oxidoreductase gene. It can be manufactured advantageously.

As the transformant used for the production of the enzyme, the transformant using the above recombinant vector can be used as it is. The insert fragment of the recombinant vector may be subcloned into another new expression vector, or a transformant obtained by introducing the recombinant vector into a new host cell can be used. In these cases, the selection of appropriate host cells and vectors, the method of use, etc. are known to those skilled in the art, and among them, a system suitable for expressing the oxidoreductase gene of the present invention can be arbitrarily selected. You can For example, the vector and host cell can be selected from those exemplified in the above gene cloning step.

The culture conditions for the transformant can be appropriately selected depending on the host cell and are not limited to particular conditions. Culturing can be carried out under the medium and culture conditions usually used in this technical field. For example, glucose, glycerol, starch and the like can be used as the carbon source, and ammonium salts such as ammonium sulfate, ammonium nitrate and ammonium acetate, nitrates, tryptone and peptone can be used as the nitrogen source. In addition, trace amounts of inorganic metals, vitamins and growth promoting factors such as yeast extract containing thiamine and biotin may be added. These medium components may have any concentrations as long as they do not inhibit the growth of the present microorganism. The medium is usually adjusted to pH 7.0 to 7.5 and sterilized before use. The range of culture temperature may be any temperature as long as the microorganism can grow, and it is usually preferable to culture at 30 to 37 ° C. When the present microorganism is liquid-cultured, shaking culture or aeration-agitation culture is preferable. The culturing time varies depending on various culturing conditions, but in the case of shaking culturing or aeration stirring culturing, it is suitable to cultivate for 12 to 36 hours. Alternatively, after culturing at 15 to 37 ° C. until the absorbance at 550 to 600 nm becomes 1 to 6, isopropylthiogalactoside (hereinafter, abbreviated as IPTG) is added and further cultivated to induce the expression of the enzyme. Good.

After completion of the culture, a crude enzyme solution in which insoluble matters such as cell walls of cells are removed from the culture solution can be obtained by a usual method such as centrifugation, filtration or coprecipitation. Alternatively, a concentrated solution of the crude enzyme solution can be obtained by means known to those skilled in the art. The crude enzyme solution thus obtained is
Further, it can be purified by using a usual enzyme purification method such as ammonium sulfate fractionation method, organic solvent fractionation method, dialysis, isoelectric focusing method or column chromatography, alone or in combination. In addition, a commercially available protein purification system such as IMPACT (trade name) -CN (trade name,
New England BioLabs (registered trademark)) may be used for purification.

The redox reaction using the enzyme can be carried out by a known method. The product obtained by the reaction can also be obtained by a well-known method. For example, an oxidoreductase can be expressed by culturing the transformant, and a substrate can be added thereto to carry out an oxidoreduction reaction. The product of the reaction can be obtained, for example, by column chromatography. Furthermore, the product can be highly purified by reverse layer chromatography. The yield and optical purity of the product can be obtained by known methods.

[0021]

The present invention will be described with reference to the following examples.
The present invention is not limited to this embodiment.

Example 1 Two types of primers were designed based on the nucleotide sequence of the Bacillus subtilis yueD gene (SEQ ID NO: 2) and synthesized by the phosphite-triester method. The sequence of the synthesized primer is 5'-GGCGAAGCTTGGAAC.
TTTTATCATCATC-3 '(SEQ ID NO: 5) and 5'-CGCGGATCTCACAAAAACTCT
TTAATATC-3 '(SEQ ID NO: 6). AAGCTT in SEQ ID NO: 5 and GGATCC in SEQ ID NO: 6 are each a restriction enzyme identification sequence.

Example 2 Based on the nucleotide sequence of the ORF region of Saccharomyces cerevisiae YIR036C (SEQ ID NO: 4), two types of primers were designed and synthesized by the phosphite-triester method. The sequence of the synthesized primer is 5'-GGCGAA
GCTTGGGCAAGGTTTATTTTGAT-3 '
(SEQ ID NO: 7) and 5'-CGCGGATCCTAG
CCCTGCACCGGCCC-3 ′ (SEQ ID NO: 8). AAGCTT in SEQ ID NO: 7 and GGATCC in SEQ ID NO: 8 are identification sequences of restriction enzymes, respectively.

Example 3 (1) Preparation of Bacillus subtilis DNA A standard strain of Bacillus subtilis (S.
The cells of registration number BG13959 in ubilist are liquid medium (Hear infusion broth (Hear
t infusion broth (manufactured by Difco) 20g
/ L) for overnight culture. After completion of the culture, centrifugation is performed, and the supernatant is removed to obtain bacterial cells, and the bacterial cells are GenT of Gen Torukun (trademark, manufactured by Takara Shuzo Co., Ltd.) for yeast and Gram-positive bacteria.
Immediately after adding 180 μl of LE solution I, the mixture was vigorously stirred for 20 seconds by vortexing. Then GenTLE Solution I
20 μl of I was added and vortexed for 5 seconds,
After incubating at 70 ° C. for 10 to 15 minutes to lyse the cells, 100 μl of GenTLE solution III was added and mixed well. After allowing to stand in ice for 5 minutes, centrifuge, transfer the supernatant to another tube, add an equal amount of isopropanol,
Well mixed. Then, the mixture was further centrifuged to remove the supernatant, the precipitate was washed with 70% ethanol, and briefly centrifuged for a few seconds to remove the supernatant. The precipitate was washed with TE buffer (10 mM Tris-HCl,
Dissolve in 300 μl of 1 mM EDTA, pH 7.5),
7.5 M ammonium acetate 150 μl and ethanol 45
0 μl was added and mixed, and the filamentous DNA precipitate was entangled with the tip of a pipette tip and dissolved in 300 μl of TE buffer.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment The oxidoreductase gene was amplified using the two kinds of oligonucleotides prepared in Example 1 as primers. That is, 1 μg of the DNA prepared in (1), 2.5
4 μm of dNTP in mM, 2 μm each of 25 μM primer
1, 2 units of TaKaRaEx Taq (trade name, manufactured by Takara Shuzo Co., Ltd.), 0.4 μl of anti-Taqhigh (trade name, manufactured by Toyobo Co., Ltd.) and 10 μl of 10 times concentrated ExTaq buffer, and pure water was added to make 100 μm in total.
1 and PCR was performed. The PCR was carried out by incubating at 94 ° C for 1 minute and then at 94 ° C for 1 minute and 7 minutes.
10 cycles of 3 minutes at 2 ° C as one cycle,
1 cycle at 94 ° C for 1 minute, 2 minutes at 65 ° C and 1 minute at 72 ° C for 10 cycles, then 1 cycle at 94 ° C for 1 minute, 2 minutes at 60 ° C and 1 cycle at 72 ° C. This was carried out by repeating this for 15 cycles.

Next, 0.5 μg of the PCR product, 20 units each of restriction enzymes HindIII and BamHI, and a buffer solution (200 mM Tris-HCl (pH 8.5), 1
Pure water is added to 5 μl of 00 mM magnesium chloride, 5 mM dithiothreitol, 660 mM potassium acetate to make a total volume of 50 μl. The PCR product was digested by incubating the reaction solution at 37 ° C. for 2 hours. Then, the PCR product was further developed by electrophoresis using 1.5% agarose gel to obtain the desired DN.
The fraction containing the A fragment was excised from the gel and prep-A-
Gene DNA Purification Kit
(Trade name, manufactured by Bio-Rad) was used to collect the PCR product. That is, an agarose gel containing a DNA fragment was dissolved in 1 ml of Prep-A-Gene binding buffer, and P
DNA was added to 20 μl of rep-A-Gene matrix.
Was adsorbed. Then, after washing twice with 600 μl of Prep-A-Gene washing solution, it was incubated in 30 μl of TE buffer at 50 ° C. for 5 minutes to elute and collect the DNA fragment.

(3) Preparation of vector 2 μg of pUC19 DNA (manufactured by Takara Shuzo), 20 units each of restriction enzymes BamHI and HindIII, buffer (200 mM Tris-HCl (pH 8.5),
5 mM of 100 mM magnesium chloride, 5 mM dithiothreitol, 660 mM potassium acetate) and E. coli-derived alkaline phosphatase (Takara Shuzo Co., Ltd.)
Add pure water to 0.4 unit to make a total volume of 50 μl.
The vector was digested by reacting for 2 hours at ° C. Then, the vector was purified by phenol extraction and further concentrated by the ethanol precipitation method.

(4) Ligation of PCR product and pUC19 vector PCR product prepared in (2) and pUC prepared in (3)
19 in a 2: 1 molar ratio and mixed with DNA Ligati
on Kit Ver. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.) buffer A is added in an amount 4 times that of the DNA solution, and the enzyme solution B is added in the same amount as the DNA solution. 1 at 16 ° C
Ligation was performed for a time to obtain a recombinant vector.

(5) Escherichia coli DH5α strain (Gibco BRL Life Technologies (Gibc)
oBRL Life Technologies)) were transformed (Methods Enzymol., Vol. 204, 63-).
113, 1991). That is, 10 μl of the ligation solution containing the recombinant vector and 200 μl of competent cells of the DH5α strain were gently mixed, and then 30
Let stand for a minute. Then, it was incubated at 42 ° C. for 60 seconds and cooled in ice for 1 minute to obtain a transformant.

(6) The transformant obtained in the method (5) for producing oxidoreductase was charged with SOC medium (tryptone 2).
0 g / l, yeast extract 5 g / l, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM glucose)
1 ml was added and incubated at 37 ° C. for 1 hour. After the incubation, the bacterial cell solution of the transformant was treated with ampicillin-containing LB agar medium (trypton 10 g /
1, yeast extract 5 g / l, sodium chloride 10 g / l,
It was applied to agar 15 g / l, pH 7.4) and cultured overnight at 37 ° C. to obtain colonies of oxidoreductase gene-containing transformant. The colony was cultured with shaking in an LB liquid medium containing ampicillin at 37 ° C. overnight. After culturing, the cells collected by centrifugation were disrupted by ultrasonication and centrifuged again. The supernatant was fractionated with ammonium sulfate, and the protein-containing fraction was dialyzed to obtain the oxidoreductase.

Example 4 (1) Amplification of Gene by PCR and Preparation of Insertion Fragment Two types prepared in Example 2 using S288C strain-derived Saccharomyces cerevisiae genomic DNA (manufactured by Promega Corporation) as DNA Example 3 except that the primer of Example 1 was used as a primer.
PCR was carried out in the same manner as (2). Then the P
Example 3 except that the CR product was used as the PCR product
The PCR product was digested with HindIII and BamHI in the same manner as in (2). Furthermore, DNA was purified in the same manner as in Example 3 (2) except that the PCR product treated with the restriction enzyme was used as the PCR product.

(2) Preparation of recombinant vector pUC19 was treated in the same manner as in Example 3 (3) to prepare a vector. Then, ligation was performed in the same manner as in Example 3 (4) except that the PCR product obtained in (1) was used as a PCR product to obtain a recombinant vector.

(3) A transformant was obtained in the same manner as in Example 3 (5) except that the recombinant vector prepared in the method (2) for producing a oxidoreductase gene-containing trait was used. It was

(4) Redox enzyme was obtained in the same manner as in Example 3 (6) except that the transformant obtained in the method (3) for producing redox enzyme was used as the transformant.

Example 5 (1) Synthesis of oligonucleotides Two kinds of oligonucleotides were synthesized by the known phosphorous acid-triester method. The sequence of the synthesized oligonucleotide is 5'-GGTGGTTGCTCTTCCAAC.
ATGGAACTTTATTATCATCACC-3 '
(SEQ ID NO: 9) and 5'-CGGCTGCAGCTA
CAAAAAACTCTTTAATATC-3 '(SEQ ID NO: 10). GCTCTTC in SEQ ID NO: 9,
And CTGCAG in SEQ ID NO: 10 are identification sequences of restriction enzymes.

(2) Amplification of oxidoreductase gene PCR was performed using Bacillus subtilis DNA as a sample in the same manner as in Example 3 (2) except that the two kinds of primers prepared in (1) were used as primers. went. The PCR product was developed by electrophoresis using 1.5% agarose gel, and the fraction containing the target DNA fragment was cut out from the gel. Then, in the same manner as in Example 3 (2) except that this fraction was used as a fraction containing the target DNA fragment, Prep-A-Gene Purificatio was prepared.
PC with n Kit (trade name, manufactured by Bio-Rad)
The R product was purified. 0.5 μg of the PCR product, Pst
I10 unit and NEBuffer3 (trade name, manufactured by New England BioLabs) (1M sodium chloride, 500 mM Tris-HCl (pH 7.9), 1
Pure water was added to 5 μl of 00 mM magnesium chloride and 10 mM dithiothreitol to make a total volume of 50 μl. The PCR product was digested by incubating the reaction solution at 37 ° C. for 2 hours. After the restriction enzyme treatment, the PCR product was purified and recovered by the phenol extraction method and the ethanol precipitation method. The recovered PCR product contains Sa
pI 9 unit and NEBuffer 4 (trade name,
New England BioLabs) (500 mM
Potassium acetate, 200 mM trisacetic acid (pH 7.
9), 100 mM magnesium acetate, 10 mM dithiothreitol) (5 μl) was added, and pure water was further added to make a total volume of 50 μl. The PCR product was digested by incubating the reaction solution at 37 ° C. for 2 hours. After the restriction enzyme treatment with SapI, the reaction solution was added to 1.5
Electrophoresis was performed using a% agarose gel, and the fraction containing the target DNA fragment was cut out from the gel. Then, the Prep-A-Gene DNA Purific was prepared in the same manner as in Example 3 (2) except that the fraction was used.
The DNA was purified by an ation kit (trade name, manufactured by Bio-Rad).

(3) Preparation of vector Instead of 0.5 μg of the PCR product of (2), IMPACT was used.
(Product Name) -CN (New England BioLabs
The vector was digested in the same manner as (2) except that 2 μg of pTYB-11 vector in the kit (registered trademark) was used. The reaction solution was developed by electrophoresis using agarose gel, and the fraction containing the target DNA fragment was cut out from the gel. Then, in the same manner as in Example 3 (2) except that the fraction was used, Prep-A-Gene was used.
DNA Purification Kit (trade name,
The vector was prepared by BioRad.

(4) Preparation of transformant Same as Example 3 (4) except that the PCR product prepared in (2) was used as the PCR product and the pTYB-11 vector prepared in (3) was used as the vector. Was ligated to obtain a recombinant vector. Next, using the recombinant vector as a recombinant vector,
In the same manner as in Example 2 (5) except that the E. coli ER2566 strain in the T (trade name) -CN kit was used as a host,
A transformant was obtained.

(5) 1 ml of SOC medium was added to the transformant obtained in the method for producing oxidoreductase (4),
Incubated at 37 ° C for 1 hour. After the incubation, the bacterial cell solution of the transformant was applied to an LB agar medium containing ampicillin and cultured overnight at 37 ° C. to obtain a colony of a transformant containing an oxidoreductase gene. Then, the colony was shake-cultured at 37 ° C. in an LB liquid medium containing ampicillin until the absorbance at 600 nm became 5. After culturing, the collected bacterial cells were disrupted by ultrasonic waves and centrifuged again to obtain a supernatant. Then, a chitin affinity column (New England BioLabs (registered trademark)) was applied to a column buffer (20 mM Tris-HCl (pH 8.
0), 500 mM sodium chloride, 1 mM EDT
After equilibration with A), the supernatant was applied to a column to form an intein-chitin complex in the column. Then, after washing with a column buffer, a buffer containing 30 mM dithiothreitol (20 mM Tris-HCl (pH 8.0), 500 m) for inducing intein self-splicing.
The oxidoreductase protein was cleaved from the intein by an overnight incubation at 4 ° C. in M sodium chloride, 1 mM EDTA, 50 mM dithiothreitol). The next day, only the enzyme protein was eluted to obtain a purified protein.

Example 6 (1) Synthesis of oligonucleotides Two kinds of oligonucleotides were synthesized by the known phosphorous acid-triester method. The sequence of the synthesized oligonucleotide is 5'-GGTGGTTGCTCTTCCAAC.
ATGGGGCAAGGTTTATTTTGATT-3 '
(SEQ ID NO: 11) and 5'-CGGCTGCAGCT
AGCCCTGCACCGGCCCCAG-3 ′ (SEQ ID NO: 12). GCCTT in SEQ ID NO: 11
C and CTGCAG in SEQ ID NO: 12 are identification sequences of restriction enzymes, respectively.

(2) Amplification of oxidoreductase gene PCR was performed in the same manner as in Example 4 (1) except that the two kinds of primers prepared in (1) were used as primers. Then, the PCR product was developed by electrophoresis in the same manner as in Example 3 (2) except that the PCR product was used as the PCR product, and the Prep-A-Gene DNA was prepared.
The PCR product was recovered using the Purification Kit (trade name, manufactured by Bio-Rad). PC concerned
R product 0.5 μg, PstI 10 units and NE
Buffer3 (trade name, New England BioL
Abs) (1M sodium chloride, 500 mM Tris-hydrochloric acid (pH 7.9), 100 mM magnesium chloride, 10 mM dithiothreitol) (5 μl) was added with pure water to a total volume of 50 μl. The PCR product was digested by incubating the reaction solution at 37 ° C. for 2 hours. After the restriction enzyme treatment, the PCR product was purified and recovered by the phenol extraction method and the ethanol precipitation method.
The recovered PCR product contains SapI 9 units and N
EBuffer4 (brand name, New England Bio
Made by Labs) (500 mM potassium acetate, 200 m
5 μl of M tris acetic acid (pH 7.9), 100 mM magnesium acetate, 10 mM dithiothreitol) was added, and pure water was added to make a total volume of 50 μl. The PCR product was digested by incubating the reaction solution at 37 ° C. for 2 hours. After the restriction enzyme treatment with SapI, the reaction solution was developed by electrophoresis using 1.5% agarose gel, and the fraction containing the target DNA fragment was cut out from the gel. Then, in the same manner as in Example 3 (2) except that the fraction was used, Prep-A-Gene was used.
The DNA was purified by the DNA Purification Kit (trade name, manufactured by Bio-Rad).

(3) Preparation of vector Instead of 0.5 μg of the PCR product of (2), IMPACT was used.
(Product Name) -CN (New England BioLabs
The vector was digested in the same manner as in (2) except that 2 μg of pTYB-11 vector in the kit (registered trademark) was used. The reaction solution was developed by electrophoresis using agarose gel, and the fraction containing the target DNA fragment was cut out from the gel. Then, in the same manner as in Example 3 (2) except that the fraction was used, Prep-A-Gene was used.
DNA Purification Kit (trade name,
The vector was prepared by BioRad.

(4) Preparation of transformant Same as Example 3 (4) except that the PCR product prepared in (2) was used as the PCR product and the pTYB-11 vector prepared in (3) was used as the vector. Was ligated to obtain a recombinant vector. Next, using the recombinant vector as a recombinant vector,
In the same manner as in Example 3 (5) except that the E. coli ER2566 strain in the T (trade name) -CN kit was used as a host,
A transformant was obtained.

(5) Redox enzyme gene-containing transformant was obtained in the same manner as in Example 5 (5) except that the transformant obtained in the method (4) for producing oxidoreductase was used as the transformant. A colony of bodies was obtained. Then, the colony was cultured in the same manner as in Example 5 (5) except that the colony was used. After culturing, the collected bacterial cells were disrupted by ultrasonic waves and centrifuged again to obtain a supernatant. A purified oxidoreductase was obtained in the same manner as in Example 5 (5) except that the supernatant was used as the supernatant.

Example 7 An SOC medium was added to the transformant obtained in Example 3 (5) for 1 m.
1 was added and incubated at 37 ° C. for 1 hour. After the incubation, the bacterial cell solution of the transformant was applied to an LB agar medium containing ampicillin and cultured overnight at 37 ° C. to obtain a colony of a transformant containing an oxidoreductase gene. LB liquid medium containing ampicillin 20
Inoculate 0 ml and do 3 until the absorbance at 600 nm becomes 5.
Cultured at 7 ° C. Then, IPTG and benzyl as a substrate were added to the culture solution so that the final concentrations were 0.5 mM and 1 mM, respectively, and the mixture was further cultured at 37 ° C. for 24 hours. After the completion of the culture, 40 ml of ethyl acetate was added to the culture solution, the mixture was stirred, and the ethyl acetate layer was separated by centrifugation. The operation was repeated twice more, and the obtained ethyl acetate layer 1
20 ml was concentrated. The obtained product was applied to a silica gel column using hexane: ether: acetic acid (volume ratio 45: 5: 1) as a developing solvent to fractionate a benzoin fraction. In addition, benzoin was highly purified by reverse phase high performance liquid chromatography. The yield was 81%.

Example 8 An oxidoreductase gene-containing transformant colony was obtained in the same manner as in Example 6 except that the transformant obtained in Example 4 (3) was used as the transformant. Culture was performed in the same manner as in Example 6 except that the colony was used as a colony. Then, IPTG and benzyl as a substrate were added to the culture solution so that the final concentrations were 0.5 mM and 1 mM, respectively, and the mixture was further cultured at 37 ° C. for 24 hours. After the completion of the culture, 40 ml of ethyl acetate was added to the culture solution, the mixture was stirred, and the ethyl acetate layer was separated by centrifugation. This operation was repeated twice more, and the resulting ethyl acetate layer 120 ml
Was concentrated. The resulting product was hexane: ether:
A column of silica gel using acetic acid (volume ratio of 45: 5: 1) as a developing solvent was applied to collect a benzoin fraction. In addition, benzoin was highly purified by reverse phase high performance liquid chromatography. Yield 85%, optical purity 94
% Ee (S) -benzoin.

[0047]

INDUSTRIAL APPLICABILITY According to the present invention, an oxidoreductase derived from Bacillus subtilis having a reducing ability for an aromatic diketone compound and, for example, asymmetric reduction of benzyl (S)
-Saccharomyces capable of producing benzoin
C. cerevisiae-derived redox enzymes, genes encoding these enzymes, vectors containing the genes, transformants transformed with the vectors, and methods for producing the enzymes are provided.

The present invention can be used to oxidize or reduce aromatic diketone compounds. In particular, if the oxidoreductase derived from Saccharomyces cerevisiae of the present invention is used, for example, benzyl can be asymmetrically reduced to produce (S) -benzoin.

[0049]

[Sequence list free text] SEQ ID NO: 5: 5'primer for cloning yueD gene SEQ ID NO: 6: 3'primer for cloning yueD gene SEQ ID NO: 7: 5'for cloning open reading frame of YIR036C Primer SEQ ID NO: 8: 3'primer for cloning the open reading frame of YIR036C SEQ ID NO: 9: 5'primer for cloning yueD gene SEQ ID NO: 10: 3'primer for cloning yueD gene SEQ ID NO: 11: 5'primer for cloning the open reading frame of YIR036C SEQ ID NO: 12: 3'primer for cloning the open reading frame of YIR036C

[Sequence list]                                SEQUENCE LISTING <110> Arakawa Kagaku Kogyo Kabushiki Kaisha <120> Oxido-reductases, Gene encoding the enzymes,       Transformants having the genes and Preparing the       enzymes <130> JP-12420 <140> <141> <160> 12 <170> PatentIn Ver. 2.0 <210> 1 <211> 243 <212> PRT <213> Bacillus subtilis <400> 1 Met Glu Leu Tyr Ile Ile Thr Gly Ala Ser Lys Gly Leu Gly Gln Ala   1 5 10 15 Ile Ala Leu Gln Ala Leu Glu Lys Gly His Glu Val His Ala Leu Ser              20 25 30 Arg Thr Lys Thr Asp Val Ser His Lys Lys Leu Thr Gln His Gln Ile          35 40 45 Asp Leu Ile Asn Leu Glu Glu Ala Glu Gln Gln Phe Glu Thr Leu Leu      50 55 60 Ser Ser Ile Asp Pro Asp Arg Tyr Ser Gly Ile Thr Leu Ile Asn Asn  65 70 75 80 Ala Gly Met Val Thr Pro Ile Lys Arg Ala Gly Glu Ala Ser Leu Asp                  85 90 95 Glu Leu Gln Arg His Tyr Gln Leu Asn Leu Thr Ala Pro Val Leu Leu             100 105 110 Ser Gln Leu Phe Thr Lys Arg Phe Ala Ser Tyr Ser Ser Lys Lys Thr         115 120 125 Val Val Asn Ile Thr Ser Gly Ala Ala Lys Asn Pro Tyr Lys Gly Trp     130 135 140 Ser Ala Tyr Cys Ser Ser Lys Ala Gly Leu Asp Met Phe Thr Arg Thr 145 150 155 160 Phe Gly Phe Glu Gln Glu Asp Glu Glu Leu Pro Val Asn Met Ile Ser                 165 170 175 Phe Ser Pro Gly Val Met Asp Thr Glu Met Gln Ala Val Ile Arg Ser             180 185 190 Ser Ser Lys Lys Asp Phe His His Ile Glu Arg Phe Arg Lys Leu Asn         195 200 205 Glu Thr Gly Ser Leu Arg Ser Pro Asp Phe Ile Ala Gly Thr Leu Leu     210 215 220 Ser Leu Leu Glu Lys Gly Thr Glu Asn Gly Arg Ile Tyr Asp Ile Lys 225 230 235 240 Glu Phe Leu <210> 2 <211> 732 <212> DNA <213> Bacillus subtilis <400> 2 atggaacttt atatcatcac cggagcgtca aaagggctgg gtcaagccat tgcattacag 60 gctttagaaa aggggcatga agtccatgcc ttatccagaa cgaaaacaga tgtctctcac 120 aaaaaactaa cgcagcatca aatagacctc atcaatctcg aagaagctga acagcaattt 180 gaaacattgc tctcatccat cgatccagat cgttattctg gtattaccct tatcaataac 240 gccggaatgg taacgccgat caaacgtgcc ggcgaagcgt ctcttgacga gcttcagcgc 300 cattatcagc tgaacctgac tgcgcccgtg cttttgagtc agctgtttac aaaacggttt 360 gcttcataca gcagcaaaaa gacggttgtc aacattactt caggagccgc caaaaatcca 420 tataagggat ggagcgcgta ttgcagttca aaagccgggc tcgacatgtt tacgaggaca 480 ttcggatttg aacaggagga tgaagagctg ccggtgaaca tgatttcgtt ctcacctgga 540 gtgatggaca ctgagatgca ggccgtcatc cgttcttcat cgaaaaagga tttccaccac 600 attgaacgat tccggaaatt aaatgaaaca ggaagccttc gcagtccgga ctttattgcc 660 ggcacgctgc tttctttact agaaaaaggg acggaaaacg gccgcattta tgatattaaa 720 gagtttttgt ag 732 <210> 3 <211> 263 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Gly Lys Val Ile Leu Ile Thr Gly Ala Ser Arg Gly Ile Gly Leu   1 5 10 15 Gln Leu Val Lys Thr Val Ile Glu Glu Asp Asp Glu Cys Ile Val Tyr              20 25 30 Gly Val Ala Arg Thr Glu Ala Gly Leu Gln Ser Leu Gln Arg Glu Tyr          35 40 45 Gly Ala Asp Lys Phe Val Tyr Arg Val Leu Asp Ile Thr Asp Arg Ser      50 55 60 Arg Met Glu Ala Leu Val Glu Glu Ile Arg Gln Lys His Gly Lys Leu  65 70 75 80 Asp Gly Ile Val Ala Asn Ala Gly Met Leu Glu Pro Val Lys Ser Ile                  85 90 95 Ser Gln Ser Asn Ser Glu His Asp Ile Lys Gln Trp Glu Arg Leu Phe             100 105 110 Asp Val Asn Phe Phe Ser Ile Val Ser Leu Val Ala Leu Cys Leu Pro         115 120 125 Leu Leu Lys Ser Ser Phe Val Gly Asn Ile Val Phe Val Ser Ser     130 135 140 Gly Ala Ser Val Lys Pro Tyr Asn Gly Trp Ser Ala Tyr Gly Cys Ser 145 150 155 160 Lys Ala Ala Leu Asn His Phe Ala Met Asp Ile Ala Ser Glu Glu Pro                 165 170 175 Ser Asp Lys Val Arg Ala Val Cys Ile Ala Pro Gly Val Val Asp Thr             180 185 190 Gln Met Gln Lys Asp Ile Arg Glu Thr Leu Gly Pro Gln Gly Met Thr         195 200 205 Pro Lys Ala Leu Glu Arg Phe Thr Gln Leu Tyr Lys Thr Ser Ser Leu     210 215 220 Leu Asp Pro Lys Val Pro Ala Ala Val Leu Ala Gln Leu Val Leu Lys 225 230 235 240 Gly Ile Pro Asp Ser Leu Asn Gly Gln Tyr Leu Arg Tyr Asn Asp Glu                 245 250 255 Arg Leu Gly Pro Val Gln Gly             260 <210> 4 <211> 792 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgggcaagg ttattttgat tacaggtgcc tcccgtggga ttggcctgca attggtgaaa 60 actgttatcg aagaggacga tgaatgcatc gtctacggcg tagcaagaac ggaagctggt 120 ctgcagtctt tgcaaagaga atacggtgca gacaaatttg tctatcgtgt cctcgacatc 180 acggacaggt ctcgaatgga agcgttggtg gaggaaatcc ggcaaaagca tggaaaactg 240 gacggtattg tcgcaaatgc ggggatgcta gaaccggtga agtccatctc ccagtccaac 300 tccgaacacg acatcaagca gtgggaacgg ctgttcgatg tgaacttttt cagcattgtc 360 tctttggtgg cactgtgttt acccctcttg aagagctcgc catttgtagg caacattgtc 420 ttcgtcagct ctggagccag tgtgaaacca tataacggat ggtcggcgta cggctgctcg 480 aaagccgcat taaaccactt tgccatggac attgccagtg aagagcccag tgataaagtg 540 cgtgccgtgt gtattgcacc gggcgtcgtt gacacgcaga tgcagaaaga tattagggaa 600 acattgggtc ctcagggcat gacacccaag gctctcgaga ggtttactca attgtacaag 660 acttcgtcac tgctggaccc aaaggtgcct gcggcggtac tagcgcaact cgtcctgaaa 720 ggtattcccg actctttgaa cggtcaatat ctccgctaca acgatgagcg actggggccg 780 gtgcagggct ag 792 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5'primer for cloning yueD gen e <400> 5 ggcgaagctt ggaactttat atcatcac 28 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3'primer for cloning yueD gen e <400> 6 cgcggatcct acaaaaactc tttaatatc 29 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5'primer for cloning open rea ding frame of YIR036C <400> 7 ggcgaagctt gggcaaggtt attttgat 28 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3'primer for cloning open rea ding frame of YIR036C <400> 8 cgcggatcct agccctgcac cggccc 26 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5'primer for cloning yueD gen e <400> 9 ggtggttgct cttccaacat ggaactttat atcatcacc 39 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3'primer for cloning yueD gen e <400> 10 cggctgcagc tacaaaaact ctttaatatc 30 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5'primer for cloning the open  reading frame of YIR036C <400> 11 ggtggttgct cttccaacat gggcaaggtt attttgatt 39 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3'primer for cloning the open  reading frame of YIR036C <400> 12 cggctgcagc tagccctgca ccggccccag 30

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Maruyama Reiji 52 Teramachi Keyadai, Teramachi, Toyama City No. 203 Tsukasa Heights Teramachi (72) Inventor Yasushi Itoi 21-502 Minamishinda, Daito City, Osaka Prefecture (56) References Ribonoctide re ductase in the arc haeon Pyrococcus furiosus: A critical enzyme in the eviction of D, PNAS, USA, January 1997, vol. 94, 475-478, NCBI, USA, June 20, 2000, http: // www. ncbi. nl m. nih. gov: 80 / entrez / query. fcgi? cmd = Retrieve & db = protein & list_uids = 7475814 & dopt = GenP (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 ZNA C12N 1/21 C12N 9/02 SwissProt / PIR / GeneBanKenSeq / / DDBJ / GeneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (11)

(57) [Claims] 1. An oxidoreductase comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. 2. An oxidoreductase encoded by the base sequence shown in SEQ ID NO: 4 in the sequence listing. 3. An oxidoreductase gene containing the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. 4. An oxidoreductase gene encoding the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing. 5. A stringent DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 4 of the Sequence Listing.
A protein or polypeptide obtained by expressing a DNA that hybridizes under conditions and having redox ability. 6. A recombinant vector containing the gene according to claim 3 or 4. 7. The recombinant vector according to claim 6, wherein the vector is pUC19. 8. A transformant transformed with the recombinant vector according to claim 6 or 7. 9. The transformant according to claim 8, wherein the host cell is a microorganism. 10. The transformant according to claim 9, wherein the microorganism is Escherichia coli. 11. A method for producing an oxidoreductase, which comprises culturing the transformant according to claim 8, 9 or 10 and collecting the oxidoreductase from the culture.