JP2000245471A - Formate dehydrogenase gene, recombinant vector containing the same, transformant containing the recombinant vector and production of formate dehydrogenase using the transformant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new formate dehydrogenase gene which encodes a formate dehydrogenase containing a specific amino acid sequence and is useful in the inexpensive production or the like of the above enzyme that possesses high specific activity, shows a small Km value to formic acid and NAD+ and exerts the broad temperature or pH stability. SOLUTION: This formate dehydrogenase gene is a new gene which encodes a formate dehydrogenase comprising of the amino acid sequence represented by the formula or the amino acid sequence in which one or plural amino acids are deleted, substituted or added to the amino sequence represented by the formula and is useful for producing the formate dehydrogenase that possesses high specific activity, shows a small Km value to formic acid and nicotinamide adenine dinucleotide (NAD+) and exerts the broad temperature or pH stability conveniently in a large quantity and at a low cost. This gene is obtained by separating a chromosomal DNA from the genus Hyphomicrobium strain JT-17 (FERM P-16973) by a conventional method, preparing a chromosomal DNA library by the use of this chromosomal DNA and then screening out this library with a probe comprising the partial sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a formate dehydrogenase gene, a recombinant vector containing the gene, a transformant using the recombinant vector, and a method for producing formate dehydrogenase using the transformant. Things.

[0002]

2. Description of the Related Art Formate dehydrogenase [EC1.2.1.2], which can use nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ) as an electron acceptor, produces NADH from formic acid and NAD ⁺ by using carbonic acid as a by-product. To do, NA
Promising as an enzyme for NADH regeneration in an enzyme reaction system requiring DH. In addition, there is an advantage that by-products do not accumulate in the reaction system because the reaction product is carbonic acid,
It is attracting attention as an enzyme for regenerating NADH on an industrial scale. Furthermore, an enzyme having a low Km value for formic acid is also useful for specific detection of formic acid and microquantification. Until now, the above-mentioned formate dehydrogenase has been used for Moraxella sp.
J. Bacteriol. Vol. 170, 3189, 1988) and Achromobacter parvulus: Eur.
J. Biochem vol. 99, 569, 1979), Pseudomonas oxalaticus: Eur.
J. Biochem. Vol. 83, 485, 1978). However, these strains have low productivity of formate dehydrogenase, and the resulting formate dehydrogenase has low specific activity, a large Km value of formate dehydrogenase for formic acid and NAD ⁺ , temperature stability and pH. Because of its narrow stability range, it was not suitable for industrial use.

[0003] In order to solve such a problem, the present inventors have conducted extensive separation of methanol-assimilating bacteria known to have formate dehydrogenase, and as a result, the genus Hyphomicrobium ( Hyphomicrobium sp.) Has a high specific activity, has a low Km value for formic acid and NAD ⁺ , and produces formate dehydrogenase having a wide range of temperature stability and pH stability. A patent has already been filed for a dehydrogenase and a method for producing the same (Japanese Patent Application No. 10-250930).

[0004]

According to the above invention, a formate dehydrogenase having a high specific activity, a small Km value for formic acid and NAD ⁺ , and a wide range of temperature stability and pH stability can be obtained. And its purification can be easily performed. However, when producing formate dehydrogenase by culturing 30 L in the above invention, it is necessary to use a special medium containing methanol as a single carbon source and culture the produced cells for a long period of 6 days, Furthermore, the problem that the production efficiency is very poor due to the small amount of formate dehydrogenase produced by this microorganism and it is difficult to mass-produce formate dehydrogenase has not been solved yet.

[0005] The present invention has been made in view of the above circumstances, and a genetic engineering material for mass-producing the above-described formate dehydrogenase by genetic engineering, and a formate dehydrogenase using the material. It is intended to provide a method for producing an enzyme.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve such problems, and as a result, have found that the formate dehydrogenase gene produced by a microorganism of the genus Hyphomicrobium sp. And the DNA of the gene
The present invention has been achieved by elucidating the nucleotide sequence. That is, the first invention relates to the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 1.
Alternatively, the gist is a gene encoding formate dehydrogenase comprising an amino acid sequence in which several amino acids have been deleted, substituted or added. Further, the second invention has a DNA comprising a base sequence represented by SEQ ID NO: 2 or a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 2, In addition, the gist is a gene encoding formate dehydrogenase. Further, a third invention is directed to a recombinant vector containing the above gene. A fourth invention is characterized in that a transformant containing the above-described recombinant vector is cultivated, and a fifth invention is that this transformant is cultured in a medium, and formate dehydrogenase is collected from the culture. The gist of the present invention is a method for producing formate dehydrogenase.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The formate dehydrogenase according to the present invention comprises:
It is a protein having the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids are deleted, substituted or added. Therefore, the gene of the present invention is a gene encoding the above amino acid sequence, and specifically, is a gene represented by DNA consisting of the nucleotide sequence of SEQ ID NO: 2.

[0008] Isolation of such a gene and preparation of a recombinant vector containing this gene, preparation of a transformant using the recombinant vector, culturing of the transformant, and the like are performed by known methods such as molecular cloning (cold culturing). Spring Harbor Press, 1989). For example, a gene is obtained by synthesizing a partial sequence of the base sequence represented by SEQ ID NO: 2 and isolating it from a DNA library using this synthetic DNA as a probe. Chromosomal DNA as primer
Can be obtained by, for example, a method of amplifying a target gene by a PCR method using as a template.

[0009] The recombinant vector of the present invention contains the gene encoding formate dehydrogenase obtained as described above, and the vector encodes the formate dehydrogenase obtained as described above. Can be obtained by ligating the genes. The vector is not particularly limited. For example, the plasmid vector pKK223-3
(Pharmacia), pUC19 (Takara Shuzo), pBluesc
riptKS (+) (manufactured by Stratagene), pBR322 (manufactured by Toyobo) and the like. In addition, a transformant capable of expressing formate dehydrogenase can be obtained by introducing the thus obtained recombinant vector into, for example, Escherichia coli, Bacillus subtilis, yeast, or the like by a transformation method. As a method for producing formate dehydrogenase using the transformant thus produced, the transformant is cultured in a medium suitable for the production of formate dehydrogenase and suitable for the growth of each host microorganism, After harvesting, the transformant can be produced by lysing the cells with ultrasonic waves or lysozyme and centrifuging. Further, the centrifuged supernatant can be purified using a commercially available ion exchange resin, affinity resin or the like.

[0010]

Next, the present invention will be described specifically with reference to examples.

Example 1: Preparation of hybridization probe (1) Analysis of N-terminal amino acid sequence of formate dehydrogenase Hyphomicrobium sp. Strain JC-17 (FERM P-
16973) was purified from a gas-phase peptide sequencer 473A (Applied Biosytems).
After being subjected to Edman degradation using a PTH amino acid derivative, the PTH amino acid derivative analyzer 120A (Applied
This derivative was analyzed using Biosytems). As a result, it was found that the sequence of the N-terminal 20 amino acid residues of formate dehydrogenase was the amino acid sequence represented by SEQ ID NO: 3.

(2) Analysis of FDH Conserved Region When the amino acid sequences of formate dehydrogenase derived from eight different microorganisms were compared, the amino acid sequence represented by SEQ ID NO: 4 was found as a completely conserved region. .

(3) Partial amplification of the formate dehydrogenase gene by PCR and digoxigenin (DIG) labeling A sense primer is designed from the N-terminal amino acid sequence analyzed above, and an antisense primer is designed from the amino acid sequence of the conserved region. The DNA shown in FIG. 1 was synthesized.
Hyphomicrobium sp. JC-17 strain (FERM P-
16973) was isolated by a known method (Saito & Miura,
After lysing with lysozyme (manufactured by Seikagaku Corporation) according to Biochim. Biophys., Acta, vol. 72, p619, 1963), DNA was extracted with an SDS-containing alkaline buffer and phenol.
Further, RNA was digested with RNase to purify 1 mg of chromosomal DNA. Known methods (Simpson et al., Bioche
In accordance with m. Biophys. Res. Commun., vol. 151. p487, 1988), 10 ng of the obtained chromosomal DNA was used as a template, and the above primers and a DNA polymerase derived from Thermus Aquaticus (manufactured by Takara Shuzo) were used. PCR was performed for 30 cycles at 94 ° C for 1 minute, 60 ° C for 90 seconds, and 72 ° C for 90 seconds. The amplified DNA fragment (about 9
00bp) was separated on an agarose gel, and then extracted and purified using β-agarase (Takara Shuzo). This DNA
The fragment was subjected to DIG by a random prime method using a DIG labeling kit (Boehringer Mannheim).
It was labeled and used as a probe for the following experiments.

Example 2: Identification of formate dehydrogenase gene (1) Southern blot analysis 1 µg of the chromosomal DNA prepared above was completely digested with a restriction enzyme SalI (Takara Shuzo), electrophoresed on an agarose gel, and then subjected to nylon membrane NY13N. (Schliecher & S
chuel). After the above-mentioned DIG-labeled probe was hybridized to this nylon membrane at 60 ° C. overnight, it was washed at room temperature for 5 minutes, and further washed twice at 60 ° C. for 15 minutes. As a result of detecting chemiluminescence by CSPD using a DIG detection kit (manufactured by Boehringer Mannheim) with an RX film (manufactured by Fuji Photo), a positive band of about 5 kb was obtained.

(2) Hyphomicrobium JC-17
Preparation of Strain (FERM P-16973) Chromosomal DNA Library 10 μg of the chromosomal DNA obtained above was subjected to restriction enzyme SalI.
After decomposing with agarose gel (manufactured by Takara Shuzo) and separating by agarose gel, a fragment of about 5 kb was extracted and purified using β-agarase (manufactured by Takara Shuzo). Apart from that, the vector pBluescr
1 μg of iptII (+) (manufactured by Takara Shuzo) was completely digested with a restriction enzyme SalI, and treated with bacterial alkaline phosphatase (manufactured by Takara Shuzo) to obtain 0.8 μg of a vector DNA fragment.
0.28 μg of the obtained chromosomal DNA fragment and vector DN
A ligation reaction was performed with 0.1 μg of the A fragment using a DNA ligase derived from T4 phage (Takara Shuzo) at 16 ° C. for 30 minutes to obtain a recombinant DNA. The obtained recombinant DNA was transformed into E. coli HB101 competent cell 200.
μg (manufactured by Takara Shuzo), left at 0 ° C. for 1 hour, and then heated at 42 ° C. for 120 seconds to perform transformation.

The resulting transformant was inoculated into 1 ml of L medium and cultured at 37 ° C. for 1 hour, and the culture was spread on an L agar plate medium containing ampicillin to obtain an ampicillin-resistant strain. L-containing these strains containing 50 μg / ml of ampicillin
After inoculating the medium, the cells were cultured overnight, and after the cells were collected, the plasmid was prepared by the alkali-SDS method to obtain a Hyphomicrobium chromosome DNA library.

(3) Isolation of formate dehydrogenase gene The transformant obtained by the above-mentioned Hyphomicrobium chromosome DNA library was cultured overnight on a nitrocellulose filter placed on an L agar plate medium containing ampicillin. . The filter was transferred onto an L agar plate medium containing 200 μg / ml chloramphenicol, cultured overnight, and then lysed with SDS and alkali. The nitrocellulose filter was subjected to colony hybridization under the same conditions as in the Southern blot analysis described above. As a result, about 2,000 colonies were examined, and four positive clones were obtained.

(4) Determination of nucleotide sequence of formate dehydrogenase gene One strain was selected from the above four positive clones and inoculated into 100 ml of L medium containing 50 μg / ml of ampicillin.
After overnight culture at 37 ° C., a plasmid was prepared by the alkali-SDS method, and this plasmid was designated as pFSI. pFSI
1 μg is transcribed by a known method (Sanger, Nicklen & Coulson, P
roc. Natl. Acad. Sci., vol. 74, p5463, 1977), and subjected to nucleotide sequence analysis in a dideoxy chain termination reaction. Taq dye primer cycle S for reaction
The equencing Kit (Applied Biosystems) was used.
The above reaction product was subjected to DNA sequencer 373A (Applied Bi
osystems), and the nucleotide sequence of 1197 base pairs shown in SEQ ID NO: 2 was determined.

Example 3 Construction of Formate Dehydrogenase Expression Plasmid Ec was added to the 5 'end of the sequence from the 1st base upstream to the 19th base of the initiation codon of the formate dehydrogenase gene represented by SEQ ID NO: 5.
At the 5 ′ end of the DNA linked with the oRI recognition sequence and the antisense sequence from the 98th base to the 117th base downstream of the stop codon of the formate dehydrogenase gene represented by SEQ ID NO: 6
Synthesizing each DNA linked HindIII recognition sequence,
PCR primers were used. Using 10 ng of plasmid pFSI as a template, PCR was performed at 94 ° C. for 1 minute, at 60 ° C. for 90 seconds, and at 72 ° C. for 1 minute using the above-mentioned primers and Thermus Aquaticus-derived DNA polymerase (manufactured by Takara Shuzo).
30 cycles were performed at 90 ° C. for 90 seconds. Amplified DN
The A fragment (about 1300 bp) was separated on an agarose gel and then extracted and purified using β-agarase (Takara Shuzo). This DNA fragment, 150ng and pT7Blue T-vector
100 ng (Novagen) and mixed with T4 phage-derived DNA ligase (Takara Shuzo) at 16 ° C.
Ligation for minutes. The obtained plasmid was subjected to restriction enzymes EcoRI (Takara Shuzo) and HindIII (Takara Shuzo).
After separation with agarose gel, a DNA fragment containing a formate dehydrogenase gene was extracted and purified using β-agarase (Takara Shuzo). On the other hand, pKK223-3 (manufactured by Pharmacia) was digested with an EcoRI recognition site located downstream of the SD sequence and a HindIII recognition site of a multiple cloning site.

DNA containing the above formate dehydrogenase gene
Fragment 150ng and vector DNA (pKK223-3) fragment 10
Ligation reaction of 0 ng was carried out at 16 ° C. for 30 minutes using T4 phage-derived DNA ligase (manufactured by Takara Shuzo) to obtain a recombinant vector pKKFDH containing a formate dehydrogenase gene. FIG. 1 is a schematic diagram showing the construction of this recombinant vector pKKFDH. Then, pKKFDH was transferred to E. coli JM109 competent cell 2
After mixing at 0 ° C. for 1 hour, the mixture was heated at 42 ° C. for 120 seconds to perform transformation.
The resulting transformant was inoculated into 1 ml of L medium, and
After culturing for 1 hour, the culture solution was treated with 50 μg of ampicillin.
/ Ml of L-agar plate, and 100 colonies of transformed Escherichia coli containing the formate dehydrogenase gene were obtained. This was designated as Escherichia coli (Escherichia coli) JM109 / pKKFDH.

Example 4: Production of formate dehydrogenase in E. coli The transformed E. coli JM109 / pKKFDH prepared in Example 3 was inoculated into 300 ml of L medium containing 50 μg / ml of ampicillin and then incubated at 37 ° C. overnight. Culture was performed. The preculture is inoculated into 30 L of L medium containing 50 μg / ml of ampicillin,
After culturing at 37 ° C. for 8 hours, 1 mM of isopropyl β-thiogalactopyranoside was added, and the cells were further cultured for 10 hours and then collected. The obtained cells were cultured in 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol.
After suspending in 00 ml, it was subjected to ultrasonic treatment using Insonator210M (manufactured by KUBOTA). From the supernatant excluding the crushed cells, D
The formate dehydrogenase was purified using EAE-Sephacel column chromatography, phenyl sepharose CL-4B column chromatography and 5′AMP-Sepharose 4B column chromatography, and 3,000 units of formate dehydrogenase were recovered.

From the above results, the culture time for culturing 30 L of Hyphomicrobium sp. JC-17 strain (FERM P-16973) was about 150 hours including the preculture,
Since the enzyme activity amount is 1500 units, the unit time,
It can be seen that the amount of formate dehydrogenase produced per unit culture volume is improved 10 times. When the properties of the obtained formate dehydrogenase were examined, the residual activity after treatment at 50 ° C. for 1 hour was 100%, the optimum pH was 6.0 to 7.5, and the Km for formic acid and NAD ⁺ was 1.08 mM each,
It was 0.05 mM. From these results, it was confirmed that the formate dehydrogenase obtained by the method of the present invention had the same properties as the formate dehydrogenase derived from Hyphomicrobium sp. Strain JC-17 (FERM P-16973). Was done.

The method for measuring the activity of formate dehydrogenase and the method for indicating the activity are as follows. That is, the composition of the reaction solution used for activity measurement was 50 μmol KPB, 5 μmol
The final volume was adjusted to 1 ml with formic acid, 1 μmol NAD ⁺ and an appropriate amount of distilled water. The reaction is started by the addition of the enzyme solution,
NAD generated by enzymatic reaction at 30 ° C for 1 minute
The initial rate of H increase was measured with a spectrophotometer (340 nm). The molecular extinction coefficient of NAD ⁺ was 6220 M ⁻¹ cm ^−1, and the amount of enzyme that can consume 1 μmol of formic acid per minute was 1
Unit.

[0024]

According to the present invention, a large amount of formate dehydrogenase having a high specific activity, a small Km value for formic acid and NAD ⁺ , and a wide range of temperature stability and pH stability can be produced by a simple method. In addition, it can be manufactured at low cost.

[Brief description of the drawings]

FIG. 1 is a diagram showing a procedure for constructing a plasmid pKKFDH containing a formate dehydrogenase gene of the present invention.

[Sequence List] <110> UNITIKA LTD. <120> NUCLEIC ACIDS ENCODING FORMATE DEHYDROGENASE, VECTOR AND TRANSFORM ANT THEREOF, AND METHOD FOR PRODUSING FORMATE DEHYDROGENASE THEREWITH <130> P0017863 <160> 6 <210> 1 <211> 399 <212 > PRT <213> Hyphomicrobium sp. <400> 1 Met Ala Lys Ile Val Cys Val Leu Tyr Asp Asp Pro Val Asn Gly Tyr 1 5 10 15 Pro Lys Ser Tyr Ala Arg Asp Asp Ile Pro Lys Ile Thr Lys Tyr Pro 20 25 30 Asp Gly Gln Thr Thr Pro Thr Pro Gln Ala Ile Asp Phe Val Pro Gly 35 40 45 His Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu 50 55 60 Glu Ser Asn Gly His Thr Leu Val Val Thr Ser Asp Lys Asp Gly Ala 65 70 75 80 Asn Ser Arg Leu Asp Gln Glu Leu Pro Asp Ala Glu Ile Val Ile Ser 85 90 95 Gln Pro Phe Trp Pro Ala Tyr Met Thr Ala Glu Arg Ile Ala Lys Ala 100 105 110 Pro Lys Leu Lys Met Ile Val Thr Ala Gly Ile Gly Ser Asp His Thr 115 120 125 Asp Leu Gln Ala Ala Met Asp Arg Gly Ile Thr Val Ala Glu Val Thr Thr 130 135 140 Tyr Cys Asn Ser Asn Ser Val Ala Glu His Val Val Met Gln Met Leu 145 150 155 160 Ser Leu Val Arg Asn Tyr ile Pro Ser Tyr Asn Trp Val Ile Lys Gly 165 170 175 Gly Trp Asn Ile Ala Asp Cys Val Glu Arg Ser Tyr Asp Ile Glu Gly 180 185 190 Met His Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Arg Val Leu 195 200 205 Arg Leu Leu Lys Pro Phe Asp Val His Leu His Tyr Met Asp Arg Tyr 210 215 220 Lys Leu Pro Asp Ala Val Glu Lys Glu Leu Asn Leu Thr His His Thr 225 230 235 240 Ser Leu Glu Ser Leu Thr Lys Ala Cys Asp Val Val Thr Leu Asn Cys 245 250 255 Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Lys Thr Leu Lys 260 265 270 Asn Phe Lys Arg Gly Ala Tyr Leu Val Asn Thr Ala Arg Gly Lys Leu 275 280 285 Cys Asp Arg Asp Ala Ile Val Arg Ala Leu Glu Ser Gly Gln Leu Ala 290 295 300 Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Gln Asp His 305 310 315 320 Pro Trp Arg Lys Met Pro His His Gly Met Thr Pro His Ile Ser Gly 325 330 335 Thr Ser Leu Ser Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile 340 345 350 Leu Glu Cys Tyr Phe Asp Lys Lys Pro Ile Arg Asn Glu Tyr Leu Ile 355360 365 Val Gln Gly Gly Lys Leu Ala Gly Val Gly Ala His Ser Tyr Ser Ala 370 375 380 Gly Asn Ala Thr Ser Gly Ser Glu Glu Ala Ala Lys Phe Lys Arg 385 390 395 <210> 2 <211> 1197 <212> DNA <213> Hyphomicrobium sp. <400> 2 atg gct aag atc gta tgt gtg ctc tac gac gac ccc gtc aac gga tat 48 Met Ala Lys Ile Val Cys Val Leu Tyr Asp Asp Pro Val Asn Gly Tyr 1 5 10 15 ccg aaa tcc tac gcg cgg gac gac atc ccc aaa att acg aaa tac ccc 96 Pro Lys Ser Tyr Ala Arg Asp Asp Ile Pro Lys Ile Thr Lys Tyr Pro 20 25 30 gat gga caa acg acc ccg acg ccg cag gcg atc gat ttc gtt cca ggg 144 Asp Gly Gln Thr Thr Pro Thr Pro Gln Ala Ile Asp Phe Val Pro Gly 35 40 45 cat ctt ctc ggt agc gta tcg ggc gaa ctt ggg ctt cgc aaa tac cta 192 His Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu 50 55 60 gag agc aat ggc cat acg ctg gtt gtc acc tcc gac aag gat ggt gcc 240 Glu Ser Asn Gly His Thr Leu Val Val Thr Ser Asp Lys Asp Gly Ala 65 70 75 80 aat tcc aga ctg gac caa gaa ctg ccg gac gct gag atc gtc atc tcg 288 Asn Ser Arg Leu Asp Gln Glu Leu Pro Asp Ala Glu Ile Val Ile Ser 85 90 95 caa ccg ttc tgg cca gca tac atg acg gcg gag cga att gcg aaa gcg 336 Gln Pro Phe Trp Pro Ala Tyr Met Thr Ala Glu Arg Ile Ala Lys Ala 100 105 110 ccg aaa ctg aaa atg att gtt act gct ggc atc ggc tcc gat cac acc 384 Pro Lys Leu Lys Met Ile Val Thr Ala Gly Ile Gly Ser Asp His Thr 115 120 125 gac ctg cag gcg gcc atg gat cgc ggc att acc gtc gca gaa gtc acg 432 Asp Leu Gln Ala Ala Met Asp Arg Gly Ile Thr Val Ala Glu Val Thr 130 135 140 tac tgc aac tcc aac agc gtc gcc gag cat gtc gtc atg cag atg ctg 480 Tyr Cys Asn Ser Asn Ser Ala Glu His Val Val Met Gln Met Leu 145 150 155 160 tcg ctc gtc cgc aac tac atc ccc tcg tat aat tgg gta atc aag ggt 528 Ser Leu Val Arg Asn Tyr ile Pro Ser Tyr Asn Trp Val Ile Lys Gly 165 170 175 ggg tgg aac atc gcc gat tgc gtt gag cga tct tac gac atc gag ggt 576 Gly Trp Asn Ile Ala Asp Cys Val Glu Arg Ser Tyr Asp Ile Glu Gly 180 185 190 atg cac gtc ggt aca gtt gcg gca ggc cgc atc ggc ttg 62 4 Met His Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Arg Val Leu 195 200 205 cgt ctg ctg aaa ccc ttc gat gtg cat ctt cac tat atg gat cgc tac 672 Arg Leu Leu Lys Pro Phe Asp Val His Leu His Tyr Met Asp Arg Tyr 210 215 220 aag ctg ccc gat gca gtt gaa aag gaa ctc aac ctc acg cat cac acg 720 Lys Leu Pro Asp Ala Val Glu Lys Glu Leu Asn Leu Thr His His Thr 225 230 235 240 agc ctg gaa agc ctc accag gct tgc gac gtc gta acg ttg aac tgc 768 Ser Leu Glu Ser Leu Thr Lys Ala Cys Asp Val Val Thr Leu Asn Cys 245 250 255 ccg ctt cat ccc gaa acc gag cat atg atc aac gat aag acc ctg aaa 816 Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Lys Thr Leu Lys 260 265 270 aac ttc aag cgc ggc gca tac ctt gtg aac aca gct cgc ggc aaa ctc 864 Asn Phe Lys Arg Gly Ala Tyr Leu Val Asn Thr Ala Arg Gly Lys Leu 285 tgc gat cgg gac gcc atc gta cgc gcg ctc gaa agc ggg cag ctt gca 912 Cys Asp Arg Asp Ala Ile Val Arg Ala Leu Glu Ser Gly Gln Leu Ala 290 295 300 ggt tat gcg ggt gac gtt tgg ttgcag ca a gac cat 960 Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Gln Asp His 305 310 315 320 cct tgg cgg aag atg ccg cat cac ggc atg aca ccg cac att tcc ggc 1008 Pro Trp Arg Lys Met Pro His His Gly Met Thr Pro His Ile Ser Gly 325 330 335 acg tcg ctt tcg gca caa gca agg tac gca gcc gga acg cgg gaa att 1056 Thr Ser Leu Ser Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile 340 345 350 ctt gaa tgt tat ttc gac aag aag ccc atc cgc aac gag tac ttg att 1104 Leu Glu Cys Tyr Phe Asp Lys Lys Pro Ile Arg Asn Glu Tyr Leu Ile 355 360 365 gtt caa ggc ggc aag ctc gcc ggt gtc ggc gc g cg gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gg Gln Gly Gly Lys Leu Ala Gly Val Gly Ala His Ser Tyr Ser Ala 370 375 380 ggc aac gca act tcc ggc tcg gaa gaa gcc gcg aag ttt aaa cgc 1197 Gly Asn Ala Thr Ser Gly Ser Glu Glu Ala Ala Lys Phe Lys Arg 390 395 <210> 3 <211> 20 <212> PRT <213> Hyphomicrobium sp. <400> 3 Ala Lys Ile Val Cys Val Leu Tyr Asp Asp Pro Val Asn Gly Tyr Pro 1 5 10 15 Lys Ser Tyr Ala 20 < 210> 4 <211> 10 <212> PRT <213> Hyp homicrobium sp. <400> 4 Gly Asp Val Trp Phe Pro Gln Pro Ala Pro 1 5 10 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <400> 5 gaattcgatg gctaagatcg tatgtg 26 <210> 6 < 211> 26 <212> DNA <213> Artificial Sequence <400> 6 aagcttctaa acggtgctgc ggagaa 26

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/02 C12N 5/00 A F term (Reference) 4B024 AA03 BA08 CA03 DA05 DA06 EA04 4B050 CC03 DD02 FF09E FF11E FF14E LL05 4B065 AA01X AA01Y AA26X AB01 AC14 CA28 CA60

Claims (5)

[Claims] 1. A gene encoding a formate dehydrogenase comprising an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. . 2. A D sequence comprising the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 2.
A gene having NA and encoding formate dehydrogenase. 3. A recombinant vector containing the gene according to claim 1. A transformant comprising the recombinant vector according to claim 3. 5. A method for producing formate dehydrogenase, comprising culturing the transformant according to claim 4 in a medium, and collecting formate dehydrogenase from the culture.