JP2002112783A - Oxydoreductase enzyme gene, cloning of the gene and method for producing the enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligonucleotide, to provide a method for amplification and cloning of a new oxydoreductase gene using the oligonucleotide as a primer, to obtain a recombinant vector containing the gene and a transformant transformed by the recombinant vector, and to provide a method for producing an oxydoreductase using the transformant. SOLUTION: The oligonucleotide is synthesized based on an oxydoreductase gene of Bacillus cereus FERM P-14210 that is provided by the inventors in the Patent Application Number 2000-257829, the new oxydoreductase gene is amplified by the PCR method using the oligonucleotide as a primer, and the recombinant vector containing the amplified product obtained by amplifying the new oxydoreductase gene by the PCR method is produced. Next, the transformant transformed by using the recombinant vector is cultured and the oxydoreductase is obtained by collection of the oxydoreductase from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel oligonucleotide, a method for amplifying and cloning a novel oxidoreductase gene using the oligonucleotide as a primer, a recombinant vector containing the gene, and transformation with the recombinant vector. And a method for producing an oxidoreductase using the transformant.

[0002]

2. Description of the Related Art Conventionally, Saccharomyces cerevisiae has been known as an oxidoreductase-producing microorganism capable of reducing diketone compounds. However, in the Saccharomyces cerevisiae, for example, one of aromatic diketone compounds, benzyl (be
When nzil) is reduced, racemic benzoin is produced. For this reason, the microorganism-producing enzyme has a disadvantage of low stereoselectivity (Chemistry Letters, 1191).
-1192, 1988). Xanthomonas oryzae is also known as a microorganism that produces an oxidoreductase capable of asymmetric reduction of benzyl, an aromatic diketone compound. When benzyl is asymmetrically reduced by the enzyme-producing microorganism, (R) -benzoin is stereoselectively produced as a reduction product (Chem
istry Letters, 1111-1112, 1985).

[0003] Saccharomyces cerevisiae reduces 4-chloroacetoacetate, an aliphatic diketone compound, to 4-hydroxybutanoate.
It is known that they produce various kinds of enzymes.
Not all genes of the seed enzymes have been isolated (Chemistry and Biology, No. 35, pp. 590-598, 1997), and their amino acid sequences also differ for the two enzymes.
It is only inferred from the genome sequence of S. cerevisiae (Biosci. Biotech. Biochem., No. 60, 1538-1539).
P. 1996).

[0004] Bacillus cereus
Is known to exhibit a reducing ability for the carbonyl group of 9-fluorenone which is a non-diketone compound (Biosci. Biotechnol. Biochem., No. 62, 814-815).
P. 1998), the reducing ability of the microorganism to aromatic diketone compounds has not been reported at all.

[0005]

SUMMARY OF THE INVENTION The present inventors have conducted various studies under the above-mentioned circumstances, and as a result, have found that Bacillus cellius T
im-r01 (Research Institute of Biotechnology, Industrial Technology Institute, Ministry of International Trade and Industry, Accession No. FERM P-14210) has a reducing ability for aromatic diketone compounds, for example, asymmetric reduction of benzyl to give (S) -benzoin To produce a novel oxidoreductase that produces However, when a viable cell of the strain is used, the expression level of the oxidoreductase is greatly affected by the culture conditions. In addition, when the oxidation-reduction reaction is carried out for a long time using the viable cells, a reverse reaction occurs and the yield of asymmetric reduction products is reduced.

Therefore, the inventors of the present application, in the earlier application,
A novel gene encoding oxidoreductase from chromosomal DNA of Bacillus cellius strain FERM P-14210 and an open reading frame (hereinafter referred to as O
After abbreviated as "RF", an oxidoreductase derived from the microorganism, a novel gene encoding the enzyme, a vector containing the gene, and a transformant transformed with the vector were provided. -25782
9).

The inventors of the present invention have conducted further studies based on such research results, and as a result, have obtained a prior application (Japanese Patent Application No. 2000-2).
If a specific sequence site of the novel oxidoreductase gene whose base sequence was determined in 57829) is used as a primer, an oxidoreductase gene can be obtained from various microorganisms, and a transformant containing the gene can be used. For example, they have found that an oxidoreductase exhibiting an asymmetric reducing ability for an aromatic diketone compound can be industrially advantageously produced, and have completed the present invention.

Accordingly, the present invention provides a novel oligonucleotide which can be used for amplification of an oxidoreductase gene, a method for amplifying and cloning a novel oxidoreductase gene using the oligonucleotide, a recombinant vector containing the gene, An object of the present invention is to provide a transformant transformed with a vector, and a method for producing an oxidoreductase using the transformant.

[0009]

Means for Solving the Problems The present inventors have solved the above-mentioned problems by providing the following inventions.

(1) An oligonucleotide having a nucleotide sequence substantially the same as the nucleotide sequence of 2 to 22 nucleotides at the 5 'end of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing. (2) an oligonucleotide having a nucleotide sequence substantially complementary to the nucleotide sequence of 2 to 23 nucleotides at the 3 'end of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing; (3) An oligonucleotide comprising a recognition sequence for a restriction enzyme at the 5 'end of the oligonucleotide according to (1). (4) An oligonucleotide comprising a recognition sequence for a restriction enzyme at the 5 'end of the oligonucleotide according to (2). (5) an oligonucleotide having the base sequence of SEQ ID NO: 1, 2, 3, or 4 in the sequence listing; (6) (a) Target oxidation-reduction contained in a sample, using as a primer at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) above Amplifying the enzyme gene, (b)
A method for producing a oxidoreductase gene-containing transformant, which comprises (c) obtaining a transformant transformed with the vector after inserting the amplified gene into a vector to prepare a recombinant vector. (7) The sample containing the oxidoreductase gene is a chromosomal DNA obtained from a strain belonging to the genus Bacillus, a DNA library of the chromosome, a cDNA library, or a recombinant vector containing the gene. The method for producing the oxidoreductase gene-containing transformant according to the above (6). (8) A sample containing an oxidoreductase gene was obtained from chromosomal DNA obtained from Bacillus
The method for producing the oxidoreductase gene-containing transformant according to the above (6), which is an NA library, a cDNA library, or a recombinant vector containing the gene. (9) The oxidoreductase gene-containing transformant according to (6), (7) or (8), wherein the two oligonucleotides according to (3) and (4) are used as primers. Manufacturing method. (10) The oxidoreductase gene-containing transformant according to (6), (7) or (8), wherein the two oligonucleotides described in SEQ ID NOs: 2 and 4 in the sequence listing are used as primers. Manufacturing method. (11) The above (6), (7), (8), (9) or (1), wherein the host cell to be transformed is a microorganism.
0) The method for producing the oxidoreductase gene-containing transformant according to the above. (12) The above (6), wherein the vector is pUC19 and the host cell to be transformed is Escherichia coli.
(7) The method for producing the oxidoreductase gene-containing transformant according to (8), (9) or (10). (13) A sequence obtained by a polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) as a primer, An oxidoreductase gene having the nucleotide sequence shown in SEQ ID NO: 5, 7, 10, 12, 14, 16, 18 or 20 (14) A sequence obtained by polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) as a primer, An oxidoreductase gene encoding the amino acid sequence shown in SEQ ID NO: 6, 8, 11, 13, 15, 17, 19 or 21 (15) An oxidoreductase defined by the gene according to (13) or (14). (16) A DNA or oligonucleotide that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to (13). (17) A protein or polypeptide having an amino acid sequence encoded by a DNA that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to (13). (18) A protein or polypeptide obtained by expressing a DNA that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to (13). (19) A recombinant vector containing the gene according to (13) or (14). (20) The recombinant vector according to the above (19), wherein the vector is pUC19. (21) A transformant transformed by the recombinant vector according to (19) or (20). (22) The transformant according to (21), wherein the host cell is a microorganism. (23) The transformant according to (21), wherein the microorganism is Escherichia coli. (24) The above (6), (7), (8), (9), (10),
(11) A method for producing an oxidoreductase, which comprises culturing an oxidoreductase gene-containing transformant obtained by the method according to (12) and collecting the oxidoreductase from the culture.

The oligonucleotide according to the present invention,
An oxidoreductase gene, a recombinant vector containing the gene, a transformant transformed with the recombinant vector, and an oxidoreductase can be obtained based on the embodiments described below. The method for producing the transformant and the method for producing the oxidoreductase can also be carried out based on the following embodiments. Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The novel oligonucleotides according to the present invention, that is, the oligonucleotides described in the sections (1) and (2) of "Means for Solving the Problems" have been disclosed by the present inventors in the earlier application ( (Japanese Patent Application 2000-257829)
Can be set based on the oxidoreductase gene of SEQ ID NO: 9 whose base sequence has been determined. In particular, in order to clone the full length ORF of the oxidoreductase gene, it is preferable to set based on about 20 bases at the 5 'or 3' end of SEQ ID NO: 9, for example, the oligonucleotide shown in SEQ ID NO: 1 or 3 is used. Can be set.

These oligonucleotides may further contain a recognition sequence for a restriction enzyme, a tag such as a histidine tag or a flag (FLAG) tag, or a green fluorescent protein at the 5 'end. When the amplified gene is cut out with a restriction enzyme in the subsequent steps, it is preferable to add a type II restriction enzyme recognition sequence to the 5 'end of the oligonucleotide. When the oligonucleotide contains a recognition sequence for a restriction enzyme, the recognition sequence for the restriction enzyme to be added can be appropriately selected according to the cloning site of the vector. The restriction enzyme is a type II restriction enzyme such as a restriction enzyme BamHI, EcoRI, HindIII, K, which can correctly digest a DNA strand at a position where a recognition sequence appears.
pnI, NcoI, NdeI, PstI, SacI, S
alI, SapI, SmaI, SphI or XbaI
It is preferable to select a recognition sequence such as In addition to the restriction enzyme recognition sequence, several bases called clamps that assist digestion by the restriction enzyme may be added. Further, for example, pUC-based vectors, pTYB-based vectors (New England BioLabs (registered trademark) (NE
W ENGLAND BioLabs) or pGEX-based vector (Amersham Pharmacia Biotech)
Depending on the type of vector used, a few bases may be added between the recognition sequence of the restriction enzyme and the oligonucleotide encoding the oxidoreductase. Specific examples of such an oligonucleotide according to the present invention include an oligonucleotide having the base sequence shown in SEQ ID NO: 2 or 4.

The oligonucleotide according to the present invention can be synthesized by a method known per se, for example, a phosphorous acid-triester method. For primer synthesis, a primer synthesizer can be used.
A synthesizer Model 380A, Model 381A or Model 394 (trade name, manufactured by PE Applied Biosystems) can be used.

As a sample for amplifying a gene according to the present invention, any sample containing a target oxidoreductase gene can be used without limitation. For example, chromosomal DNA prepared from a microbial cell containing the target oxidoreductase gene by lysing the cell, if necessary, for example, using Gentoru-kun (trade name, manufactured by Takara Shuzo Co., Ltd.). A chromosomal DNA library and a cDNA library of the strain, a vector containing the target gene, and the like can be used as appropriate. As an example of the sample, a strain belonging to the genus Bacillus can be used. Specifically, for example, Bacillus Serius FERM P-
14210, Bacillus cellius IFO3001, IFO3002, I available from the Fermentation Research Institute
FO3003, IFO3132, IFO3457,
IFO3466, IFO3514, IFO356
3, IFO3836, IFO13494, IFO
13597, IFO 13690 and IFO 153
05, and IAM1029, IAM1110, and IAM1 available from the Institute for Molecular and Cell Biology of the University of Tokyo.
656 and chromosomal DN obtained from IAM1729
A, a DNA library of the same chromosome, a cDNA library or a plasmid containing the same gene. When the benzyl reduction activity of the cells was examined, all the cells had the same asymmetric benzyl reduction ability as the FERMP-14210 cells.

According to the present invention, the desired oxidoreductase gene can be amplified by a method known per se, for example, the polymerase chain reaction (hereinafter abbreviated as PCR). When amplifying by the PCR method, the sample containing the target gene contains an oligonucleotide as a primer for gene amplification, a thermostable polymerase, dNTPs (deoxy ATP, deoxy TTP, deoxy GTP and deoxy CTP) and a reaction buffer. To the solution,
The target gene can be amplified by annealing the oligonucleotide to the target gene. Here, the thermostable polymerase is a DNA-dependent 5 ′ → 3 ′ DNA synthase that has an optimum temperature of 70 ° C. or higher and is hardly inactivated even at 90 ° C. The PCR conditions of the gene include, for example, incubation at 94 ° C. for 1 minute, 10 cycles of 94 ° C. for 1 minute and 72 ° C. for 3 minutes, and further 94 cycles.
1 minute at 65 ° C, 2 minutes at 65 ° C and 1 minute at 72 ° C.
This is repeated for 10 cycles and 94
1 minute at 60 ° C, 2 minutes at 60 ° C and 1 minute at 72 ° C.
This can be carried out by performing 15 cycles as a cycle (Genes to Cells, No. 5, pp. 111-125,
year 2000).

The gene amplification reaction can be performed using one of the oligonucleotides described above. Further, two or more oligonucleotides can be used in combination. As a combination of two or more oligonucleotides, for example, [A] (1) an oligonucleotide having a nucleotide sequence substantially the same as the nucleotide sequence of 2 to 22 nucleotides at the 5 'end of the oxidoreductase shown in SEQ ID NO: 9 (2) 3 ′ terminal 2-2 of oxidoreductase gene shown in SEQ ID NO: 9
Combination with an oligonucleotide having a base sequence substantially complementary to three base sequences, or preferably [B]
(1) 5 ′ terminal 2 to 2 of the oxidoreductase shown in SEQ ID NO: 9
An oligonucleotide having a base sequence substantially the same as the 22 base sequences and containing a recognition sequence for a restriction enzyme on the 5 'end side, and (2) an oxidoreductase represented by SEQ ID NO: 9 It can be carried out using a combination of an oligonucleotide having a base sequence substantially complementary to the 23 base sequences and containing a recognition sequence for a restriction enzyme at the 5 'end. Specific examples of the combination of the oligonucleotides include, for example, a combination of an oligonucleotide having the base sequence shown in SEQ ID NO: 2 and an oligonucleotide having the base sequence shown in SEQ ID NO: 4. Further, the gene amplification reaction can be performed using a commercially available thermostable polymerase. Specific examples of such a polymerase include, for example, TaKaRa Ex Taq (trade name, manufactured by Takara Shuzo Co., Ltd.). If the target oxidoreductase gene cannot be amplified by PCR using the polymerase, the cause may be a mismatch between the base sequence of the target gene and the base sequence of the primer. In such a case, a DNA polymerase having an excellent proofreading function, for example, KOD DNA p
oligomerase (trade name, manufactured by Toyobo Co., Ltd.),
KOD DNA plus DNA polymerase
se (trade name, manufactured by Toyobo Co., Ltd.) or Pylob
est (trade name) DNA polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.), preferably KOD DNA p
rus DNA polymerase (trade name, manufactured by Toyobo Co., Ltd.) or Pyrobest (trade name) D
NA polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.), more preferably Pyrobest (trade name) DN
The cause can be avoided by using A polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.). For example, IFO3001, IFO3002, IFO
3003, IFO3563, IFO3836, IFO1
3597 and IFO15305, and IAM16
When 56 and IAM1729 cells are used as samples, the target gene can be amplified by TaKaRa Ex Taq (trade name, manufactured by Takara Shuzo Co., Ltd.). However, IFO3132, IFO3457, IFO346
6, IFO3514, IFO13494, IFO136
90, IAM1029 and IAM1110 are TaKa
It cannot be amplified by Ra Ex Taq (trade name, manufactured by Takara Shuzo Co., Ltd.). Of these, IFO3466, IF
O13494 and IAM1029 are KOD DNA
By using a polymerase (trade name, manufactured by Toyobo Co., Ltd.), the target gene can be amplified from the strain. IFO3132, IFO3457, I
FO3514, IFO13690 and IAM1110
Indicates that even when KOD DNA polymerase was used, the target gene could not be amplified,
yrobest (trade name) DNA Polymeras
e (trade name, manufactured by Takara Shuzo Co., Ltd.).

After the gene amplification reaction, the PCR product is developed by, for example, electrophoresis using a 1.5% agarose gel, and a fraction containing a band of about 800 bp is cut out. Then, for example, Prep-A-Gene DNA Pur
By using an identification kit (trade name, manufactured by Bio-RAD), a target PCR fragment can be purified.

When an oligonucleotide containing a recognition sequence for a restriction enzyme is used as a primer, a DNA fragment of the amplified gene is reacted with a restriction enzyme that recognizes a recognition sequence for the restriction enzyme contained in the oligonucleotide primer. Thus, a desired gene fragment can be obtained.

The recombinant vector containing the oxidoreductase gene can be prepared by mixing and ligating the above gene fragment and vector at a molar ratio of 2 to 5: 1. That is, first, a vector is treated with a restriction enzyme to substantially completely digest the vector to obtain a vector for inserting a DNA fragment. The reaction conditions such as the reaction temperature and the reaction time of the digestion reaction with the restriction enzyme can be appropriately set according to the selected enzyme.

There are no restrictions on the vectors that can be used for recombination.
For example, any of a plasmid vector, a bacteriophage vector, a retrovirus vector, a baculovirus vector or a papillomavirus vector can be used. Specifically, for example, pUC19
DNA (manufactured by Takara Shuzo Co., Ltd.), pTYB-1 or pT
It is preferable to use YB-11 (manufactured by New England BioLabs (registered trademark)). These vectors can be purified by a purification means such as phenol extraction if necessary, and further concentrated by a concentration means such as an ethanol precipitation method. Further, in order to prevent self-ligation of the vector fragment, phosphatase treatment with, for example, alkaline phosphatase derived from calf small intestine may be performed. Next, the amplified gene obtained in the previous step and the vector are mixed, and a ligase such as T4 DNA ligase is allowed to act on the mixed gene to obtain a recombinant vector. Specifically, a ligation kit, for example, DNA Ligation Kit Ve
r. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.) or Ligat
By performing ligation using ion high (trade name, manufactured by Toyobo Co., Ltd.) under specified conditions, a recombinant vector can be obtained.

When an oligonucleotide not containing a restriction enzyme recognition sequence is used as a primer, PC
A commercially available vector suitable for cloning the amplified fragment by R, for example, pGEM®-T Ea
pGEM (registered trademark) -T Easy Vector, which is included in sy Vector System I (trade name, manufactured by Promega) can be used. With this vector and T4 ligase, P
Ligation can be performed without restriction enzyme treatment of the amplification product and vector by CR, and a recombinant vector containing an oxidoreductase gene can be prepared.

When a combination of an oligonucleotide containing a recognition sequence for a restriction enzyme and an oligonucleotide not containing the recognition sequence is used as a primer,
By using a fragment cloning vector, a recombinant vector containing an oxidoreductase gene can be prepared without restriction enzyme treatment as described above.

The above-mentioned recombinant vector is introduced into a host by a known transformation method. This transformation can be suitably performed by a conventional method (for example, Methods Enzymol., No. 204, pp. 63-113, 1991). The host cell in the present invention can be used without limitation as long as the recombinant vector is replicable. Specific examples of host cells include, for example, microorganisms such as Escherichia coli and yeast, insect cells such as sf9 strain, and animal cells such as COS cells. In particular,
E. coli DH5α, JM109, ER2566 or Ep
icultianColi (R) BL21-Cod
onPlus (brand name) Competent Cell
It is preferable to use s (trade name, manufactured by Toyobo Co., Ltd.).

Next, if necessary, the nucleotide sequence of the oxidoreductase gene contained in the thus obtained transformant and the amino acid sequence of the oxidoreductase encoded by the gene can be determined appropriately. Specifically, for example, after culturing a transformant, FlexiPrep (trade name)
Using Kit (trade name, manufactured by amersham pharmacia biotech), plasmid DNA was prepared from the cells, and ABI PRIZM was prepared.
(Product name) BigDye (Product name) Termina
tor Cycle Sequencing Read
The base sequence can be determined by y Reaction Kit (trade name, P.E. Applied Biosystems). In accordance with the sequencing method, we have identified Bacillus serius IFO3001, IFO
3003, IFO3132, IFO3563, IFO1
3597, IFO15305, IAM1029 and I
The nucleotide sequence of the oxidoreductase genes obtained from the AM1110 strain was determined, and at the same time, the amino acid sequence encoded by the oxidoreductase encoded by those genes was estimated.

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 the recombinant vector containing the oxidoreductase gene. It can be advantageously manufactured.

As the transformant used for the production of the enzyme, the transformant using the above-mentioned recombinant vector can be used as it is. Further, the insert of the recombinant vector may be subcloned into another new expression vector, or a transformant having the recombinant vector introduced into a new host cell may be used. In these cases, the selection of appropriate host cells and vectors, and methods of use are known to those skilled in the art, and a system suitable for expressing the oxidoreductase gene of the present invention can be arbitrarily selected from them. Can be. For example, the vector and the host cell can be selected from those exemplified in the gene cloning step.

Culture conditions for the transformant can be appropriately selected depending on the host cell, and are not limited to specific conditions. The cultivation can be carried out under the medium and culturing 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, nitrate, tryptone, peptone and the like can be used as the nitrogen source. Other trace amounts of inorganic metals, vitamins, growth promoting factors such as thiamine and yeast extract containing biotin may be added. These medium components need only have a concentration that does not inhibit the growth of the present microorganism. The medium is usually adjusted to pH 7.0 to 7.5 and used after sterilization. The range of the culture temperature may be any temperature at which the present microorganism can grow, and it is usually preferable to culture at 30 to 37 ° C. When the present microorganism is subjected to liquid culture, shaking culture or aeration-agitation culture is preferred. The culturing time varies depending on various culturing conditions, but in the case of shaking culturing or aeration and stirring culturing, culturing for 12 to 36 hours is appropriate. Alternatively, enzyme expression may be induced by culturing at 15 to 37 ° C. until the absorbance at 550 to 600 nm becomes 1 to 6, followed by adding isopropylthiogalactoside (IPTG) and further culturing.

After completion of the cultivation, a crude enzyme solution from which insoluble substances such as cell walls have been removed from the culture solution can be obtained by a conventional 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, the enzyme can be purified by a conventional enzyme purification method such as ammonium sulfate fractionation method, organic solvent fractionation method, dialysis, isoelectric point precipitation method or column chromatography, alone or in combination. Also, commercially available protein purification systems such as IMPACT (trade name) -CN (trade name,
New England BioLabs (registered trademark)).

In the present invention, the nucleotide sequence of the oligonucleotide used for gene amplification is set based on the nucleotide sequence of the oxidoreductase gene shown in SEQ ID NO: 9, and Was determined by the following procedure, as described in Japanese Patent Application No. 2000-257829, which was a prior application of the present inventors.

That is, Bacillus serius FERM
Cells of the P-14210 strain are lysed, and chromosomal DNA is recovered by a known method. After the obtained chromosomal DNA was digested with the restriction enzyme Sau3AI, a 2 to 4 kb DNA fragment mixture was obtained by agarose gel electrophoresis. The DN
For the A fragment, a part of the protruding end of DNA was filled in with a Klenow fragment in order to prevent ligation by two or more inserted fragments.

Next, the vector p obtained by previously digesting completely with the restriction enzyme SalI and pretreating with the Klenow fragment was obtained.
UC19 and the DNA fragment mixture were mixed, and a known ligation was performed to obtain a recombinant vector.

The thus obtained recombinant vector is used in a conventional manner (Methods Enzymol., No. 204, pp. 63-113, 1991).
E. coli was transformed according to

Next, a transformant colony was formed on an agar medium containing benzyl, and a transformant containing the oxidoreductase gene DNA was selected by a halo formed around the colony.

From the transformant thus selected, a recombinant vector (the present inventors named the recombinant vector pUC-BC-B1) was recovered, and then the Bacillus cellius contained in the vector was recovered. The nucleotide sequence of the chromosomal DNA fragment was determined using a commercially available kit EZ :: TN (trade name) <
KAN-1> Insertion Kit (trade name, Epicentre Technologies)
Corporation)) by the dideoxy method.

Several ORFs considered from the base sequence
The amino acid sequence for each was determined by DDBJ / EMB
The homology with the known amino acid sequence was searched in the amino acid database of L / GenBank international base sequence database. As a result, the amino acid sequence consisting of 249 residues has the amino acid sequence encoded by the yueD gene of Bacillus subtilis having similarity to sepiapterin reductase, and the ORF Y of Saccharomyces cerevisiae.
It was found that they had 40% and 30% similarity to the amino acid sequence encoded by IR036C, respectively. On the other hand, the kit EZ :: TN (trade name) <K
When the transposon was inserted into the ORF encoding the 249 amino acid residues by insertion of the transposon using AN-1> Insert Kit, no halo was formed. It is thought to encode an enzyme. The base sequence shown in SEQ ID NO: 9 shows the base sequence of the ORF including the stop codon.

[0037]

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

Example 1 Two oligonucleotides were designed based on the nucleotide sequences at the 5 'and 3' ends in the nucleotide sequence of SEQ ID NO: 9 and synthesized by the phosphorous acid-triester method. The sequence of the synthesized oligonucleotide was 5'-GGCGAAG
CTTGCGCTACGTTATCATAACAG-3
'(SEQ ID NO: 2) and 5'-CGCGGATCCCTA
TTCATCAATTCTAATAAC-3 ′ (SEQ ID NO: 4). A in the nucleotide sequence shown in SEQ ID NO: 2
AGCTT and GGATCC in the nucleotide sequence shown in SEQ ID NO: 4 are each a recognition sequence of a restriction enzyme.

Example 2 (1) Preparation of recombinant vector for oxidoreductase gene Escherichia coli DH5 transformed with pUC-BC-B1
The α strain was used in a 1.5 ml LB medium (medium composition: tryptone 10 g / l, yeast extract 10 g / l, sodium chloride 5
g / l, pH 7.0) and cultured with shaking at 37 ° C for 24 hours. After culturing, the bacterial solution is centrifuged to collect the cells, and a commercially available plasmid DNA preparation kit, FlexiPrep
(Trade name) Plasmid DNA was purified using Kit (trade name, manufactured by Amersham Pharmacia Biotech).
That is, the cells were collected after the culture, and the cells were added to the solution I (1
00 mM Tris-HCl (pH 7.5), 10 mM ED
Add 200 μl of TA, 400 μg / ml RNase I), and suspend with vortex. Then, solution II
(1M sodium hydroxide, 5.3% (weight / volume) S
DS) After adding 200 μl and mixing gently, the solution II
200 μl of I (3M potassium, 5M acetic acid) was added and mixed. After isopropanol precipitation was performed on the supernatant obtained by centrifugation, a DNA precipitate was obtained by centrifugation. Sepha
glass (trade name) FP (7M guanidine-hydrochloric acid, 50
mM Tris-HCl (pH 7.5), 10 mM EDT
A) 150 μl was added and suspended to dissolve the DNA. Then, the supernatant was removed by centrifugation, and the washing buffer (20 mM
Tris-HCl (pH 7.5), 2 mM EDTA, 20
0 mM sodium chloride, 60% ethanol) 200 μl
Was added and suspended, and a precipitate was obtained by centrifugation again. 300 μl of 70% ethanol was added to the precipitate, and the mixture was gently stirred with a vortex. Centrifugation again yielded a precipitate, vortexed briefly and dried the precipitate in the room for 10 minutes.
Subsequently, the DNA was recovered by elution with a TE buffer, and the DNA was further concentrated by isopropanol precipitation.

(2) Amplification of oxidoreductase gene and preparation of inserted fragment The oxidoreductase gene was amplified using the two types of oligonucleotides prepared in Example 1 as primers. That is, 20 ng of the sample prepared in (1), 2 mM
5 μl of dNTP, 4 μl of each of 5 μM primers, KO
2 units of D DNA polymerase (trade name, manufactured by Toyobo Co., Ltd.) and 10-fold concentrated KOD DN
Pure water was added to 5 μl of A polymerase PCR buffer to make a total volume of 50 μl, and PCR was performed. PCR
After incubation at 94 ° C. for 1 minute, 98
C. for 15 seconds, 65.degree. C. for 2 seconds and 74.degree. C. for 30 seconds as one cycle, followed by 5 cycles. Further, 98.degree. C. for 15 seconds, 60.degree. C. for 2 seconds and 74.degree.
This is repeated for 10 cycles and 98
C. for 15 seconds, 55.degree. C. for 2 seconds and 74.degree. C. for 30 seconds.
Incubation was performed at 4 ° C. for 30 seconds.

After completion of the PCR, the reaction solution was developed by electrophoresis using a 1.5% agarose gel, and a fraction containing the PCR product of the oxidoreductase gene was cut out from the gel.
rep-A-Gene DNA Purificati
On (trade name, manufactured by Bio-Rad) 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,
20 μl of Prep-A-Gene matrix with DN
A was adsorbed. Then, after washing twice with 600 μl of Prep-A-Gene washing solution, the mixture was incubated in 30 μl of TE buffer at 50 ° C. for 5 minutes to elute and collect DNA fragments.

Then, 0.5 μg of the PCR product, 20 units each of the restriction enzymes HindIII and BamHI, and a buffer (200 mM Tris-HCl (pH 8.5),
Pure water is added to 5 μl of 00 mM magnesium chloride, 5 mM dithiothreitol, and 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. Next, the PCR product was further developed by electrophoresis using a 1.5% agarose gel, a fraction containing the target DNA fragment was cut out from the gel, and the above Prep-A
-DNA was purified using Gene DNA Purification (trade name, manufactured by Bio-Rad).

(3) Preparation of Vector Next, 2 μg of pUC19 DNA (manufactured by Takara Shuzo Co., Ltd.), restriction enzymes BamHI and HindIII were added in 2 parts each.
0 units, buffer solution (200 mM Tris-HCl (pH
8.5), 100 mM magnesium chloride, 5 mM dithiothreitol, 660 mM potassium acetate) 5 μl
Pure water is added to 0.4 units of Escherichia coli-derived alkaline phosphatase (Takara Shuzo Co., Ltd.), and the total volume is 50 μl.
The vector was digested by reacting at 37 ° C. for 2 hours. Subsequently, the vector was prepared by phenol treatment and ethanol precipitation.

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

(5) Method for producing a oxidoreductase gene-containing transformant The recombinant vector prepared in (4) was used for the E. coli DH5α strain (Gibco BRL Life Technologies, Inc.
oBRL Life Technologies) was transformed (Methods Enzymol. No. 204, 63-
113, 1991). That is, after gently mixing 10 μl of the ligation solution containing the recombinant vector and 200 μl of the competent cells of the DH5α strain,
Let stand for minutes. Then, the mixture was incubated at 42 ° C. for 60 seconds and cooled in ice for 1-2 minutes to obtain a transformant.

(6) Production method of oxidoreductase An SOC medium (medium composition: tryptone 20 g / l, yeast extract 5 g / l, 10 mM sodium chloride, 2.5 mM potassium chloride, 1 ml of 10 mM magnesium chloride, 10 mM magnesium sulfate, and 20 mM glucose) was added, and the mixture was incubated at 37 ° C. for 1 hour. After the incubation, the cell solution of the transformant was added to an LB agar medium containing ampicillin (medium composition: tryptone 10 g / l, yeast extract 5 g / l, sodium chloride 1).
0 g / l, agar 15 g / l, pH 7.4).
The cells were cultured overnight at ℃ to obtain colonies of the oxidoreductase gene-containing transformant. The colonies were transformed with L containing ampicillin.
The cells were cultured with shaking at 37 ° C. overnight in liquid B medium. After culture
The cells collected by centrifugation were disrupted by ultrasonic waves and centrifuged again. The supernatant was fractionated with ammonium sulfate, and the protein-containing fraction was dialyzed to obtain the oxidoreductase.

Example 3 (1) Preparation of Chromosomal DNA of IFO3001 Strain A single colony of Bacillus serius IFO3001 strain was placed in a 10 ml PNE medium (medium composition: polypeptone 10 g / l, skipjack fish meat extract 10 g / l, sodium chloride 2 g / 1, pH 7.4) and inoculated at 37 ° C for 2 hours.
The cells were cultured with shaking for 4 hours. After the culture is completed, yeast cells
The chromosomal DNA of the bacterial cells was collected using Gentorukun for Gram-positive bacteria (trade name, manufactured by Takara Shuzo Co., Ltd.). That is, after completion of the culture, the cells were obtained by removing the supernatant by centrifugation, and after adding 180 μl of GenTLE solution I (Gentra, trade name, manufactured by Takara Shuzo Co., Ltd.) to the cells, immediately vortexed for 20 seconds. Stirred. Then G
After adding 20 μl of enTLE solution II and vortexing for 5 seconds, incubating at 70 ° C. for 10 to 15 minutes to lyse, and then adding 100 μl of GenTLE solution III
1 was added and mixed well. After allowing to stand in ice for 5 minutes, the mixture was centrifuged. The supernatant was transferred to another tube, an equal amount of isopropanol was added, and the mixture was mixed well. Thereafter, the supernatant was further removed by centrifugation, the precipitate was washed with 70% ethanol, and the supernatant was removed by lightly centrifuging for several seconds. The precipitate was washed with TE buffer (10 mM
Tris-hydrochloric acid, 1 mM ethylenedinitrolotetraacetic acid, pH
7.5) Dissolve in 300 μl, add 150 μl of 7.5 M ammonium acetate and 450 μl of ethanol and mix,
The filamentous DNA precipitate is entangled with the tip of a pipette tip, and T
Dissolved in 300 μl of E buffer.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment PCR was performed using the two oligonucleotides synthesized in Example 1 as primers. That is, 1 μg of the chromosomal DNA prepared in (1), 2.5 mM dNTP4
μl, 5 μM each of primers 4 μl, 5 U / μl of Ta
KaRa ExTaq (trade name, manufactured by Takara Shuzo Co., Ltd.)
Pure water was added to 0.4 μl, 10 μl of 10-fold concentrated Ex Taq buffer and 0.4 μl of 1 mg / ml anti-Taq high (trade name, manufactured by Toyobo Co., Ltd.) to make a total volume of 100 μl, and PCR was performed. PCR at 94 ° C
After 1 minute incubation at 94 ° C for 1 minute, 72 ° C for 3 minutes as one cycle, 10 cycles, further at 94 ° C for 1 minute, 65 ° C for 2 minutes and 7 minutes.
One cycle at 2 ° C. for 1 minute is 15 cycles,
Furthermore, at 94 ° C. for 1 minute, at 60 ° C. for 2 minutes and at 7 ° C.
This was performed for 10 cycles, one cycle at 2 ° C. for 1 minute, followed by a 1 minute incubation at 72 ° C.

(3) Preparation of recombinant vector pUC19 (manufactured by Takara Shuzo Co., Ltd.) was treated in the same manner as in Example 2 (3) to prepare a vector. Then, the PCR product obtained in (2) and the pUC19 were mixed at a molar ratio of 2: 1 to prepare a half of the DNA solution of Ligation Hi.
gh (trade name, manufactured by Toyobo Co., Ltd.) was added and ligated at 16 degrees to obtain a recombinant vector.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product The transformation was performed in the same manner as in Example 2 (5) except that the recombinant vector prepared in (3) was used as the recombinant vector. I got a body. 1 ml of an SOC medium was added to the transformant and incubated at 37 ° C. for 1 hour. After the incubation, the bacterial cell solution of the transformant was washed with LB containing ampicillin.
The cells were plated on an agar medium and cultured at 37 ° C. overnight to obtain a colony of the oxidoreductase gene-containing transformant. Then, the colonies were cultured in an LB liquid medium containing ampicillin with shaking at 37 ° C. overnight. After the culture, the cells were collected by centrifugation. Next, FlexiPrep (brand name) Kit
(Amersham Pharmacia Biotech), and plasmid DNA was prepared from the cells in the same manner as in Example 2 (1) except that the cells were used as the cells.

The nucleotide sequence of the gene is M13 Prim
er RV (trade name, manufactured by Takara Shuzo Co., Ltd.) and ABI
PRIZM (trade name) BigDye (trade name) T
emitter Cycle Sequencer
g Ready Reaction Kit (product name,
It was determined from the 5 'side of the gene by the dideoxy method using P. Applied Biosystems.
The nucleotide sequence was also determined from the 3 'side of the gene by the dideoxy method using M13 Primer M4 (trade name, manufactured by Takara Shuzo Co., Ltd.) and the kit. As a result, it was confirmed that the nucleotide sequences determined from both sides were the same.

The nucleotide sequence of the PCR product in the IFO3001 strain was 750 bp. The nucleotide sequence derived from the IFO3001 strain is shown in SEQ ID NO: 5, and the deduced amino acid sequence is shown in SEQ ID NO: 6.

Example 4 (1) Preparation of Chromosomal DNA of IFO15305 Strain Chromosome D of the bacterial cell was prepared in the same manner as in Example 3 (1) except that Bacillus serius IFO15305 was used as the strain.
NA was prepared.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment A PCR product treated with restriction enzymes was prepared in the same manner as in Example 2 (2) except that 1 μg of the chromosomal DNA prepared in (1) was used as a sample. Obtained.

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

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product In the same manner as in Example 3 (4) except that the recombinant vector obtained in (3) was used as a recombinant vector, A transformant was prepared. The PCR was carried out in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The nucleotide sequence of the product was determined.

As a result, P in the IFO15305 strain
The nucleotide sequence of the CR product was found to be 750 bp. SEQ ID NO: 7
And the deduced amino acid sequence is shown in SEQ ID NO: 8.

Examples 5 to 7 (1) Preparation of chromosomal DNA of bacterial strain Chromosomal DNA of bacterial cells was prepared in the same manner as in Example 3 (1) except that Bacillus cellius IFO3003, IFO3563 or IFO13597 was used as a bacterial strain. .

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment Except that 1 μg of the chromosomal DNA prepared in (1) was used as a sample, the same operation as in Example 2 (2) was performed for each, A processed PCR product was obtained.

(3) Preparation of recombinant vector pUC19 was treated in the same manner as in Example 2 (3) to prepare a vector. Subsequently, ligation was carried out in the same manner as in Example 2 (4) except that the PCR product obtained in (2) was used as a PCR product, to obtain respective recombinant vectors.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product In the same manner as in Example 3 (4) except that the recombinant vector obtained in (3) was used as a recombinant vector, Was prepared. Also, the nucleotide sequence of each PCR product was determined in the same manner as in Example 3 (4) except that the transformant was used as a transformant.

The nucleotide sequence of IFO3003 was changed to SEQ ID NO: 1.
0, the nucleotide sequence of IFO3563
The nucleotide sequence of FO13597 is shown in SEQ ID NO: 14. In addition, the deduced amino acid sequence of IFO3003 is
1. The deduced amino acid sequence of IFO3563 is
SEQ ID NO: 15 and the deduced amino acid sequence of IFO13597.

Example 8 (1) Preparation of Chromosomal DNA of IAM1029 Strain The chromosomal DNA of the bacterial cell was prepared in the same manner as in Example 3 (1) except that Bacillus cellius IAM1029 was used as the strain.
A was prepared.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment The oxidoreductase gene was amplified using the two oligonucleotides prepared in Example 1. That is,
2 μg of the sample prepared in (1), 5 μm of 2 mM dNTP
l, 5 μM each of primers 5 μl, KOD plus
1 unit of DNA polymerase (trade name, manufactured by Toyobo Co., Ltd.), 2 μm of 25 mM magnesium sulfate
l and KOD plus DNA polymerase
Pure water is added to 5 μl of a 10-fold concentrated buffer solution for a total volume of 50
μl, and PCR was performed. The PCR was incubated at 94 ° C for 2 minutes, followed by 60 seconds at 94 ° C for 15 seconds.
10 cycles of 30 ° C. for 30 seconds and 1 minute at 68 ° C., and 55 ° C. at 94 ° C. for 15 seconds.
For 20 seconds and 68 ° C. for 1 minute as 10 cycles, and further for 5 seconds at 94 ° C. for 5 seconds.
This was carried out for 10 cycles, one cycle at 0 ° C. for 10 seconds and one cycle at 68 ° C., and a final incubation at 68 ° C. for 1 minute.

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

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product In the same manner as in Example 3 (4) except that the recombinant vector obtained in (3) was used as a recombinant vector, A transformant was prepared. The PCR was carried out in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The nucleotide sequence of the product was determined.

The nucleotide sequence of IAM1029 was changed to SEQ ID NO: 16.
And the deduced amino acid sequence is shown in SEQ ID NO: 17.

Example 9 (1) Preparation of Chromosomal DNA of Bacterial Cell Chromosome DN of bacterial cell was prepared in the same manner as in Example 3 (1) except that Bacillus serius IFO3132 was used as the bacterial strain.
A was prepared.

(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. That is,
1 μg of the sample prepared in (1), 2.5 mM dNTP4
μl, 10 μM each of primers 5 μl, Pyrobes
t (trade name) DNA Polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.) 1.25 units and Pyrobe
pure water is added to 10 μl of a 10-fold buffer of st (trade name) DNA polymerase to make a total volume of 100 μl.
CR was performed. PCR was carried out at 94 ° C. for 1 minute, followed by 10 cycles of 94 ° C. for 1 minute and 72 ° C. for 3 minutes, followed by 94 ° C.
For 1 minute, 65 ° C. for 2 minutes and 72 ° C. for 1 minute for 15 cycles, and further at 94 ° C.
This was repeated 10 times, with 1 cycle at 60 ° C. for 2 minutes and 72 ° C. for 1 minute, followed by a final incubation at 72 ° C. for 1 minute.

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

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product In the same manner as in Example 3 (4) except that the recombinant vector obtained in (3) was used as a recombinant vector, A transformant was prepared. The PCR was carried out in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The nucleotide sequence of the product was determined.

The nucleotide sequence of IFO3132 was changed to SEQ ID NO: 18.
And the deduced amino acid sequence is shown in SEQ ID NO: 19.

Example 10 (1) Preparation of chromosomal DNA of IAM1110 strain The chromosomal DNA of the bacterial cell was prepared in the same manner as in Example 3 (1) except that Bacillus cellius IAM1110 was used as the strain.
A was prepared.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment Except that 1 μg of the chromosomal DNA prepared in (1) was used as a sample, the same operation as in Example 15 (2) was performed for each, A processed PCR product was obtained.

(3) Preparation of recombinant vector pUC19 was treated in the same manner as in Example 2 (3) to prepare a vector. Subsequently, ligation was carried out in the same manner as in Example 2 (4) except that the PCR product obtained in (2) was used as a PCR product, to obtain respective recombinant vectors.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product In the same manner as in Example 3 (4) except that the recombinant vector obtained in (3) was used as a recombinant vector, Was prepared. The nucleotide sequence of the PCR product was determined in the same manner as in Example 3 (4) except that the transformant was used as a transformant.

The nucleotide sequence of IAM1110 was changed to SEQ ID NO: 20.
And the deduced amino acid sequence is shown in SEQ ID NO: 21.

Example 11 (1) Synthesis of Oligonucleotides Two kinds of oligonucleotides were synthesized by a known phosphorous acid-triester method. The sequence of the synthesized oligonucleotide is GGTGGTTGCTCTCTCCAACATG
CGCTACGTTATCATAAC (SEQ ID NO: 22)
And CGGCTGCAGTTATTCATCAATT
CTAATAAC (SEQ ID NO: 23). SEQ ID NO: 2
GCTCTTC in No. 2 and CTGCAG in SEQ ID NO: 23 are each a recognition sequence of a restriction enzyme.

(2) Amplification of oxidoreductase gene The procedure of Example 2 (2) was repeated, except that the two oligonucleotides prepared in (1) were used as primers.
PCR was performed. Then, the same procedure as in Example 2 (2) was repeated except that the PCR product was used as a PCR product.
The R product was recovered. 0.5 μg of the PCR product, Pst
I 10 units and NEBuffer3 (trade name,
New England BioLabs) (1 M sodium chloride, 500 mM Tris-HCl (pH 7.9),
Pure water was added to 5 μl of 100 mM magnesium chloride, 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 treatment with the restriction enzyme, the P
The CR product was purified and recovered by a phenol extraction method and an ethanol precipitation method. Sap was added to the recovered PCR product.
I 9 units and NEBuffer 4 (trade name, manufactured by New England BioLabs) (500 mM
Potassium acetate, 200 mM Tris acetic acid (pH 7.
9), 5 μl of 100 mM magnesium acetate, 10 mM dithiothreitol), 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. Sa
After restriction enzyme treatment with pI, the reaction solution was developed by electrophoresis using a 1.5% agarose gel,
The fraction containing the A fragment was cut out of the gel. Then, in the same manner as in Example 2 (2) except that the fraction was used, Pr was used.
ep-A-Gene DNA Purificatio
n (trade name, manufactured by Bio-Rad) to purify the DNA.

(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 the pTYB-11 vector in the kit was used. The reaction solution was developed by electrophoresis using an agarose gel, and a fraction containing the target DNA fragment was cut out from the gel. Then, Prep-A-Gene D was prepared in the same manner as in Example 2 (2) except that the fraction was used.
The vector was prepared by NA Purification (trade name, manufactured by Bio-Rad).

(4) Preparation of Transformant Same as Example 2 (4) except that the PCR product prepared in (2) was used as a PCR product and the pTYB-11 vector prepared in (3) was used as a vector. And ligation was performed to obtain a recombinant vector. Next, the recombinant vector was used as a recombinant vector, and
Except that E. coli ER2566 strain in T (trade name) -CN kit was used as a host, the procedure was the same as in Example 2 (5).
A transformant was obtained. 1 ml of SOC medium was added to the transformant.
In addition, the mixture was incubated at 37 ° C. for 1 hour. After the incubation, the bacterial cell solution of the transformant was treated with L containing ampicillin.
The cells were sown on a B agar medium and cultured at 37 ° C. overnight to obtain a colony of the oxidoreductase gene-containing transformant. Then, 60
The colonies were shake-cultured at 37 ° C. in an LB liquid medium containing ampicillin until the absorbance at 0 nm reached 5.
After culturing, the collected cells were disrupted by ultrasonic waves and centrifuged again to obtain a supernatant. Then, a chitin affinity column (New England BioLabs (registered trademark)) was added to a column buffer (20 mM Tris-HCl (pH 8.0), 5
(00 mM sodium chloride, 1 mM ethylenedinitrolotetraacetic acid), the supernatant was applied to a column, and an intein-chitin complex was formed in the column. Then, after washing with a column buffer, a buffer containing 30 mM dithiothreitol (20 mM Tris-HCl (pH 8.0), 50 mM) to induce intein self-splashing was added.
The oxidoreductase protein was cleaved from the intein by incubating overnight at 4 ° C. in 0 mM sodium chloride, 1 mM ethylene dinitrolotetraacetic acid, 50 mM dithiothreitol. The next day, only the enzyme protein was eluted to obtain a purified protein.

Example 12 The transformant obtained in Example 2 (5) was inoculated into 200 ml of LB liquid medium containing ampicillin, and cultured at 37 ° C. until the absorbance at 600 nm reached 5. Then, isopropylthiogalactoside (IPTG) and benzyl as a substrate were added at final concentrations of 0.5 mM and 1 mM, respectively, and then cultured at 37 ° C. for 24 hours. After 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 further repeated twice, and the obtained ethyl acetate layer (120 ml) was obtained.
Was concentrated. The product obtained is converted to hexane: ether:
The mixture was applied to a silica gel column using acetic acid (volume ratio: 45: 5: 1) as a developing solvent, and the benzoin fraction was collected. In addition, benzoin was highly purified by reverse-layer high performance liquid chromatography. The yield is 80% and the optical purity is 88
% Ee.

REFERENCE EXAMPLE 1 (1) Method for recovering chromosomal DNA from bacterial cells A single colony of Bacillus serius FERM P-14210 strain was inoculated into 10 ml of a PNE medium.
Shaking culture was performed at 24 ° C. for 24 hours. After completion of the culture, chromosomal DNA was recovered using Gen Toru-kun (trade name, manufactured by Takara Shuzo Co., Ltd.) for the cells. The recovered chromosomal DNA is 3
Dissolved in 00 μl of TE buffer. The amount of the obtained DNA was 220 μg for several times.

(2) Preparation of Chromosomal DNA Insertion Fragment The restriction enzyme Sau3A was added to the chromosomal DNA prepared in (1).
The chromosomal DNA was partially digested by the action of I (Takara Shuzo). Then, after phenol extraction of the partially digested reaction solution, isopropanol precipitation was performed twice to purify the DNA fragment.

The Klenow fragment (manufactured by Takara Shuzo Co., Ltd.) was allowed to act on the DNA fragment obtained by the above method to obtain Sa.
Half of the 5 'overhangs generated by digestion with u3AI were filled in. Therefore, in the chromosomal DNA fragment, GA becomes a 5 'protruding end. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, so that a fraction containing the DNA fragment of 2 to 4 kb was cut out from the gel.
ene DNA Purification (trade name,
(Bio-Rad)).

(3) Preparation of Vector Next, the vector was digested by allowing pUC19 DNA (manufactured by Takara Shuzo) to act on the restriction enzyme SalI (manufactured by Takara Shuzo). Then, the Klenow fragment was allowed to act on the vector to fill in half of the 5 'protruding end generated by digestion with SalI. Therefore, the pUC1
The 5 'protruding end in 9 is TC.

(4) Ligation of Insert Fragment and Vector The chromosomal DNA whose GA is a 5′-protruding end and the pUC19 DNA whose TC is a 5′-protruding end are mixed at a molar ratio of 2: 1 to obtain a DNA Ligation Kit Ve.
r. Ligation was performed using No. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.). Thus, a chromosomal DNA library of Bacillus cellius was completed. Using 25 ng of this library, E. coli DH5
α strain (Gibco BRL Life Technologies, Inc.)
fe Technologies)) (Methods Enzymol., No. 204, 63-113).
1991).

(5) Screening of oxidoreductase gene The transformed Escherichia coli was dissolved in dimethyl sulfoxide at a concentration of 40 μl per plate with a 0.1 M benzyl solution.
Seed on LB agar medium containing ampicillin, and
And 2710 colonies were observed. It was left at room temperature and waited for halo formation. That is, when benzyl is reduced and benzoin is generated around the colonies of Escherichia coli, the solubility is increased and a halo is formed. This was used as an index for screening clones having benzyl reductase. As a result, there were 7 colonies that formed a halo. In the earliest colony, a halo was formed 30 hours after the transformed bacterial solution was inoculated.

The obtained seven colonies were inoculated into 2 ml of a liquid culture LB medium and cultured with shaking at 37 ° C. for 12 hours. 20 μl of 0.1 M benzyl substrate (final concentration 1 m
M) After the addition, the cells were further cultured for 24 hours. The substrate and the product were extracted from the bacterial solution with ethyl acetate, developed by thin-layer chromatography using hexane: ether: acetic acid (volume ratio: 45: 5: 1) as a solvent, and the benzyl reduction activity was confirmed again. As a result, only the cells that formed the halo earliest showed activity. Therefore, the present inventors named the recombinant vector contained in the cells as pUC-BC-B1.

(6) Determination of base sequence of oxidoreductase gene The base sequence of the inserted fragment of pUC-BC-B1 was determined by E.
Z :: TN (trade name) <KAN-1> Insertion
n Kit (trade name, manufactured by EpiCenter Technologies). By this decision, pUC-BC-
The B1 insert was found to be 2438 bp.
Next, the nucleotide sequence of the DNA fragment was determined using DDBJ / EMB.
A homology with a known base sequence was searched for in the base sequence database of the L / GenBank international base sequence database, but a base sequence having high homology with the base sequence was not obtained. Therefore, for each possible ORF, the amino acid sequence encoded by that ORF is converted to DDBJ / EMB.
A homology search was performed on the amino acid sequence database of the L / GenBank International Base Sequence Database, and the amino acid sequence consisting of 249 residues shown in SEQ ID NO: 2 was found to have the amino acid sequence encoded by the yueD gene of Bacillus subtilis (243 amino acid residues) And 40% homology. Although the function of the YueD protein is unknown, its amino acid sequence was highly homologous to sepiapterin reductase.

Also, OR of Saccharomyces cerevisiae
The amino acid sequence encoded by F YIR036C is 3
The finding of having 0% similarity was also obtained. This YIR0
Although the function of the 36C protein is unknown, it is known that its amino acid sequence has similarity to short-chain alcohol dehydrogenase which is a kind of oxidoreductase.

The OR estimated from the YuD protein
In order to confirm that F has a redox function, pUC-BC-B1 having a transposon inserted into the ORF in the process of determining the base sequence was placed on an agar plate containing ampicillin coated with benzyl at 25 ° C. for 36 hours. ~ 4
The cells were cultured for 8 hours. Observation after culturing revealed no halo-forming colonies. Therefore, the OR
F was determined to encode benzyl reductase. The ORF, including the stop codon, is shown in SEQ ID NO: 9.

[0093]

Industrial Applicability The present invention provides a novel oligonucleotide, a method for amplifying and cloning a novel oxidoreductase gene using the oligonucleotide as a primer, a recombinant vector containing the gene, and a trait transformed by the recombinant vector. Provided are a transformant and a method for producing an oxidoreductase using the transformant. By using the primer of the present invention, an oxidoreductase gene of an unknown Bacillus serius species can be isolated. In addition, the use of the gene of the present invention makes it possible to easily clone a novel oxidoreductase having an asymmetric reducing ability for an aromatic diketone compound, and to express it in various host cells.

[0094]

[Sequence List Free Text] SEQ ID NO: 1: Synthetic oligonucleotide. SEQ ID NO: 2: Synthetic oligonucleotide. SEQ ID NO: 3: Synthetic oligonucleotide. SEQ ID NO: 4: Synthetic oligonucleotide.

[0095]

[Sequence List] SEQUENCE LISTING <110> Arakawakagakukougyoukabushikigaisha <120> oxido-reductase genes, Cloning the genes and Preparing the enzymes <130> JP-12297 <140> <141> <160> 23 <170> PatentIn Ver. 2.0 <210 > 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 1 tgcgctacgt tatcataaca g 21 <210> 2 <211> 30 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 2 ggcgaagctt gcgctacgtt atcataacag 30 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 3 tattcatcaa ttctaataac 20 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 4 cgcggatcct attcatcaat tctaataac 29 < 210> 5 <211> 750 <212> DNA <213> Bacillus cereus <400> 5 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgccacacaa 6 0 ttgttagaaa aaaatacaac tgtcatctct atttctagaa gagaaaataa agagcttacg 120 aaactcgcag aacaatataa tagcaattgt gttctccact ccttagatct tcaagatgta 180 cataatttag aaacgaactt taacaaaatc atttcatcta ttcaagaaga cagtgtatct 240 tctattcatt taattaataa tgcgggtacg gttgctccta tgaagccaat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattctcacc 360 tccactttca tgaaacatac gaaagaatgg aaagtagata aacgtgttat aaacatttca 420 tctggtgcag gaaaaaatcc atacttcgga tggggcgctt attgtacaac gaaagctggc 480 gtaaatatgt ttacacagtg cgtagcaacc gaagaagcag caaaagaatt tccagtaaaa 540 atcgtcgctt ttgcacctgg cgttgttgat acaaatatgc aagcacaaat tcgggaaaca 600 aatagagaag acttcacaaa tttagatcga ttcatcgcat taaaagaaga aggaaggcta 660 ttatcacccg aatacgttgc gaaagctatt cgtaacttac tagaaactga agacttccct 720 caaggcgagg ttattagaat tgatgaatag 750 <210> 6 <211> 249 <212> PRT <213> Bacillus cereus < 400> 6 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Lys Asn Thr Thr Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Lys Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Val Leu His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Asn Lys Ile Ile Ser Ser Ile Gln Glu Asp Ser Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Ala Ala Lys Glu 165 170 175 Phe Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Asn Arg Glu Asp Phe Thr Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Arg Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Asp Phe Pro 225 230 235 24 0 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 7 <211> 750 <212> DNA <213> Bacillus cereus <400> 7 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggcaat cgctacgcaat cgctacgcaat cgctacgcaat gagcatcagcaat tagatacatcagcaat tga ccctagatct tcaagatgta 180 cataacttag agactaactt taaagagatc atttcatcta ttaaaaaaga caatgtatcc 240 tctattcatt taattagtaa cgcgggtaca gttgcaccta tgaagccaat tgaaaaagct 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacattta tgaaacatac gaaagactgg aaagtaggtg aacgcgttat taacatttca 420 tctggtgcag gaaaaaaccc ttactttggc tggggcgctt attgtacaac gaaagctagt 480 gtaaatgtgt ttacacagtg cgtagcaact gaagaagtgg aaaaagaata tccagtaaaa 540 atcgtcgctt ttgcacctgg cgttgttgat acaaatatgc aatcacaaat tcgcgaaaca 600 aataaagaag atttcataaa tttggaccgc ttcattgcat taaaggaaga aggaaaacta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggacttccct 720 caaggcgagg ttattagaat tgatgaatag 750 <210> 210 <210> PRT <213> Bacillus cereus <400> 8 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Lys Asn Thr Thr Val Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Gln Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Ile Phe His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Lys Glu Ile Ile Ser Ser Ile Lys Lys Asp Asn Val Ser 65 70 75 80 Ser Ile His Leu Ile Ser Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Asp Trp Lys Val Gly Glu Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Ser 145 150 155 160 Val Asn Val Phe Thr Gln Cys Val Ala Thr Glu Glu Val Glu Lys Glu 165 170 175 Tyr Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ser Gln Ile Arg Glu Thr Asn Lys Glu Asp Phe Ile Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Asp Phe Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 9 <211> 750 <212> DNA <213> Bacillus cereus <400> 9 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgccactcaa 60 ttgttagaag aaagtacaac tgtcatctct atttctagaa gagaaaataa agaacttact 120 aaacttgcag agcaatataa cagcaattgc atttttcatt ccttagatct tcaagatgta 180 cataacttag aaactaactt taaagaaatc atttcatcca ttaaagaaga caatgtatcc 240 tctattcatt taattaataa tgcaggtaca gttgcaccta tgaagccgat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacgttta tgaaacatac gaaagaatgg aaagtagata aacgcgttat aaatatttca 420 tctggtgcag gaaaaaatcc ttatttcgga tggggcgctt attgtacgac gaaagctggt 480 gtaaatatgt ttacacaatg cgtagcaact gaagaagtgg aaaaagaata tccagtaaaa 540 atcgtcgctt ttgcccccgg tgttgttgat acaaatatgc aagcacaaat tcgtgaaaca 600 gctaaagaag acttcacaaa tttagaccgc t tcattgcat taaaagaaga aggaaagcta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggaattccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 10 <211> 750 <212> DNA <213> Bacillus cereus <400> 10 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgccacacaa 60 ttgttagaga aaaatacaac tgtcatctct atttctagaa gagaaaataa agagcttacg 120 aaactcgcag aacaatataa tagcaattgt gttctccact ccttagatct tcaagatgta 180 cataatttag aaacgaactt taacaaaatc atttcatcta ttcaagaaga cagtgtatct 240 tctattcatt taattaataa tgcgggtacg gttgctccta tgaagccaat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattctcacc 360 tccactttca tgaaacatac gaaagaatgg aaagtagata aacgtgttat aaacatttca 420 tctggtgcag gaaaaaatcc atacttcgga tggggcgctt attgtacaac gaaagctggc 480 gtaaatatgt ttacacagtg cgtagcaacc gaagaagcag taaaagaatt tccagtaaaa 540 atcgtcgctt ttgcacctgg tgttgttgat acaaatatgc aagcacaaat tcgggaaaca 600 aatagagaag acttcgcaaa tttagatcga ttcatcgcat taaaagaaga aggaaagcta 660 ttatcacccg aatacgttgc gaaagc tatt cgtaacttac tagaaactga agacttccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 11 <211> 249 <212> PRT <213> Bacillus cereus <400> 11 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 Ile Ala Thr Gln Leu Leu Glu Lys Asn Thr Thr Val Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Lys Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Val Leu His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Asn Lys Ile Ile Ser Ser Ile Gln Glu Asp Ser Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Ala Val Lys Glu 165 170 175 Phe Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Asn Arg Glu Asp Phe Ala Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Asp Phe Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 12 <211> 750 <212> DNA <213> Bacillus cereus <400> 12 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgccactcag 60 ttgttagaag aaagtacaac tgtcatctct atttctagaa gagaaaataa agaacttact 120 aaacttgcag agcaatataa cagcaattgc atttttcatt ccttagatct tcaagatgta 180 cataacttag aaactaactt taaagaaatc atttcatcca ttaaagaaga caatgtatcc 240 tctattcatt taattaataa tgcaggtaca gttgcaccta tgaagccgat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacgttta tgaaacatac gaaagaatgg aaagtagata aacgcgttat aaatatttca 420 tctggtgcag gaaaaaatcc ttatttcgga tggggcgctt attgtacgac gaaagctggt 480 gtaaatatgt ttacacaatg cgtagcaact gaagaagtgg aaaaagaata tccagtaaaa 54 0 atcgtcgctt ttgcccccgg tgttgttgat acaaatatgc aagcacaaat tcgtgaaaca 600 gctaaagaag acttcacaaa tttagaccgc ttcattgcat taaaagaaga aggaaagcta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggaattccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 13 <211> 249 <212> PRT <213> Bacillus cereus <400> 13 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Glu Ser Thr Thr Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Lys Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Ile Phe His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Lys Glu Ile Ile Ser Ser Ile Lys Glu Asp Asn Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe G ly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Val Glu Lys Glu 165 170 175 Tyr Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Ala Lys Glu Asp Phe Thr Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Glu Phe Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 14 <211> 750 <212> DNA <213> Bacillus cereus <400> 14 atgcgctacg ttatcataac aggaacttca caaggtttag gtgagga tgccca tgccca aaagtacaac tgtcatctct atttctagaa gagaaaatag agaacttact 120 aaacttgcag agcaatataa cagcaattgc atttttcatt ccctagatct tcaagatgta 180 cataacttag aaactaactt taaagagatc atttcatcta ttaaaaaaga caatgtatcc 240 tctattcatt taattaataa tgcgggtaca gttgcaccta tgaagccaat cgaaaaagct 300 gaaagcgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacattta tgaaacatac gaaagaatgg aaagt agata aacgcgttat aaatatttca 420 tctggtgcag gaaaaaatcc ttatttcgga tggggcgctt attgtacgac gaaagctggt 480 gtaaatatgt ttacacaatg cgtagcaact gaagaagtgg aaaaagagtg tccagtaaaa 540 atcgtcgctt ttgcccccgg tgttgttgat acaaatatgc aagcacaaat tcgtgaaaca 600 aataaagaag acttcacaaa tttagaccgc ttcattgcat taaaagaaga aggaaagcta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggaattccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 15 <211> 249 <212> PRT <213> Bacillus cereus <400> 15 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Glu Ser Thr Thr Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Arg Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Ile Phe His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Lys Glu Ile Ile Ser Ser Ile Lys Lys Asp Asn Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Val Glu Lys Glu 165 170 175 Cys Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Asn Lys Glu Asp Phe Thr Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Glu Plu Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 16 <211> 750 <212> DNA <213> Bacillus cereus <400> 16 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgcca tagacca tagattacagta tagaca tagaca tagattacagta tagattacagta tagattacagta tagattagag cagcaattgc atttttcatt ccttagatct tcaagatgta 180 cataacttag aaactaactt taaagaaatc atttcatcca ttaaagaaga caatgtatcc 240 tctattc att taattaataa tgcaggtaca gttgcaccta tgaagccgat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacgttta tgaaacatac gaaagaatgg aaagtagata aacgcgttat aaatatttca 420 tctggtgcag gaaaaaatcc ttatttcgga tggggcgctt attgtacgac gaaagctggt 480 gtaaatatgt ttacacaatg cgtagcaact gaagaagtgg aaaaaggata tccagtaaaa 540 atcgtcgctt ttgcccccgg tgttgttgat acaaatatgc aagcacaaat tcgtgaaaca 600 gctaaagaag acttcacaaa tttagaccgc ttcattgcat taaaagaaga aggaaagcta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggaattccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 17 <211> 249 <212> PRT <213> Bacillus cereus <400> 17 Met Arg Tyr Val Ile Ile Thr Gly 1 Glu Glu Glu Le 15 Ile Ala Thr Gln Leu Leu Glu Glu Ser Thr Thr Val Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Lys Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Ile Phe His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Lys Glu Ile Ile Ser Ser Ile Lys Glu Asp Asn V al Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Val Glu Lys Gly 165 170 175 Tyr Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Ala Lys Glu Asp Phe Thr Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Glu Plu Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 18 <211> 750 <212> DNA <213> Bacillus cereus <400> 18 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggcaat cgctacgcaa 60 ttattagaaa aaaatacaac tgtcat ctga atttctaga gaaaatca agagcttaca 120 aaacttgcag agcaatataa cagcaattgc atttttcatt ccctagatct tcaagatgta 180 cataacttag aaactaactt taaagagatc atttcatcta ttaaaaaaga caatgtatcc 240 tctattcatt taattaataa tgcgggtaca gttgcaccta tgaagccaat tgaaaaagct 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattcttaca 360 tctacattta tgaaacatac gaaagactgg aaagtagata aacgcgttat taacatttca 420 tctggtgcag gaaaaaaccc ttactttggc tggggcgctt attgtacaac gaaagctggt 480 gtaaatatgt ttacacagtg cgtagcaact gaagaagtgg aaaaagaata tccagtaaaa 540 atcgtcgctt ttgcacctgg cgttgttgat acaaatatgc aatcacaaat tcgcgaaaca 600 aataaagaag atttcataaa tttggaccgc ttcattgcat taaaagaaga aggaaaacta 660 ttatcacctg aatatgttgc gaaagctatt cgtaacttac tagaaactga ggacttccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 19 <211> 249 <212> PRT <213> Bacillus cereus <400> 19 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Lys Asn Thr Thr Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Gln Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Ile Phe His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Lys Glu Ile Ile Ser Ser Ile Lys Lys Asp Asn Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Asp Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Val Glu Lys Glu 165 170 175 Tyr Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ser Gln Ile Arg Glu Thr Asn Lys Glu Asp Phe Ile Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys Glu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Asp Phe Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210 > 20 <211> 750 <212> DNA <213> Bacillus cereus <400> 20 atgcgctacg ttatcataac aggaacttca caaggtttag gtgaggctat tgccacacaa 60 ttgttagaaa aaaatacaac tgtcatctct atttctagaa gagaaaataa agagcttacg 120 aaactcgcag aacaatataa tagcaattgt gttctccact ccttagatct tcaagatgta 180 cataatttag aaacgaactt taacaaaatc atttcatcta ttcaagaaga cagtgtatct 240 tctattcatt taattaataa tgcgggtacg gttgctccta tgaagccaat tgaaaaagca 300 gaaagtgaac aattcattac gaacgttcac attaatttac ttgcacctat gattctcacc 360 tccactttca tgaaacatac gaaagaatgg aaagtagata aacgtgttat aaacatttca 420 tctggtgcag gaaaaaatcc atacttcgga tggggcgctt attgtacaac gaaagctggc 480 gtaaatatgt ttacacagtg cgtagcaacc gaagaagcag caaaagaatt tccagtaaaa 540 atcgtcgctt ttgcacctgg tgttgttgat acaaatatgc aagcacaaat tcgggaaaca 600 aatagagaag acttcacaaa tttagatcga ttcatcgcat taaaagaaga aggaaagcta 660 ttatcacccg aatacgttgc gaaagctatt cgtaacttac tagaaactga agacttccct 720 caaggcgagg ttattagaat tgatgaataa 750 <210> 21 <211> 249 <212> PRT <213> Bacillus cereus <400> 21 Met Arg Tyr Val Ile Ile Thr Gly Thr Ser Gln Gly Leu Gly Glu Ala 1 5 10 15 Ile Ala Thr Gln Leu Leu Glu Lys Asn Thr Thr Val Ile Ser Ile Ser 20 25 30 Arg Arg Glu Asn Lys Glu Leu Thr Lys Leu Ala Glu Gln Tyr Asn Ser 35 40 45 Asn Cys Val Leu His Ser Leu Asp Leu Gln Asp Val His Asn Leu Glu 50 55 60 Thr Asn Phe Asn Lys Ile Ile Ser Ser Ile Gln Glu Asp Ser Val Ser 65 70 75 80 Ser Ile His Leu Ile Asn Asn Ala Gly Thr Val Ala Pro Met Lys Pro 85 90 95 Ile Glu Lys Ala Glu Ser Glu Gln Phe Ile Thr Asn Val His Ile Asn 100 105 110 Leu Leu Ala Pro Met Ile Leu Thr Ser Thr Phe Met Lys His Thr Lys 115 120 125 Glu Trp Lys Val Asp Lys Arg Val Ile Asn Ile Ser Ser Gly Ala Gly 130 135 140 Lys Asn Pro Tyr Phe Gly Trp Gly Ala Tyr Cys Thr Thr Lys Ala Gly 145 150 155 160 Val Asn Met Phe Thr Gln Cys Val Ala Thr Glu Glu Ala Ala Lys Glu 165 170 175 Phe Pro Val Lys Ile Val Ala Phe Ala Pro Gly Val Val Asp Thr Asn 180 185 190 Met Gln Ala Gln Ile Arg Glu Thr Asn Arg Glu Asp Phe Thr Asn Leu 195 200 205 Asp Arg Phe Ile Ala Leu Lys G lu Glu Gly Lys Leu Leu Ser Pro Glu 210 215 220 Tyr Val Ala Lys Ala Ile Arg Asn Leu Leu Glu Thr Glu Asp Phe Pro 225 230 235 240 Gln Gly Glu Val Ile Arg Ile Asp Glu 245 <210> 22 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 22 ggtggttgct cttccaacat gcgctacgtt atcataac 38 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 23 cggctgcagt tattcatcaa ttctaataac 30

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Reiji Maruyama 52, Kayakidai, Teramachi, Toyama 203 Tsukasa Heights Teramachi 203 (72) Inventor Yasushi Itoi 1-121-502 Minamishinda, Daito-shi, Osaka F-term (reference) 4B024 AA03 BA08 CA04 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL05 4B065 AA16Y AA26X AB01 AC14 BA02 CA28

Claims (24)

[Claims] 1. An oligonucleotide having a nucleotide sequence substantially the same as the nucleotide sequence of 2 to 22 5′-terminal of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing. 2. An oligonucleotide having a nucleotide sequence substantially complementary to the nucleotide sequence of 2 to 23 nucleotides at the 3 ′ end of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing. 3. The oligonucleotide of claim 1, wherein
'An oligonucleotide containing a recognition sequence for a restriction enzyme on the terminal side. 4. The oligonucleotide of claim 2, wherein
'An oligonucleotide containing a recognition sequence for a restriction enzyme on the terminal side. 5. An oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4 in the sequence listing. (A) amplifying a target oxidoreductase gene contained in a sample, using as a primer at least one oligonucleotide selected from the oligonucleotides according to claims 1, 2, 3, 4 and 5; b) inserting the amplified gene into a vector to prepare a recombinant vector, and then (c) obtaining a transformant transformed with the vector, the transformant comprising an oxidoreductase gene. Manufacturing method. 7. A sample containing an oxidoreductase gene,
The oxidoreductase gene-containing transformant according to claim 6, which is a chromosomal DNA obtained from a strain belonging to the genus Bacillus, a DNA library of the chromosome, a cDNA library, or a recombinant vector containing the gene. Recipe. 8. A sample containing an oxidoreductase gene,
The method for producing an oxidoreductase gene-containing transformant according to claim 6, which is a chromosomal DNA obtained from Bacillus cellius, a chromosome DNA library, a cDNA library, or a recombinant vector containing the gene. 9. The method for producing the oxidoreductase gene-containing transformant according to claim 6, 7 or 8, wherein the two oligonucleotides according to claim 3 and 4 are used as primers. 10. 2 according to SEQ ID NOs: 2 and 4 in the sequence listing
9. The method for producing a oxidoreductase gene-containing transformant according to claim 6, wherein a species of oligonucleotide is used as a primer. 11. The method for producing a oxidoreductase gene-containing transformant according to claim 6, wherein the host cell to be transformed is a microorganism. 12. The method for producing the oxidoreductase gene-containing transformant according to claim 6, wherein the vector is pUC19 and the host cell to be transformed is Escherichia coli. 13. A polynucleotide obtained by a polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides according to claim 1, 2, 3, 4, and 5 as a primer, wherein SEQ ID NOs: 5, 7, and 1
An oxidoreductase gene having the nucleotide sequence of 0, 12, 14, 16, 18 or 20. 14. A polynucleotide obtained by a polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides according to claim 1, 2, 3, 4, and 5 as a primer, wherein SEQ ID NOs. 1
An oxidoreductase gene encoding the amino acid sequence shown in 1, 13, 15, 17, 19 or 21. An oxidoreductase defined by the gene according to claim 13 or 14. A DNA or oligonucleotide that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to claim 13. 17. A protein or polypeptide having an amino acid sequence encoded by a DNA that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to claim 13. 18. A protein or polypeptide obtained by expressing a DNA that hybridizes to a DNA having a nucleotide sequence complementary to the nucleotide sequence of the gene according to claim 13. 19. A recombinant vector containing the gene according to claim 13 or 14. 20. The vector according to claim 1, wherein the vector is pUC19.
10. The recombinant vector according to 9. 21. A transformant transformed by the recombinant vector according to claim 19 or 20. 22. The transformant according to claim 21, wherein the host cell is a microorganism. 23. The transformant according to claim 21, wherein the microorganism is Escherichia coli. 24. A method comprising culturing a transformant containing an oxidoreductase gene obtained by the method according to claim 6, 7, 8, 9, 10, 11, or 12, and collecting oxidoreductase from the culture. A method for producing an oxidoreductase.