JP3518501B2 - Oxidoreductase gene, cloning of the gene and method for producing the enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel oligonucleotide, a novel oxidoreductase gene amplification / cloning method 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 by the transformant.

[0002]

2. Description of the Related Art Saccharomyces cerevisiae has been known as an oxidoreductase-producing microorganism capable of reducing diketone compounds. However, in the Saccharomyces cerevisiae, for example, benzyl (be
When nzil) is reduced, racemic benzoin is produced. Therefore, the microorganism-produced enzyme has a drawback of low stereoselectivity (Chemistry Letters, 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 (Chem
istry Letters, pages 1111-1112, 1985).

Further, Saccharomyces cerevisiae reduces 4-chloroacetoacetic acid ester, which is an aliphatic diketone compound, to 4-hydroxybutanoic acid ester 7.
It is known to produce a variety of enzymes, but these 7
All the genes for seed enzymes have not been isolated yet (Chemicals and Biology, No. 35, pp. 590-598, 1997), and their amino acid sequences are also about Saccharomyces for two types of enzymes.
It is only deduced from the genomic sequence of S. cerevisiae (Biosci. Biotech. Biochem., 60, 1538-1539.
Page, 1996).

Bacillus cereus
Is known to exhibit reducing ability for the carbonyl group of 9-fluorenone, which is a non-diketone compound (Biosci. Biotechnol. Biochem., No. 62, 814-815.
(1998, 1998), the reducing ability of the microorganism for aromatic diketone compounds has never been reported.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have conducted various studies under the above circumstances, and as a result, have found that the Bacillus cerius T
im-r01 (Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Life Science and Technology, Accession No. FERM P-14210) has a reducing ability for aromatic diketone compounds, for example, by asymmetric reduction of benzyl (S) -benzoin. It was found that it produces a novel oxidoreductase that produces However, when live cells of the strain are used, the expression level of the oxidoreductase is greatly affected by the culture conditions. Further, when the redox reaction is carried out for a long time using the living cells, there is a drawback that a reverse reaction occurs and the yield of the asymmetric reduction product decreases.

[0006] Therefore, the present inventors in the previous application,
A novel gene encoding an oxidoreductase from the chromosomal DNA of the Bacillus serius FERM P-14210 strain and an open reading frame of the gene (hereinafter referred to as O
(Hereinafter, abbreviated as "RF"), the 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 (Japanese Patent Application No. 2000-2000). -25782
9).

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

Therefore, the present invention provides a novel oligonucleotide that can be used for amplification of an oxidoreductase gene, a novel oxidoreductase gene amplification / cloning method using the same oligonucleotide, a recombinant vector containing the same gene, and the same recombination. It is an object to provide a transformant transformed with a vector, and a method for producing an oxidoreductase by the transformant.

[0009]

The inventors of the present invention solve the above problems by providing the following inventions.

(1) An oligonucleotide having a nucleotide sequence substantially the same as the nucleotide sequence of 2 to 22 5'ends of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing. (2) An oligonucleotide having a base sequence that is substantially complementary to the 2'to 3'base sequences of the oxidoreductase gene shown in SEQ ID NO: 9 in the sequence listing. (3) An oligonucleotide containing a recognition sequence for a restriction enzyme on the 5'end side of the oligonucleotide according to (1) above. (4) An oligonucleotide containing a recognition sequence for a restriction enzyme on the 5'end side of the oligonucleotide according to (2). (5) An oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4 in the sequence listing. (6) (a) Target redox contained in a sample using at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) as a primer. Amplify the enzyme gene, (b)
A method for producing a transformant containing an oxidoreductase gene, 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 characterized in that it 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 a transformant containing an oxidoreductase gene according to (6) above. (8) The sample containing the oxidoreductase gene is a chromosomal DNA obtained from Bacillus cerius, D of the chromosome.
The method for producing a transformant containing an oxidoreductase gene according to (6) above, 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), characterized in that the two kinds of oligonucleotides according to (3) and (4) above are used as primers. Manufacturing method. (10) The transformant containing the oxidoreductase gene according to (6), (7) or (8) above, wherein the two kinds of oligonucleotides shown in SEQ ID NOS: 2 and 4 of the Sequence Listing are used as primers. Manufacturing method. (11) The above-mentioned (6), (7), (8), (9) or (1) wherein the transformed host cell is a microorganism.
0) A method for producing a transformant containing a redox enzyme gene described in (12) The above-mentioned (6), wherein the vector is pUC19 and the host cell to be transformed is Escherichia coli.
A method for producing the oxidoreductase gene-containing transformant according to (7), (8), (9) or (10). (13) Sequence listing obtained by polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) above as a primer. An oxidoreductase gene having the base sequence shown in SEQ ID NO: 5, 7, 10, 12, 14, 16, 18, or 20. (14) Sequence listing obtained by polymerase chain reaction using at least one oligonucleotide selected from the oligonucleotides described in (1), (2), (3), (4) and (5) above as a primer. A redox enzyme 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) above. (16) A DNA or an oligonucleotide that hybridizes to a DNA having a base sequence complementary to the base sequence of the gene described in (13) above. (17) A protein or polypeptide having an amino acid sequence encoded by a DNA that hybridizes to a DNA having a base sequence complementary to the base sequence of the gene described in (13) above. (18) A protein or polypeptide obtained by expressing a DNA that hybridizes to a DNA having a base sequence complementary to the base sequence of the gene described in (13) above. (19) A recombinant vector containing the gene according to (13) or (14). (20) The recombinant vector according to (19) above, wherein the vector is pUC19. (21) A transformant transformed with the recombinant vector according to (19) or (20). (22) The transformant according to the above (21), wherein the host cell is a microorganism. (23) The transformant according to the above (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 the oxidoreductase gene-containing transformant obtained by the method according to (12) and collecting the oxidoreductase from the culture.

The above-mentioned oligonucleotide according to the present invention,
The oxidoreductase gene, the recombinant vector containing the gene, the transformant transformed with the recombinant vector, and the oxidoreductase can be obtained based on the embodiments described below. The method for producing the transformant and the method for producing the redox enzyme can also be carried out based on the embodiments described below. 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 paragraphs (1) and (2) of "Means for Solving the Problems", were previously filed by the present inventors ( (Japanese Patent Application 2000-257829)
The base sequence can be set based on the oxidoreductase gene of SEQ ID NO: 9 determined in. In particular, in order to clone the full-length ORF of the oxidoreductase gene, it is preferable to set it based on about 20 bases on the 5'or 3'terminal side 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 include a recognition sequence for a restriction enzyme, a tag such as a histidine tag or a FLAG tag, a green fluorescent protein or the like on the 5'end side thereof. When the amplified gene is cut out with a restriction enzyme in the subsequent process, it is preferable to add a recognition sequence for a type II restriction enzyme to the 5'end of the oligonucleotide. When the oligonucleotide contains a restriction enzyme recognition sequence, the restriction enzyme recognition sequence to be added can be appropriately selected according to the cloning site of the vector. Examples of the restriction enzyme include a type II restriction enzyme such as restriction enzymes BamHI, EcoRI, HindIII, and K, in that the DNA chain can be accurately digested at the position where the 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 a clamp that aids digestion by the restriction enzyme may be added. In addition, for example, pUC-based vectors, pTYB-based vectors (New England BioLabs (registered trademark) (NE
W ENGLAND BioLabs) or pGEX vector (Amersham Pharmacia Biotech)
Some bases can be added between the recognition sequence of the restriction enzyme and the oligonucleotide encoding the oxidoreductase, depending on the type of vector used (eg, rmacia biotech). 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, the phosphite-triester method. A primer synthesizer can be used for primer synthesis.
A synthesizers Model 380A, 381A or 394 (trade name, manufactured by PE Applied Biosystems) can be used.

As a sample for gene amplification according to the present invention, any sample containing a target oxidoreductase gene can be used without limitation. For example, a chromosomal DNA prepared from a microbial cell containing a target oxidoreductase gene, if necessary, by lysing the cell, for example, using Gen Torukun (trade name, manufactured by Takara Shuzo Co., Ltd.) , A chromosomal DNA library of the strain, a cDNA library, a vector containing a target gene, and the like can be appropriately used. A strain belonging to the genus Bacillus can be used as an example of the sample. Specifically, for example, Bacillus cerius FERM P-
14210, Bacillus Celius IFO3001, IFO3002, and I available from Fermentation Research Institute
FO3003, IFO3132, IFO3457,
Same IFO 3466, Same IFO 3514, Same IFO 356
3, the same IFO 3836, the same IFO 13494, the same IFO
13597, IFO 13690 and IFO 153
05, and IAM1029, IAM1110, and IAM1 available from Institute of Molecular and Cellular Biology, University of Tokyo
Chromosomal DN from 656 and IAM1729
A, a DNA library of the same chromosome, a cDNA library or a plasmid containing the same gene. When the benzyl-reducing activity of the cells was examined, all the cells had the same degree of asymmetric reduction of benzyl as the FERMP-14210 cells.

According to the present invention, amplification of a desired oxidoreductase gene can be carried out by a method known per se, for example, polymerase chain reaction (hereinafter abbreviated as PCR). In the case of amplification by the PCR method, the sample containing the target gene contains an oligonucleotide as a primer for gene amplification, thermostable polymerase, dNTP (deoxy ATP, deoxy TTP, deoxy GTP and deoxy CTP) and a reaction buffer. Add to the solution,
Oligonucleotides can be annealed to the target gene and amplified. Here, the thermostable polymerase is a DNA-dependent 5 ′ → 3 ′ DNA synthase having an optimum temperature of 70 ° C. or higher and hardly inactivating even at 90 ° C. PCR conditions for the gene are, for example, incubation at 94 ° C. for 1 minute, 10 cycles at 94 ° C. for 1 minute and 72 ° C. for 3 minutes, and further 94 cycles.
1 minute at ℃, 2 minutes at 65 ℃ and 1 minute at 72 ℃
This is 10 cycles, and 94 more
1 min at ℃, 2 min at 60 ℃ and 1 min at 72 ℃
This can be carried out by performing 15 cycles as a cycle (Genes to Cells, No. 5, pages 111 to 125,
the year of 2000).

The gene amplification reaction can be carried out using one of the above-mentioned oligonucleotides. Also, 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 5'-terminal 2 to 22 nucleotide sequences of the oxidoreductase shown in SEQ ID NO: 9 (2) 3'ends 2 to 2 of the oxidoreductase gene shown in SEQ ID NO: 9
A combination with an oligonucleotide having a base sequence substantially complementary to the three base sequences, or preferably [B]
(1) From the 5'end of the oxidoreductase shown in SEQ ID NO: 2
An oligonucleotide having a base sequence substantially the same as 22 base sequences and containing a recognition sequence for a restriction enzyme on the 5'end side, and (2) 3'end 2 of the oxidoreductase shown in SEQ ID NO: 9. It can be carried out by using a combination with an oligonucleotide having a nucleotide sequence substantially complementary to the 23 nucleotide sequence and containing a restriction enzyme recognition sequence at the 5'end side. Specific examples of the combination of the oligonucleotides include a combination of the oligonucleotide having the base sequence shown in SEQ ID NO: 2 and the oligonucleotide having the base sequence shown in SEQ ID NO: 4. Furthermore, the gene amplification reaction can be carried out using a commercially available thermostable polymerase. Specific examples of such a polymerase include TaKaRa Ex Taq (trade name, manufactured by Takara Shuzo Co., Ltd.). When the oxidoreductase gene of interest 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
olymase (trade name, manufactured by Toyobo Co., Ltd.),
KOD DNA plus DNA polymer
se (trade name, manufactured by Toyobo Co., Ltd.) or Pyrob
est (trade name) DNA polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.), preferably KODDNA p
lus 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
By using A polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.), the cause can be avoided. 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 with 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
The target gene can be amplified from the strain by using polymerase (trade name, manufactured by Toyobo Co., Ltd.). IFO3132, IFO3457, I
FO3514, IFO13690 and IAM1110
Was unable to amplify the target gene even when KOD DNA polymerase was used.
yrbest (brand name) DNA polymeras
It can be amplified by using e (trade name, manufactured by Takara Shuzo Co., Ltd.).

After the amplification reaction of the gene, the PCR product is developed by electrophoresis using, for example, 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
The PCR fragment of interest can be purified by using the purification Kit (trade name, manufactured by Bio-Rad (BIO-RAD)).

When an oligonucleotide containing a restriction enzyme recognition sequence is used as a primer, a restriction enzyme that recognizes the restriction enzyme recognition sequence contained in the oligonucleotide primer is allowed to act on the amplified DNA fragment of the gene. The desired gene fragment can be obtained.

The recombinant vector containing the oxidoreductase gene can be prepared by mixing the above gene fragment and the vector at a molar ratio of 2 to 5: 1 and ligating. That is, first, a restriction enzyme is allowed to act on the vector to substantially completely digest it to obtain a vector for inserting a DNA fragment. 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.

There is no restriction on the vector that can be used for recombination,
For example, any of plasmid vector, bacteriophage vector, retrovirus vector, baculovirus vector, papillomavirus vector and the like can be used. Specifically, for example, pUC19
DNA (Takara Shuzo Co., Ltd.), pTYB-1 or pT
It is preferable to use YB-11 (manufactured by New England BioLabs (registered trademark)). 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. Further, 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 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 this to obtain a recombinant vector. Specifically, a ligation kit such as DNA Ligation Kit Ve
r. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.) or Ligat
The recombinant vector can be obtained by ligation using ion high (trade name, manufactured by Toyobo Co., Ltd.) under prescribed conditions.

When an oligonucleotide containing no restriction enzyme recognition sequence is used as a primer, PC
A commercially available vector suitable for cloning the amplified fragment by R, eg pGEM®-TEa.
pGEM (registered trademark) -T Easy Vector included in sy Vector System I (trade name, manufactured by Promega) can be used. Using the vector and T4 ligase, P
Ligation can be performed without treating the amplification product with CR and the vector with a restriction enzyme, and a recombinant vector containing an oxidoreductase gene can be prepared.

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

The above recombinant vector is introduced into a host by a known transformation method. This transformation can be suitably carried out by a conventional method (eg, Methods Enzymol., No. 204, 63-113, 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 host cells include 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
icurianColi (registered trademark) BL21-Cod
onPlus (trade name) Competent Cell
It is preferable to use s (trade name, manufactured by Toyobo Co., Ltd.).

Next, if necessary, the base sequence of the oxidoreductase gene contained in the transformant thus obtained and the amino acid sequence of the oxidoreductase encoded by the gene can be appropriately determined. Specifically, for example, after culturing the transformant, FlexiPrep (trade name)
Kit (trade name, manufactured by Amersham Pharmacia Biotech) was used to prepare plasmid DNA from the cells, and ABI PRIZM was prepared.
(Brand name) BigDye (Brand name) Termina
tor Cycle Sequencing Read
The base sequence can be determined by y Reaction Kit (trade name, PE Applied Biosystems). Based on the sequencing method, the present inventors have found that Bacillus cerius IFO3001, IFO
3003, IFO3132, IFO3563, IFO1
3597, IFO15305, IAM1029 and I
The nucleotide sequences of the oxidoreductase genes obtained from the AM1110 strain were determined, and at the same time, the amino acid sequences encoded by the oxidoreductases encoded by these genes were 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 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 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 specific conditions. Culturing can be performed 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, the expression of the enzyme may be induced by culturing at 15 to 37 ° C. until the absorbance at 550 to 600 nm becomes 1 to 6, then adding isopropylthiogalactoside (IPTG) and further culturing.

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.

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. Was determined by the following procedure as described in Japanese Patent Application No. 2000-257829, which is a prior application of the present inventors.

That is, Bacillus Celius FERM
The bacterial cells of the P-14210 strain are lysed, and the chromosomal DNA is recovered by a known method. The obtained chromosomal DNA was digested with the restriction enzyme Sau3AI and then a 2-4 kb DNA fragment mixture was obtained by agarose gel electrophoresis. The DN
In the A fragment, a part of the protruding end of the DNA was filled with Klenow fragment for the purpose of preventing ligation by two or more insert fragments.

Then, the vector p obtained by digestion with the restriction enzyme SalI in advance and pretreatment with Klenow fragment was obtained.
A recombinant vector was obtained by mixing UC19 and the above-mentioned DNA fragment mixture and performing known ligation.

The recombinant vector thus obtained was prepared by a conventional method (Methods Enzymol., No. 204, 63-113, 1991).
E. coli was transformed accordingly.

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

A recombinant vector (the inventors named the recombinant vector pUC-BC-B1) was recovered from the transformant selected in this manner, and then the Bacillus cerius contained in the vector was recovered. The nucleotide sequence of the chromosomal DNA fragment is obtained by using the commercially available kit EZ :: TN (trade name) <
KAN-1> Insertion Kit (trade name, Epicenter Technologies
Corporation)) and the dideoxy method.

Several ORFs considered from the base sequence
The amino acid sequences for each are shown in DDBJ / EMB
The amino acid database of the L / GenBank international nucleotide sequence database was searched for homology with known amino acid sequences. As a result, an amino acid sequence consisting of 249 residues was encoded by the yueD gene of Bacillus subtilis, which has similarity to sepiapterin reductase, and ORF Y of Saccharomyces cerevisiae.
It was found that they have 40% and 30% similarity, respectively, to the amino acid sequence encoded by IR036C. On the other hand, the kit EZ :: TN (trade name) <K
When the transposon was inserted into the ORF encoding the 249 amino acid residue by the insertion of the transposon using AN-1> Insertion Kit, the halo was not 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 with reference to the following examples.
The present invention is not limited to this embodiment.

Example 1 Two kinds of oligonucleotides were designed based on the nucleotide sequences on the 5'and 3'terminal sides in the nucleotide sequence of SEQ ID NO: 9 and synthesized by the phosphite-triester method. The sequence of the synthesized oligonucleotide is 5'-GGCGAAG
CTTGCGCTACGTTTATATAACAG-3
'(SEQ ID NO: 2) and 5'-CGCGGATCTCTA
TTCATCAATTTCTAATAAC-3 '(SEQ ID NO: 4). A in the base sequence shown in SEQ ID NO: 2
AGCTT and GGATCC in the nucleotide sequence shown in SEQ ID NO: 4 are restriction enzyme recognition sequences, respectively.

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

(2) Amplification of oxidoreductase gene and preparation of insert fragment The oxidoreductase gene was amplified using the two kinds of oligonucleotides prepared in Example 1 as primers. That is, 20 ng of the sample prepared in (1), 2 mM
DNTP 5 μl, 5 μM primer 4 μl each, KO
D DNA polymerase (trade name, manufactured by Toyobo Co., Ltd.) 2 units and 10 times concentrated KOD DN
PCR was carried out by adding pure water to 5 μl of the A polymerase PCR buffer to make the total volume 50 μl. PCR
Was incubated at 94 ° C for 1 minute, then 98
5 cycles of 15 seconds at 65 ° C., 2 seconds at 65 ° C. and 30 seconds at 74 ° C. for 5 cycles, 1 second at 98 ° C. for 15 seconds, 60 ° C. for 2 seconds and 74 ° C. for 30 seconds
10 cycles of this as a cycle, plus 98 more
10 cycles of 15 seconds at ℃, 2 seconds at 55 ℃ and 30 seconds at 74 ℃, and finally 7 cycles.
The incubation was carried out at 4 ° C. for 30 seconds.

After completion of PCR, the reaction solution was developed by electrophoresis using a 1.5% agarose gel, the PCR product-containing fraction of the oxidoreductase gene was cut out from the gel, and P
rep-A-Gene DNA Purificati
On (trade name, manufactured by Bio-Rad), the PCR product was recovered. That is, an agarose gel containing a DNA fragment was dissolved in 1 ml of Prep-A-Gene binding buffer,
DN in 20 μl of Prep-A-Gene matrix
A 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.

Then, 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, and the fraction containing the DNA fragment of interest was cut out from the gel, and the Prep-A was prepared.
-The DNA was purified using Gene DNA Purification (trade name, manufactured by Bio-Rad).

(3) Preparation of vector Next, 2 μg each of pUC19 DNA (manufactured by Takara Shuzo Co., Ltd.), the restriction enzymes BamHI and HindIII were used for each 2
0 unit, buffer (200 mM Tris-HCl (pH
8.5), 100 mM magnesium chloride, 5 mM dithiothreitol, 660 mM potassium acetate) 5 μl
And pure water was added to 0.4 unit of alkaline phosphatase derived from Escherichia coli (Takara Shuzo Co., Ltd.) to make a total volume of 50 μl.
And the vector was digested by reacting at 37 ° C. for 2 hours. Then, phenol treatment and ethanol precipitation were performed to prepare the vector.

(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, Inc. (Gibc)
oBRL Life Technologies)) were transformed (Methods Enzymol., No. 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 1-2 minutes to obtain a transformant.

(6) SOC medium (medium composition: tryptone 20 g / l, yeast extract 5 g / l, 10 mM sodium chloride, 2.5 mM potassium chloride) was added to the transformant obtained in the method (5) for producing oxidoreductase. 1 ml of 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM glucose) was added, and the mixture was incubated at 37 ° C. for 1 hour. After incubation, the microbial cell solution of the transformant was treated with ampicillin-containing LB agar medium (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 0 ° C. to obtain colonies of transformants containing the oxidoreductase gene. The colony was treated with ampicillin-containing L
It was shake-cultured in a liquid medium B at 37 ° C. overnight. After culturing,
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 cerius IFO3001 strain was mixed with 10 ml of PNE medium (medium composition: polypeptone 10 g / l, skipjack fish meat extract 10 g / l, sodium chloride 2 g / l). l, pH 7.4) and incubate at 37 ° C for 2
It was shake-cultured for 4 hours. After culturing, yeast
The chromosomal DNA of the bacterial cells was recovered using Gen Torukun for Gram-positive bacteria (trade name, manufactured by Takara Shuzo Co., Ltd.). That is, after the completion of the culture, the supernatant was removed by centrifugation to obtain bacterial cells, and 180 μl of GenTLE solution I of Gen Torukun (trade name, manufactured by Takara Shuzo Co., Ltd.) was added to the bacterial cells and immediately vortexed for 20 seconds. It was stirred. Then, G
20 μl of enTLE solution II was added, and the mixture was vortexed for 5 seconds, incubated at 70 ° C. for 10 to 15 minutes to lyse, and then GenTLE solution III 100 μm
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 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. Precipitate 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,
Twist the filamentous DNA precipitate with the tip of the pipette tip, and
It was dissolved in 300 μl of E buffer.

(2) Amplification of gene by PCR and preparation of insert fragment PCR was carried out using the two kinds of oligonucleotides synthesized in Example 1 as primers. That is, 1 μg of chromosomal DNA prepared in (1) and 2.5 mM dNTP4
μl, 5 μM primer 4 μl each, 5 U / μl Ta
KaRa ExTaq (trade name, manufactured by Takara Shuzo Co., Ltd.)
0.4 μl, 10-fold concentrated Ex Taq buffer 10 μl and 1 mg / ml anti-Taq high (trade name, manufactured by Toyobo Co., Ltd.) 0.4 μl were added with pure water to make 100 μl, and PCR was performed. PCR is 94 ℃
After 1 minute incubation at 94 ° C. for 1 minute, 72 ° C. for 3 minutes as 1 cycle, this is repeated for 10 cycles, 94 ° C. for 1 minute, 65 ° C. for 2 minutes and 7 cycles.
15 cycles for 1 minute at 2 ° C as 1 cycle,
Furthermore, 94 ° C for 1 minute, 60 ° C for 2 minutes and 7
This was carried out 10 times by setting 1 minute at 2 ° C as one cycle, and finally, incubation was performed at 72 ° C for 1 minute.

(3) Preparation of recombinant vector pUC19 (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 obtain half the amount of the light hi of the DNA solution.
gh (trade name, manufactured by Toyobo Co., Ltd.) was added and ligated at 16 ° to obtain a recombinant vector.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product Transformation was carried out in the same manner as in Example 2 (5) except that the recombinant vector prepared in (3) was used. Got the body 1 ml of 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 treated with LB containing ampicillin.
The seeds were plated on an agar medium and cultured overnight at 37 ° C. to obtain colonies of transformants containing an oxidoreductase gene. Then, the colony was cultured in an LB liquid medium containing ampicillin at 37 ° C. overnight with shaking. After culturing, cells were collected by centrifugation. Next, FlexiPrep (trade name) Kit
(Amersham Pharmacia Biotech) was used, and a 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 (product name) BigDye (product name) T
erminator Cycle Sequence
g Ready Reaction Kit (trade name,
It was determined from the 5'side of the gene by the dideoxy method using PEE 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 these two sides are the same.

The base 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 As in Example 3 (1) except that Bacillus cerius IFO15305 was used as the strain, chromosome D of the bacterial cell was obtained.
NA was prepared.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment A PCR product treated with a restriction enzyme 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 The procedure of Example 3 (4) was repeated except that the recombinant vector obtained in (3) was used as a recombinant vector. A transformant was prepared. PCR was performed in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The base sequence of the product was determined.

As a result, P in IFO15305 strain
The base sequence of the CR product was found to be 750 bp. The nucleotide sequence derived from the IFO15305 strain is represented by 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 cerius IFO3003, IFO3563 or IFO13597 was used as the bacterial strain. .

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment The same operation as in Example 2 (2) was performed except that 1 μg of the chromosomal DNA prepared in (1) was used as a sample, and the restriction enzyme was added. 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. 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 each recombinant vector.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product The procedure of Example 3 (4) was repeated except that the recombinant vector obtained in (3) was used as a recombinant vector. The transformant was prepared. In addition, 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 base sequence of IFO3003 is SEQ ID NO: 1.
0, the base sequence of IFO3563 is SEQ ID NO: 12 and I
The nucleotide sequence of FO13597 is shown in SEQ ID NO: 14. The deduced amino acid sequence of IFO3003 is shown in SEQ ID NO: 1.
1, the deduced amino acid sequence of IFO3563 is SEQ ID NO: 13
And the deduced amino acid sequence of IFO13597 is shown in SEQ ID NO: 15.

Example 8 (1) Preparation of chromosomal DNA of IAM1029 strain As in Example 3 (1) except that Bacillus cerius IAM1029 was used as the strain, chromosome DN of the bacterial cell was obtained.
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,
Sample prepared in (1) 2 μg, 2 mM dNTP 5 μ
1, 5 μM each of 5 μl, KOD plus
DNA polymerase (trade name, manufactured by Toyobo Co., Ltd.) 1 unit, 25 mM magnesium sulfate 2 μ
l and KOD plus DNA polymer
The total volume is 50 by adding pure water to 5 μl of 10 times concentrated buffer of SE.
PCR was performed using 1 μl. PCR was carried out by incubating at 94 ° C for 2 minutes and then at 94 ° C for 15 seconds for 60 seconds.
1 cycle of 30 seconds at 68 ° C and 1 minute at 68 ° C for 10 cycles, and then at 94 ° C for 15 seconds at 55 ° C.
10 cycles of 20 seconds at 68 ° C. for 1 minute at 68 ° C. and 5 seconds at 94 ° C. for 5 seconds
This was carried out by 10 cycles of 1 cycle of 0 ° C. for 10 seconds and 68 ° C. for 1 minute, and finally 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. Then, a 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 a recombinant vector.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product The procedure of Example 3 (4) was repeated except that the recombinant vector obtained in (3) was used as a recombinant vector. A transformant was prepared. PCR was performed in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The base sequence of the product was determined.

The nucleotide sequence of IAM1029 is shown in SEQ ID NO: 16.
The deduced amino acid sequence is shown in SEQ ID NO: 17.

Example 9 (1) Preparation of chromosomal DNA of bacterial strain Chromosome DN of bacterial cells was carried out in the same manner as in Example 3 (1) except that Bacillus cerius IFO3132 was used as the bacterial strain.
A was prepared.

(2) Amplification of Gene by PCR and Preparation of Insert 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, 5 μl of 10 μM each, Pyrobes
t (trade name) DNA polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.) 1.25 units and Pyrobe
st (trade name) 10 times buffer of 10 times DNA polymerase was added with pure water to make the total volume 100 μl.
CR was performed. PCR was carried out by incubating at 94 ° C for 1 minute, and then 10 cycles at 94 ° C for 1 minute and 72 ° C for 3 minutes, and then at 94 ° C.
1 cycle at 65 ° C. for 2 minutes and 72 ° C. for 1 minute as 15 cycles, then 94 ° C.
1 cycle for 1 minute, 60 ° C. for 2 minutes, and 72 ° C. for 1 minute as 10 cycles, and finally 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. A recombinant vector was obtained by ligation in the same manner as in Example 2 (4) except that the PCR product described in (2) was used as the PCR product.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product PCR was carried out 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. PCR was performed in the same manner as in Example 3 (4) except that the transformant was used as a transformant.
The base sequence of the product was determined.

The base sequence of IFO3132 is SEQ ID NO: 18
The deduced amino acid sequence is shown in SEQ ID NO: 19.

Example 10 (1) Preparation of chromosomal DNA of IAM1110 strain As in Example 3 (1) except that Bacillus cerius IAM1110 was used as the strain, chromosome DN of the bacterial cell was obtained.
A was prepared.

(2) Amplification of Gene by PCR and Preparation of Insertion Fragment The same procedure as in Example 15 (2) was performed except that 1 μg of the chromosomal DNA prepared in (1) was used as a sample, and the restriction enzyme was added. 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. 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 each recombinant vector.

(4) Preparation of Transformant and Determination of Nucleotide Sequence of PCR Product The procedure of Example 3 (4) was repeated except that the recombinant vector obtained in (3) was used as a recombinant vector. The transformant 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 the transformant.

The base sequence of IAM1110 is SEQ ID NO: 20.
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 GGTGGTTGCTCTTCCAACATG.
CGCTACGTTATCATAAC (SEQ ID NO: 22)
And CGGCTGCAGTTATTCATCATCAATT
CTAATAAC (SEQ ID NO: 23). Sequence number 2
GCTCTTC in 2 and CTGCAG in SEQ ID NO: 23 are identification sequences of restriction enzymes, respectively.

(2) Amplification of oxidoreductase gene The procedure of Example 2 (2) was repeated except that the two kinds of oligonucleotides prepared in (1) were used as primers.
PCR was performed. Then, a PC was prepared in the same manner as in Example 2 (2) 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 unit and NEBuffer3 (trade name,
New England BioLabs) (1M sodium chloride, 500 mM Tris-HCl (pH 7.9),
Pure water was added to 5 μl of 100 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 P
The CR product was purified and recovered by the phenol extraction method and the ethanol precipitation method. Sap was added to the recovered PCR product.
I 9 unit and NEBuffer4 (trade name, manufactured by New England BioLabs) (500 mM
Potassium acetate, 200 mM trisacetic acid (pH 7.
9), 100 mM magnesium acetate, 10 mM dithiothreitol) (5 μl) were 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. Sa
After the restriction enzyme treatment with pI, the reaction solution was developed by electrophoresis using 1.5% agarose gel to obtain the desired DN.
Fractions containing the A fragment were excised from the gel. Then, Pr was used in the same manner as in Example 2 (2) except that the fraction was used.
ep-A-Gene DNA Purificatio
n (trade name, manufactured by Bio-Rad) was used 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 (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, Prep-A-Gene D was performed 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 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. 1 ml of SOC medium to the transformant
In addition, it incubated at 37 degreeC for 1 hour. After incubation, the bacterial cell solution of the transformant was treated with ampicillin-containing L
The cells were plated on B agar medium and cultured overnight at 37 ° C. to obtain colonies of transformants containing an oxidoreductase gene. Then, 60
The colony was shake-cultured in an LB liquid medium containing ampicillin at 37 ° C. until the absorbance at 0 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), 5
Equilibrated with 00 mM sodium chloride, 1 mM ethylenedinitrorotetraacetic 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 solution containing 20 mM dithiothreitol (20 mM Tris-HCl (pH 8.0), 50
The oxidoreductase protein was cleaved from the intein by incubation overnight at 4 ° C. in 0 mM sodium chloride, 1 mM ethylenedinitrorotetraacetic 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 ampicillin-containing LB liquid medium and cultured at 37 ° C. until the absorbance at 600 nm reached 5. Next, isopropylthiogalactoside (IPTG) and benzyl as a substrate were added so that the final concentrations were 0.5 mM and 1 mM, respectively, and the mixture was further incubated 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. 80% yield, 88 optical purity
% Ee.

Reference Example 1 (1) Method for recovering chromosomal DNA from bacterial cells A single colony of Bacillus cerius FERM P-14210 strain was inoculated into 10 ml of PNE medium, and 37
The cells were shake-cultured at 24 ° C for 24 hours. After the culture was completed, chromosomal DNA was recovered using Gen Torukun (trade name, manufactured by Takara Shuzo Co., Ltd.) on the cells. The recovered chromosomal DNA is 3
It was dissolved in 00 μl of TE buffer. The amount of DNA obtained was 220 μg in several batches.

(2) Preparation of Chromosomal DNA Insertion Fragment The chromosomal DNA prepared in (1) was digested with the restriction enzyme Sau3A.
The chromosomal DNA was partially digested by applying I (Takara Shuzo Co., Ltd.). Then, the partially digested reaction solution was extracted with phenol and then subjected to isopropanol precipitation twice to purify the DNA fragment.

The Klenow fragment (Takara Shuzo Co., Ltd.) is allowed to act on the DNA fragment obtained by the above method to produce 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. Then, the DNA fragment is subjected to agarose gel electrophoresis to cut out a fraction containing the DNA fragment of 2 to 4 kb from the gel, and Prep-A-G
ene DNA Purification (trade name,
The DNA fragment was recovered by Bio-Rad).

(3) Preparation of vector Next, pUC19 DNA (Takara Shuzo Co., Ltd.) was digested with the restriction enzyme SalI (Takara Shuzo Co., Ltd.). Then, 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'overhang in 9 is TC.

(4) Ligation of Insertion Fragment with Vector The chromosomal DNA having 5'protruding ends of GA and the pUC19 DNA having 5'protruding ends of TC were mixed at a molar ratio of 2: 1 to prepare a DNA Ligation Kit Ve.
r. 1 (trade name, manufactured by Takara Shuzo Co., Ltd.) was used for ligation. This completed the chromosomal DNA library of Bacillus serius. Escherichia coli DH5 prepared by a conventional method using 25 ng of the library
α strain (Gibco BRL Life Technologies
fe Technologies)) were transformed (Methods Enzymol., No. 204, 63-113).
P., 1991).

(5) Screening for oxidoreductase gene [0110] Transformed Escherichia coli was dissolved in dimethyl sulfoxide, and a 0.1 M benzyl solution was added to each plate in an amount of 40 μl.
Plate on LB agar medium containing ampicillin, 37 ℃
After overnight culture at 2710 colonies, 2710 colonies were observed. Furthermore, it was left to stand at room temperature and waited for halo formation. That is, when benzyl around the colony of E. coli is reduced to produce benzoin, the solubility is increased and a halo is formed, which is used as an index for screening a clone having a benzyl reductase. As a result, there were seven colonies that formed halos, and the earliest colonies formed halos 30 hours after plating the transformed bacterial solution.

The thus obtained 7 colonies were inoculated into 2 ml of a liquid culture LB medium and cultured at 37 ° C. for 12 hours with shaking. 20 μl of 0.1 M benzyl substrate (final concentration 1 m
After the addition of M), the cells were further cultured for 24 hours. The substrate and product were extracted from the bacterial solution with ethyl acetate and developed by thin layer chromatography using hexane: ether: acetic acid (volume ratio 45: 5: 1) as a solvent to reconfirm the benzyl reduction activity. As a result, it was only the bacterial cells that formed the earliest halo that showed activity. Therefore, the present inventors named the recombinant vector possessed by this bacterium as pUC-BC-B1.

(6) Determination of nucleotide sequence of oxidoreductase gene The nucleotide sequence of the insert of pUC-BC-B1 was determined by E
Z :: TN (trade name) <KAN-1> Insertio
n Kit (trade name, manufactured by Epicenter Technologies, Inc.) was used. By this decision, pUC-BC-
The insert of B1 was found to be 2438 bp.
Next, the nucleotide sequence of the DNA fragment is set to DDBJ / EMB.
A homology with a known base sequence was searched 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 set to DDBJ / EMB.
A homology search was performed on the amino acid sequence database of the L / GenBank international nucleotide sequence database, and as a result, the amino acid sequence consisting of 249 residues shown in SEQ ID NO: 2 was encoded by the yueD gene of Bacillus subtilis (243 amino acid residues). With 40% homology. Although the function of the YueD protein is unknown, its amino acid sequence had high homology to sepiapterin reductase.

In addition, OR of Saccharomyces cerevisiae
3 with the amino acid sequence encoded by FYIR036C
The finding was also found to have 0% similarity. This YIR0
Although the function of the 36C protein is unknown, it is known that its amino acid sequence has similarity to a short-chain alcohol dehydrogenase, which is a type of oxidoreductase.

The OR inferred from the Yue D protein
In order to confirm that F has a redox function, pUC-BC-B1 having a transposon inserted on the ORF was subjected to 36 ° C. at 25 ° C. on an agar plate containing ampicillin coated with benzyl in the process of determining the nucleotide sequence. ~ 4
It was cultured for 8 hours. When observed after culture, no halo-forming colonies were found. Therefore, the OR
It was determined that F codes for 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 novel oxidoreductase gene amplification / cloning method 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 by the transformant. Using the primer of the present invention, an oxidoreductase gene of an unknown Bacillus serius species can be isolated. Further, by using the gene of the present invention, a novel oxidoreductase capable of asymmetrically reducing an aromatic diketone compound can be easily cloned and can be expressed 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 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 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 225 230 235 240 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 cgctacgcaa 60 ttattagaaa aaaatacaac tgtcatctct atttctagaa gagaaaatca agagcttaca 120 aaacttgcag agcaatataa cagcaattgc atttttcatt 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> 8 <211> 249 <212> 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 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 ttcattgcat 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 gaaagctatt 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 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 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 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> 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 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 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 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 gtgaggctat tgccactcaa 60 ttgttagaag 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 aaagtagata 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 Phe 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 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 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 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 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 Phe 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 tgtcatctct atttctagaa gagaaaatca 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 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> 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

Front page continuation (72) Inventor Maruyama Reiji 52 Teramachi Keya Kidai, Toyama City No. 203 Tsukasa Heights Teramachi (72) Inventor Yasushi Itoi 1-21-502 Minamishinda, Daito City, Osaka Prefecture (56) References JP 2002 -65267 (JP, A) Gene, 1997, Vol. 189, p. 213-219 J. Bacteriol. , 1991, Vol. 173, no. 17, p. 5385-5395 Chemistry Letters, 1988, Vol. 7, p. 1191 -1192 Chemistry Letters, 1985, Vol. 8, p. 1111 -1112 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 9/02 SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG) )

Claims (16)

(57) [Claims] [Claim 1] comprising the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4 of the Sequence Listing oligonucleotide. 2. A target oxidoreductase gene contained in a sample is amplified using (a) at least one oligonucleotide selected from the oligonucleotides according to claim 1 as a primer, and (b) the amplified gene is amplified. A method for producing a transformant containing an oxidoreductase gene, which comprises inserting the vector into a vector to prepare a recombinant vector, and then obtaining a transformant transformed with the vector. 3. A sample containing a redox 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 a transformant containing an oxidoreductase gene according to claim 2 . 4. The sample containing a redox gene is a chromosomal DNA obtained from Bacillus serius, a DNA library of the chromosome, a cDNA library or a recombinant vector containing the gene. 2. The method for producing a transformant containing an oxidoreductase gene according to 2 . 5. The method for producing a oxidoreductase gene-containing transformant according to claim 2, 3 or 4 , wherein the two kinds of oligonucleotides shown in SEQ ID NOS: 2 and 4 of the Sequence Listing are used as primers. 6. The method for producing a transformant containing an oxidoreductase gene according to claim 2, 3, 4, or 5 , wherein the host cell to be transformed is a microorganism. 7. The vector is pUC19, and the host cell to be transformed is Escherichia coli.
A method for producing a transformant containing an oxidoreductase gene according to 2, 3, 4 or 5 . 8. obtained by polymerase chain reaction using at least one oligonucleotide selected from claims 1 Symbol placement of oligonucleotides as primers,
SEQ ID NO: 5, 7, 10, 12, 14, 16, 1 in the sequence listing
Oxidoreductase gene having the nucleotide sequence shown in 8 or 20. 9. obtained by polymerase chain reaction using at least one oligonucleotide selected from claims 1 Symbol placement of oligonucleotides as primers,
Sequence ID Nos. 6, 8, 11, 13, 15, 17, 1 in the sequence listing
An oxidoreductase gene encoding the amino acid sequence shown in 9 or 21. 10. Coat the gene according to claim 8 or 9
De redox enzyme. 11. A recombinant vector containing the gene according to claim 8 or 9 . 12. The method of claim 1 vector is pUC19
The recombinant vector according to 1 . 13. transformant transformed with the recombinant vector of claim 11 or 12 wherein. 14. The transformant according to claim 13 , wherein the host cell is a microorganism. 15. The transformant according to claim 13 , wherein the microorganism is Escherichia coli. 16. The oxidoreductase gene-containing transformant obtained by the method according to claim 2 , 3 , 4 , 5 , 6 or 7 is cultured, and the oxidoreductase is collected from the culture. Method for producing oxidoreductase.