JP2000069971A - New thermostable formate dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new thermostable formate dehydrogenase, from a hyperthermophilic archaeon 'strain KOD1', which has a specific amino acid sequence, catalyzes the oxidation from formate to carbon dioxide, and can be used, for example, for the reproduction of NAD. SOLUTION: This is a new thermostable formate dehydrogenase which has the specific amino acid sequence shown by the formula or an amino acid sequence which is obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into the amino acid sequence shown by the formula, or a new modifier which functions as formate dehydrogenase, which catalyzes the oxidation from formate to carbon dioxide, and can be used, for example, for the reproduction of nicotinamide adenine dinucleotide (NAD) which is a coenzyme involved in redox enzymatic reactions to its reduced form (NADH). This enzyme is obtained by isolating chromosomal DNA from a hyperthermophilic archaeon 'strain KOD1', preparing a chromosomal DNA library using the obtained DNA, screening the obtained library, incorporating the obtained DNA into a vector, followed by introducing the obtained vector into a host cell for expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel thermostable formate dehydrogenase and a DNA encoding the same.

[0002]

2. Description of the Related Art Formate dehydrogenase is an enzyme that catalyzes the reaction of oxidizing formate ions to CO ₂ . The reaction is of the formula HCOO
^- + NAD ⁺ ⇔CO ₂ + NADH. Nicotinamide adenine dinucleotide (NAD; reduced form is NADH) is one of the coenzymes involved in the oxidoreductase reaction. Formate dehydrogenase can be used for the regeneration of NAD → NADH. Therefore, formate dehydrogenase is useful for supplying coenzymes for enzymatic reactions that require this coenzyme. For example, an enzyme reaction requiring this coenzyme and NAD by formate dehydrogenase
→ Combining the regeneration of NADH enables a continuous enzymatic reaction.

[0003] For such uses (especially when the reaction is carried out at high temperatures), a novel formate dehydrogenase which is more structurally stable than conventional room temperature organism-derived formate dehydrogenases (Especially for long-term continuous enzymatic reactions)
Was desired.

[0004] Because hyperthermophilic archaeons survive at high temperatures, the proteins (eg, enzymes) produced by the microorganism are generally highly thermostable (ie, structurally stable). In addition, the evolution of these organisms is evident from the fact that it has been proposed that the archaeon to which hyperthermophilic archaea belong is different from the conventionally known prokaryotes and eukaryotes. And different. Therefore, even though they have similar functions to known enzymes derived from prokaryotes and eukaryotes, enzymes derived from hyperthermophilic archaea are structurally and enzymatically different from conventional enzymes. Is often different. For example, the hyperthermophilic archaeon KOD1 strain (Morikawa, M. et al., Appl. En.
viron. Microbiol. 60 (12), 4559-4566 (1994)) was isolated from Escherichia coli GroE.
It has the same function as L. However, GroEL itself forms a 14-mer, and GroES forms a further 7-mer.
, Whereas the chaperonin from the KOD1 strain functions alone (Yan, Z et al., Appl.
Environ. Microbiol. 63 : 785-789).

[0005]

SUMMARY OF THE INVENTION The present invention provides a thermostable formate dehydrogenase and a DNA encoding the same, a vector containing the DNA, a host cell containing the vector, and a host cell. It is an object of the present invention to provide a method for producing a thermostable formate dehydrogenase including a step of culturing cells.

[0006]

According to the present invention, there is provided a thermostable formate dehydrogenase comprising the amino acid sequence of SEQ ID NO: 2, or one or several amino acids in the amino acid sequence of SEQ ID NO: 2 are deleted, substituted or substituted. Variants thereof comprising an added amino acid and having formate dehydrogenase activity are provided.

According to the present invention, there is provided a thermostable formate dehydrogenase comprising the amino acid sequence of SEQ ID NO: 4, or deletion of one or several amino acids in the amino acid sequence of SEQ ID NO: 4,
Variants thereof that include substituted or added amino acids and have formate dehydrogenase activity are provided.

According to the present invention, a thermostable formate dehydrogenase comprising the amino acid sequences of SEQ ID NOs: 2 and 4, or one or several amino acids are deleted, substituted or added in the amino acid sequences of SEQ ID NOs: 2 and 4 A modified form thereof comprising the amino acid and having formate dehydrogenase activity is provided.

[0009] In one embodiment, the thermostable formate dehydrogenase or its variant is derived from the hyperthermophilic archaeon KOD1 strain.

According to the present invention, there is provided a DNA encoding the thermostable formate dehydrogenase or a variant thereof.

In one embodiment, the DNA comprises the nucleotide sequence from position 1 to position 2130 of SEQ ID NO: 1.

In one embodiment, the DNA comprises the nucleotide sequence of positions 1 to 498 of SEQ ID NO: 3.

In one embodiment, the DNA is derived from the hyperthermophilic archaeon strain KOD1.

According to the present invention, there is provided a vector containing the DNA.

According to the present invention, there is provided a recombinant host cell containing the above vector.

According to the present invention, there is provided a method for producing a thermostable formate dehydrogenase or a variant thereof, comprising a step of culturing the host cell.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a thermostable formate dehydrogenase produced by a hyperthermophilic archaeon, by culturing a hyperthermophilic archaeon that originally produces the enzyme, or by encoding the enzyme. The present invention relates to a method for isolating DNA, constructing a vector containing the DNA, preparing a host cell containing the vector, and culturing the host cell to produce a thermostable formate dehydrogenase.

Formate dehydrogenase is an enzyme that catalyzes the reaction of oxidizing formate ions to CO ₂ . The reaction formula HCOO ^-
+ NAD ⁺ ⇔CO ₂ + NADH. Here, NAD (nicotinamide adenine dinucleotide; reduced form is NADH) is one of the coenzymes involved in the oxidoreductase reaction.

The formate dehydrogenase activity is measured, for example, by using NADP ⁺ (340 nm, ε = 16.22 × 10 ³ ), methyl viologen (600 nm, ε = 1.13 × 10 ⁴ ) or benzyl viologen (605 nm) as an electron acceptor. , Ε = 1.47 × 10 ⁴ ) (Andreesen, JR et al. (1974) J. Bacteriol., 120: 6-
14).

Known formate dehydrogenases include homodimers consisting only of the α subunit, heterodimers consisting of the α and β subunits and tetramers, and consisting of the α, β and γ subunits. There are 12-mer and the like.

[0021] The formate dehydrogenase of the present invention may be composed of one or more subunits. Preferably, the formate dehydrogenase of the invention consists of two subunits.

The thermostable formate dehydrogenase of the present invention has an optimum temperature of 70 ° C. or higher, preferably 80 ° C. or higher, more preferably 90 ° C.

The hyperthermophilic archaeon used in the present invention is defined as a microorganism that grows at 90 ° C. or higher. Preferably, the hyperthermophilic archaeon is a thermostable thiol protease-producing strain KOD1 (Morikawa, M. et al., Appl. Environ. Mic.) Which produces thermostable formate dehydrogenase and has been isolated by the present inventors.
robiol. 60 (12), 4559-4566 (1994)). The KOD1 strain has been deposited with the Institute of Biotechnology and Industrial Technology,
Its accession number is FERM P-15007. This KOD1
The strain was originally classified as belonging to the genus Pyrococcus, as described in the above-mentioned literature. However, when the sequence of 16S rRNA was compared using the registered data of GenBank R91.0 October, 1995 + DailyUpdate input to DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.), the KOD1 strain was found to be of the Thermococcus genus rather than the Pyrococcus genus. Has been suggested to be closely related.

The culture of the hyperthermophilic archaeon producing the thermostable formate dehydrogenase of the present invention can be performed, for example, by the method described in Appl. Environ.
icrobiol. 60 (12), 4559-4566 (1994) (supra). The culture can be either static culture or aeration and agitation culture with nitrogen gas, and can be either continuous or batch.

The conditions for culturing the recombinant host cell are appropriately selected depending on the type of the host cell used. As the host cell, any host cell that can be used in recombinant DNA technology can be used. These include, for example, bacterial cells, yeast cells, animal cells, plant cells, insect cells, and the like. Preferred host cells are bacterial cells.

After the culture, the thermostable formate dehydrogenase of the present invention can be purified from the resulting culture by a method known in the art. If the expression product is secreted extracellularly, a supernatant is obtained, for example, by centrifuging or filtering the culture,
This is purified directly or concentrated by precipitation or ultrafiltration and then purified. If the expression product accumulates in the cells, the cells are disrupted using cell wall lytic enzymes, changes in osmotic pressure, glass beads, homogenizer or sonication to obtain a cell extract, which is purified.
Purification can be performed by a combination of methods known in the art, such as ion exchange chromatography, gel filtration, affinity chromatography, and electrophoresis.

DNA encoding thermostable formate dehydrogenase
Can be obtained, for example, by isolating chromosomal DNA from a hyperthermophilic archaeon, preparing a library containing the chromosomal DNA, and screening the library.

The chromosomal DNA of the hyperthermophilic archaeon is obtained by lysing cultured bacterial cells using a surfactant (for example, N-lauryl sarcosine) and then subjecting the resulting lysate to cesium ethidium bromide equilibrium density. It can be obtained by fractionation by gradient ultracentrifugation or the like (for example, Imanaka et al.,
J. Bacteriol. 147 : 776-786 (1981)). The library is obtained by cleaving the obtained chromosomal DNA with various restriction enzymes, and then cutting the same restriction enzyme or a restriction enzyme giving a common cleavage end to a vector (such as a phage or plasmid), and then adding T4 DNA ligase or the like. And can be obtained by linking.

Screening of the library can be performed by selecting a clone containing a DNA encoding the desired thermostable formate dehydrogenase from this library.
The selection is performed using, for example, an oligonucleotide designed based on a predetermined partial amino acid sequence of thermostable formate dehydrogenase, a cloned DNA presumed to have homology with the target DNA, or the like as a probe. Can be done.
Alternatively, selection can be performed by expressing the enzyme of interest. For example, expression can be detected by detecting the activity of an expression product against a substrate added to a plate when the activity of the enzyme of interest can be easily detected, or when an antibody against the enzyme of interest is available. Is
It can be carried out by utilizing the reactivity between the expression product and the antibody.

The obtained cloned DNA can be analyzed by, for example, isolating the selected DNA, preparing a restriction map thereof, and determining the nucleotide sequence. Preparation of cloned DNA, restriction enzyme treatment,
Techniques such as subcloning and nucleotide sequence determination are well known in the art, and include, for example, “Molecular
Cloning: A Laboratory Manual Second Edition "(Sambrook, Fr
edited by itsch and Maniatis, Cold Spring Harbor Laborato
ry Press, 1989).

Next, the obtained cloned DNA is operably inserted into an expression vector compatible with the host cell to be used, and the host cell is transformed with the expression vector. By culturing, a thermostable formate dehydrogenase can be expressed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the invention is not limited by these examples.

[0033]

EXAMPLES (Example 1: DN encoding formate dehydrogenase)
A Isolation) KOD1 strain was isolated from Appl. Environ. Microbiol. 60 (1
2), 0.5 × 2216 marine broth medium described in 4559-4566 (1994) (2216 marine broth: 18.7 g / L, PIPES 3.48 g / L,
_{CaCl 2 · H 2 O 0.725g /} L, 0.4 mL 0.2% resazurin, 475 mL artificial seawater (NaCl 28.16 g / L, KCl 0.7 g / L, MgCl 2 · 6H 2 O 5.
5 g / L, MgSO ₄・ 7H ₂ O 6.9 g / L), distilled water 500 mL, pH 7.0)
1,000 ml was inoculated and cultured using a 2 liter fermenter. When culturing, the inside of the fermenter is replaced with nitrogen gas,
The internal pressure was maintained at 0.1 kg / cm ² with the same gas. Culture at a temperature of 85
The cells were cultured at ± 1 ° C. for 14 hours. The cultivation was performed by stationary cultivation, and aeration and agitation of nitrogen gas were not performed during the culturing. After the culture is completed, the culture solution (about 1,000 ml) is
The cells were collected by centrifugation for minutes.

1 g of the obtained cells was added to 10 ml of an A solution (50 mM Tr
is-HCl, 50 mM EDTA, pH 8.0) and centrifuged (8,00
After collecting cells by 0 rpm, 5 minutes, 4 ° C.), the cells were suspended in 3 ml of A solution containing 15% sucrose, incubated at 37 ° C. for 30 minutes, and then 1% N
3 ml of solution A containing lauryl sarcosine were added. To this solution, 5.4 g of cesium chloride and 300 μl of a 10 mg / ml ethidium bromide solution were added, and the mixture was added at 55,000 rpm for 16 hours.
Ultracentrifugation was performed at 8 ° C. to fractionate chromosomal DNA. After removing ethidium bromide from the obtained chromosomal DNA fraction by n-butanol extraction, a TE solution (10 mM Tris-HCl (pH 8.0),
(0.1 mM EDTA) overnight to obtain chromosomal DNA.

A primer designed by selecting an amino acid sequence of a region highly conserved between amino acid sequences of formate dehydrogenase derived from various organisms, and a chromosome DN prepared as described above.
PCR reactions were performed using A.

Using the resulting PCR product as a probe, the following KOD1 chromosomal DNA library was screened.

The chromosomal DNA prepared above was digested with the restriction enzyme EcoRI.
And ligated with phage vector λEMBL4 (Stratagene) using T4 DNA ligase. This concatenated mixture is used as a Max Plax ^™ Packaging Extract (EPICENTRE T
ECHNOLOGIES).

This phage library was prepared using Escherichia
E. coli XL1-Blue MRA (P2) strain, and the resulting plaque was screened by a conventional method using the above PCR fragment as a probe (see “Molecular Cloning: A
Laboratory Manual Second Edition "Sambrook, Fritsch and M
aniatis, Cold Spring Harbor Laboratory Press, 198
9).

The fragments in the positive clone were
By analyzing by digestion with various restriction enzymes and Southern blotting using the above probe, the position of the gene encoding formate dehydrogenase was restricted.

(Example 2: Sequence of formate dehydrogenase) The nucleotide sequence of the fragment obtained in Example 1 was determined, and the presence of two consecutive open reading frames via a 29 base pair non-coding sequence was determined. confirmed. The upstream sequence is SEQ ID NO: 1 and the downstream sequence is SEQ ID NO: 3.
Shown in The deduced amino acid sequences are shown in SEQ ID NOs: 2 and 4, respectively. These two genes are called fdhA and fd
Named hB.

The products encoded by these two genes are thought to constitute the two subunits of formate dehydrogenase.

FIG. 1 shows the results of analysis indicating that the fdhA gene product shows homology to amino acids of various formate dehydrogenases registered in the database.

(Example 3: Expression of thermostable formate dehydrogenase) The formate dehydrogenase encoded by the two open reading frames obtained above was purified from Escherichia co
The following operation was performed to express in li. fdhA
Fragments containing both fdhB and fdhB
Forward including I site

Reverse containing primer 1 and Bam HI sites
Amplification was performed using primer 1, and plasmid pET21a (+) (Novag
en) to obtain plasmid pET21a / fdhAB (FIG. 2). Using this plasmid, Escherichia coli BL21
(DE3) strain was transformed.

The resulting ampicillin-resistant transformant was transformed into an NZCYM medium (1% NZ) containing ampicillin (50 μg / ml).
Amine, 0.5% NaCl, 0.5% yeast extract, 0.1% casamino acids, was inoculated into _{0.2% MgSO 4 · 7H 2 O} (pH7)), O at 37 ° C.
Culture until _D660 reaches 0.4, then isopropyl-β-D
-Thiogalactopyranoside (IPTG, 0.1 mM)
Culture was continued at 37 ° C. for another 4 hours. After completion of the culture, the cells were collected by centrifugation, disrupted by sonication, and centrifuged again to obtain a cell extract. The cell extract was heated at 80 ° C. for 15 minutes and then centrifuged to obtain a supernatant, which was used as a crude enzyme solution.

The formate dehydrogenase activity of this crude enzyme solution was determined by a conventional method (Andreesen, JR et al. (1974) J. Bacteriol., 120: 6-1).
When measured according to 4), formate dehydrogenase activity was confirmed. Also, this enzyme had an optimal temperature of 90 ° C. (data not shown).

[0047]

According to the present invention, a thermostable formate dehydrogenase and a DNA encoding the same are provided. The present invention also provides a vector containing the DNA and a host cell containing the vector. Further, the present invention provides a method for producing a thermostable formate dehydrogenase, which comprises a step of culturing the host cell.

[0048]

[Sequence List] SEQUENCE LISTING <110> Imanaka, Tadayuki <120> Novel heat resistant formate dehydrogenase <130> J198432419 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 2138 <212> DNA <213> KOD1 <220> <221> CDS <222> (1) .. (2130) <400> 1 ATG GAT GAA AAG CTC GTC CCT GTG GTC TGC CCC TAC TGC GGT GTG GGG 48 Met Asp Glu Lys Leu Val Pro Val Val Cys Pro Tyr Cys Gly Val Gly 1 5 10 15 TGC AGG CTG TAC ATT AGG AGT TCT GAC GGC CAT CCC CTT GGC ATC GAG 96 Cys Arg Leu Tyr Ile Arg Ser Ser Asp Gly His Pro Leu Gly Ile Glu 20 25 30 TAC GCC AAT GAC ATA CCC GGA ATC TCA AAC GAA AAT GGA AAG CTC TGT 144 Tyr Ala Asn Asp Ile Pro Gly Ile Ser Asn Glu Asn Gly Lys Leu Cys 35 40 45 CCC AAG GGC AAC GCC GTT CTT GAG TAC GTC CTC GCC CGG GAC AGG CTT 192 Pro Lys Gly Asn Ala Val Leu Glu Tyr Val Leu Ala Arg Asp Arg Leu 50 55 60 AAG AAG CCA TTA AAG GCG AAG GAG CAA GGC AAG TTC GTC GAG ATA AGC 240 Lys Lys Pro Leu Lys Ala Lys Glu Gln Gly Lys Phe Val Glu Ile Ser 65 70 75 80 TGG AGT CAG GCC ATA AAG GAG GTC GCC GAG AGG CTT AAG GAG TAT TCA 288 Trp Ser Gln Ala Ile Lys Glu Val Ala Glu Arg Leu Lys Glu Tyr Ser 85 90 95 AAG GAC GAT CCG AAT TCA ATG ATG TTC TTC GGC AGT GCA AAA ACT TTC 336 Lys Asp Asp Pro Asn Ser Met Met Phe Phe Gly Ser Ala Lys Thr Phe 100 105 110 AAC GAG CCA AAC TAC CTT ATA CAG AAG CTG GCC AGA ATG CTC GGA ACC 384 Asn Glu Pro Asn Tyr Leu Ile Gln Lys Leu Ala Arg Met Leu Gly Thr 115 120 125 AAC AAC GTT GAT CAC TGC GCG AGA CTC TGC CAT GCA TCA ACA GTT TCC 432 Asn Asn Val Asp His Cys Ala Arg Leu Cys His Ala Ser Thr Val Ser 130 135 140 GGC CTC AAA GCC GTC TTC GGC GCT GGG GCG ATG ACG AAC ACA TAC AGG 480 Gly Leu Lys Ala Val Phe Gly Ala Gly Ala Met Thr Asn Thr Tyr Arg 145 150 155 160 GAC ATT GAA GAG GCG AAC GTT ATC TTC ATC ATC GGC CAC AAC TTC GCC 528 Asp Ile Glu Glu Ala Asn Val Ile Phe Ile Ile Gly His Asn Phe Ala 165 170 175 GAG ACT CAC CCG GTT GGC TTC CGC TAC GTT CTC AAG GCC AAG GAG AGG 576 Glu Thr His Pro Val Gly Phe Arg Tyr Val Leu Lys Ala Lys Glu Arg 180 185 190 GGA GCT AAG GTC ATA GTG GCC GAC CCG CGC TTT ACA AGG ACA GCC TGG 624 Gly Ala Lys Val Ile Val Ala Asp Pro Arg Phe Thr Arg Thr Ala Trp 195 200 205 TTC GCC GAC ATT TAC CTG CAG CAC TAC CCC GGT ACC GAC ATC GCC CTG 672 Phe Ala Asp Ile Tyr Leu Gln His Tyr Pro Gly Thr Asp Ile Ala Leu 210 215 220 ATA AAC GGC CTC ATT CAT GTC ATC ATA GAG GAG AGG CTC TAC GAC GAG 720 Ile Asn Gly Leu Ile His Val Ile Ile Glu Glu Arg Leu Tyr Asp Glu 225 230 235 240 AAG TTC GTC AGG GAG AGG TGC ACG GGC TTC GAT GAA CTC GCC AAG ACC 768 Lys Phe Val Arg Glu Arg Cys Thr Gly Phe Asp Glu Leu Ala Lys Thr 245 250 255 GTT GAG AAG TTC ACT CCC GGG TAC GTC GAG AAG ATA ACC GGC GTT CCA 816 Val Glu Lys Phe Thr Pro Gly Tyr Val Glu Lys Ile Thr Gly Val Pro 260 265 270 GCA GAT CTG ATA GTC CAG GCC GCG AGG ACG TTT GCA ACG GGG GGG AAG 864 Ala Asp Leu Ile Val Gln Ala Ala Arg Thr Phe Ala Thr Gly Gly Lys 275 280 285 GGC GTA ATA ACG TGG GCG ATG GGA ATA ACC CAG CAT ACC CAC GGA CAC 912 Gly Val Ile Thr Trp Ala Met Gly Ile Thr Gln His Thr His Gly His 290 295 300 GAC AAC GTG AGA CTG CTC GCA ACG CTT TCA GCC ATA TGT GGC TAC CAG 960 Asp Asn Val Arg Leu Leu Ala Thr Leu Ser Ala Ile Cys Gly Tyr Gln 305 310 315 320 GGA AGG GAA GGC TGT GGA GTA TCC CCA ATG CGC GGC CAG AAC AAC GTC 1008 Gly Arg Glu Gly Cys Gly Val Ser Pro Met Arg Gly Gln Asn Asn Val 325 330 335 CAG GGA GCC TGC GAC CTT GGA GTA CTT CCA AAC GTC TTC CCC GGC TAC 1056 Gln Gly Ala Cys Asp Leu Gly Val Leu Pro Asn Val Phe Pro Gly Tyr 340 345 345 350 AAA GCG GTT TCC GAT CCT GAA GGG AGG AAG TTC TTC AAG GAG TTC TGG 1104 Lys Ala Val Ser Asp Pro Glu Gly Arg Lys Phe Phe Lys Glu Phe Trp 355 360 365 GGA ACC GAG CTG AGC GGA GAG GTG GGA CTT ACA GTC ATC GAG GCT GCC 1152 Gly Thr Glu Leu Ser Gly Glu Val Gly Leu Thr Val Ile Glu Ala Ala 370 375 380 CAC GCC ATA GAG AAG GGG AAG GTC AGG GCC TAC TAC GTC ATG GGT GAG 1200 His Ala Ile Glu Lys Gly Lys Val Arg Ala Tyr Tyr Val Met Gly Glu 385 390 395 400 AAC CCT GTC ATA AGC GAT GCT AAC ACC AAC CAC GTC ATC AAG GCC CTC 1248 Asn Pro Val Ile Ser Asp Ala Asn Thr Asn His Val Ile Lys Ala Leu 405 410 415 CAT AAG CTT G AG TTC ATG GTC GTT CAG GAC ATA GTT CCC ACA CCG ACC 1296 His Lys Leu Glu Phe Met Val Val Gln Asp Ile Val Pro Thr Pro Thr 420 425 430 ATG GAG TTC GCG GAC ATA GTT CTC CCG GCT GCG GCT ATG CTT GAG AAC 1344 Met Glu Phe Ala Asp Ile Val Leu Pro Ala Ala Ala Met Leu Glu Asn 435 440 445 GAG GGC TCC CTC ACG AAC ACC GAG AGA AGG GTT CAG TGG AGT TTC CAG 1392 Glu Gly Ser Leu Thr Asn Thr Glu Arg Arg Val Gln Trp Ser Phe Gln 450 455 460 GCG ATC AAC CCG CCG GGG GAG GCG AGG CCG GAC TGG TGG ATA GTC AGT 1440 Ala Ile Asn Pro Pro Gly Glu Ala Arg Pro Asp Trp Trp Ile Val Ser 465 470 470 475 480 480 GAG ATA GGC AAG GCC GCT GGC TTC ACA GGA GAC GGG CCG AAG GGC TTC 1488 Glu Ile Gly Lys Ala Ala Gly Phe Thr Gly Asp Gly Pro Lys Gly Phe 485 490 495 AAC TAT TCC GGC CCA GAG GAC ATT CTG CGT GAG GTC AAC GCC TGC ACC 1536 Asn Tyr Ser Gly Pro Glu Asp Ile Leu Arg Glu Val Asn Ala Cys Thr 500 505 510 CCA CAG TAT CGG GGC ATA ACT CCA GAG AGG CTC AAG GCA AAC TTA GCT 1584 Pro Gln Tyr Arg Gly Ile Thr Pro Glu Arg Leu Lys Ala Asn Leu Ala 515 52 0 525 GGA ATA CAC TGG CCG TGC CCG AGT GAA GAC CAT CCC GGC ACG AGG GTT 1632 Gly Ile His Trp Pro Cys Pro Ser Glu Asp His Pro Gly Thr Arg Val 530 535 540 CTC TAC AAG GAG AGG TTC CTG ACC AGC GAT GGC AAG GCG CAC CTC GCG 1680 Leu Tyr Lys Glu Arg Phe Leu Thr Ser Asp Gly Lys Ala His Leu Ala 545 550 555 560 GCC GTT GAG TAC CGC GGG CCG GCT GAA ACA CCG GAC GAG GAT TAC CCG 1728 Ala Val Glu Tyr Arg Gly Pro Ala Glu Thr Pro Asp Glu Asp Tyr Pro 565 570 575 TTC CTC CTG ACG ACA GTG AGG TAC GTT GGC CAC TAC CAC ACC CTC ACG 1776 Phe Leu Leu Thr Thr Val Arg Tyr Val Gly His Tyr His Thr Leu Thr 580 585 590 ATG ACC GGA AGG AGC AGG GCC CTC GTC AAG AGG TGG CCC GAG CCC CTC 1824 Met Thr Gly Arg Ser Arg Ala Leu Val Lys Arg Trp Pro Glu Pro Leu 595 600 605 GCT GAG ATA CAC CCT GAG GAC GCC GAG AGA CTT GGA ATA AAG ACC GGG 1872 Ala Glu Ile His Pro Glu Asp Ala Glu Arg Leu Gly Ile Lys Thr Gly 610 615 620 GAC TGG GTC AAA ATC GTC ACA AGA AGA GGA GAG TAT CCG ATC AGG GCA 1920 Asp Trp Val Lys Ile Val Thr Arg Arg Gly Glu Tyr Pro Ile Arg Ala 625 630 635 640 AAG GTC ACG AGG ACA GTC AAG AAG GGG GTT ATA GCG GTT CCC TGG CAC 1968 Lys Val Thr Arg Thr Val Lys Lys Gly Val Ile Ala Val Pro Trp His 645 650 655 655 TGG GGA GCG AAC GTC GTT ACC AAC GAT GCC CTC GAC CCA GTG GCC AAG 2016 Trp Gly Ala Asn Val Val Thr Asn Asp Ala Leu Asp Pro Val Ala Lys 660 665 670 ATT CCG GAG ACC AAG GCC TGC GCC TGC AAA GTC GTC AAG ATT ACG GAG 2064 Ile Pro Glu Thr Lys Ala Cys Ala Cys Lys Val Val Lys Ile Thr Glu 675 680 685 GAA GAG GCC AGG GAG CTC ATG AAG AAG GTA CCT CCC GTT ATA CCA GAG 2112 Glu Glu Ala Arg Glu Leu Met Lys Lys Val Pro Pro Val Ile Pro Glu 690 695 700 ATT GAA ATT GTT AGG GGG TGACCTGA 2138 Ile Glu Ile Val Arg Gly 705 710 <210> 2 <211> 710 <212> PRT <213> KOD1 <400> 2 Met Asp Glu Lys Leu Val Pro Val Val Cys Pro Tyr Cys Gly Val Gly 1 5 10 15 Cys Arg Leu Tyr Ile Arg Ser Ser Asp Gly His Pro Leu Gly Ile Glu 20 25 30 Tyr Ala Asn Asp Ile Pro Gly Ile Ser Asn Glu Asn Gly Lys Leu Cys 35 40 45 Pro Lys Gly Asn Ala Val Leu Glu Tyr Val Leu Ala Arg As p Arg Leu 50 55 60 Lys Lys Pro Leu Lys Ala Lys Glu Gln Gly Lys Phe Val Glu Ile Ser 65 70 75 80 Trp Ser Gln Ala Ile Lys Glu Val Ala Glu Arg Leu Lys Glu Tyr Ser 85 90 95 Lys Asp Asp Pro Asn Ser Met Met Phe Phe Gly Ser Ala Lys Thr Phe 100 105 110 Asn Glu Pro Asn Tyr Leu Ile Gln Lys Leu Ala Arg Met Leu Gly Thr 115 120 125 Asn Asn Val Asp His Cys Ala Arg Leu Cys His Ala Ser Thr Val Ser 130 135 140 Gly Leu Lys Ala Val Phe Gly Ala Gly Ala Met Thr Asn Thr Tyr Arg 145 150 155 160 Asp Ile Glu Glu Ala Asn Val Ile Phe Ile Ile Gly His Asn Phe Ala 165 170 175 Glu Thr His Pro Val Gly Phe Arg Tyr Val Leu Lys Ala Lys Glu Arg 180 185 190 Gly Ala Lys Val Ile Val AlA Asp Pro Arg Phe Thr Arg Thr Ala Trp 195 200 205 Phe Ala Asp Ile Tyr Leu Gln His Tyr Pro Gly Thr Asp Ile Ala Leu 210 215 220 Ile Asn Gly Leu Ile His Val Ile Ile Glu Glu Arg Leu Tyr Asp Glu 225 230 235 240 Lys Phe Val Arg Glu Arg Cys Thr Gly Phe Asp Glu Leu Ala Lys Thr 245 250 255 Val Glu Lys Phe Thr Pro Gly Tyr Val Glu Lys Ile Thr Gly Val Pro 260 265 270 Ala Asp Leu Ile Val Gln Ala Ala Arg Thr Phe Ala Thr Gly Gly Lys 275 280 285 Gly Val Ile Thr Trp Ala Met Gly Ile Thr Gln His Thr His Gly His 290 295 300 Asp Asn Val Arg Leu Leu Ala Thr Leu Ser Ala Ile Cys Gly Tyr Gln 305 310 315 320 Gly Arg Glu Gly Cys Gly Val Ser Pro Met Arg Gly Gln Asn Asn Val 325 330 335 Gln Gly Ala Cys Asp Leu Gly Val Leu Pro Asn Val Phe Pro Gly Tyr 340 345 350 Lys Ala Val Ser Asp Pro Glu Gly Arg Lys Phe Phe Lys Glu Phe Trp 355 360 365 Gly Thr Glu Leu Ser Gly Glu Val Gly Leu Thr Val Ile Glu Ala Ala 370 375 380 His Ala Ile Glu Lys Gly Lys Val Arg Ala Tyr Tyr Val Met Gly Glu 385 390 395 400 400 Asn Pro Val Ile Ser Asp Ala Asn Thr Asn His Val Ile Lys Ala Leu 405 410 415 His Lys Leu Glu Phe Met Val Val Gln Asp Ile Val Pro Thr Pro Thr 420 425 430 Met Glu Phe Ala Asp Ile Val Leu Pro Ala Ala Ala Met Leu Glu Asn 435 440 445 Glu Gly Ser Leu Thr Asn Thr Glu Arg Arg Val Gln Trp Ser Phe Gln 450 455 460 Ala Ile Asn Pro Pro Gly Glu Ala Arg Pro Asp Trp Trp Ile Val Ser 465470 475 480 Glu Ile Gly Lys Ala Ala Gly Phe Thr Gly Asp Gly Pro Lys Gly Phe 485 490 495 Asn Tyr Ser Gly Pro Glu Asp Ile Leu Arg Glu Val Asn Ala Cys Thr 500 505 510 Pro Gln Tyr Arg Gly Ile Thr Pro Glu Arg Leu Lys Ala Asn Leu Ala 515 520 525 Gly Ile His Trp Pro Cys Pro Ser Glu Asp His Pro Gly Thr Arg Val 530 535 540 Leu Tyr Lys Glu Arg Phe Leu Thr Ser Asp Gly Lys Ala His Leu Ala 545 550 555 560 Ala Val Glu Tyr Arg Gly Pro Ala Glu Thr Pro Asp Glu Asp Tyr Pro 565 570 575 Phe Leu Leu Thr Thr Val Arg Tyr Val Gly His Tyr His Thr Leu Thr 580 585 590 Met Thr Gly Arg Ser Arg Ala Leu Val Lys Arg Trp Pro Glu Pro Leu 595 600 605 Ala Glu Ile His Pro Glu Asp Ala Glu Arg Leu Gly Ile Lys Thr Gly 610 615 620 Asp Trp Val Lys Ile Val Thr Arg Arg Gly Glu Tyr Pro Ile Arg Ala 625 630 635 640 Lys Val Thr Arg Thr Val Lys Lys Gly Val Ile Ala Val Pro Trp His 645 650 655 Trp Gly Ala Asn Val Val Thr Asn Asp Ala Leu Asp Pro Val Ala Lys 660 665 670 Ile Pro Glu Thr Lys Ala Cys Ala Cys Lys Val Val Lys Ile Thr Glu 675680 685 Glu Glu Ala Arg Glu Leu Met Lys Lys Val Pro Pro Val Ile Pro Glu 690 695 700 Ile Glu Ile Val Arg Gly 705 710 <210> 3 <211> 501 <212> DNA <213> KOD1 <220> <221 > CDS <222> (1) .. (498) <400> 3 ATG GCC AGA AAG ACC GTT TTT ATA GAC TTT TCA AAG TGC ATC GAG TGC 48 Met Ala Arg Lys Thr Val Phe Ile Asp Phe Ser Lys Cys Ile Glu Cys 1 5 10 15 CGC GCC TGT GAG GTA GCC TGC GAG CGC GAA CAC AAC GGA CGA TCA TTC 96 Arg Ala Cys Glu Val Ala Cys Glu Arg Glu His Asn Gly Arg Ser Phe 20 25 30 ATA AAC GTC TTT GAA TGG CAG GAA ATG GCC GCG ATG GCC CTC AAC TGC 144 Ile Asn Val Phe Glu Trp Gln Glu Met Ala Ala Met Ala Leu Asn Cys 35 40 45 CGC CAC TGT GAG AAG GCT CCC TGC CTC GAG GTC TGC CCG ACA AAC GCA 192 Arg His Cys Glu Lys Ala Pro Cys Leu Glu Val Cys Pro Thr Asn Ala 50 55 60 CTC TAT CGC GAT GGG GAT GGA GCA GTT CTG CTC GCC CCC CAG AAG TGC 240 Leu Tyr Arg Asp Gly Asp Gly Ala Val Leu Leu Ala Pro Gln Lys Cys 65 70 75 80 ATT GGC TGT CTG ATG TGT GGA ATC GTG TGC CCC TTT GGA ATA CCA GAG 288 Ile Gly Cys Leu Met Cys Gly I le Val Cys Pro Phe Gly Ile Pro Glu 85 90 95 CTT GAC TCC CTT GAC AAG ATA ATG ATG AAG TGC GAC CTC TGC GCC CAC 336 Leu Asp Ser Leu Asp Lys Ile Met Met Lys Cys Asp Leu Cys Ala His 100 105 110 AGG AGA GCC GAA GGA AAG CTC CCG GCG TGT GTG GAA ACC TGC CCG ACC 384 Arg Arg Ala Glu Gly Lys Leu Pro Ala Cys Val Glu Thr Cys Pro Thr 115 120 125 GAT GCC CTT CTC TTC GGC GAC TTC AAC GAC ATC CAA AGG ATG AGG AGA 432 Asp Ala Leu Leu Phe Gly Asp Phe Asn Asp Ile Gln Arg Met Arg Arg 130 135 140 CAA AAG TTC ACC GAA AAG GCG ATA GAG ATA GCC AAG AGC GGC GAG AGA 480 Gln Lys Phe Thr Glu Lys Ala Ile Glu Ile Ala Lys Ser Gly Glu Arg 145 150 155 160 ATA CAG TTC AGG GGT GTG TAG 501 Ile Gln Phe Arg Gly Val 165 <210> 4 <211> 166 <212> PRT <213> KOD1 <400> 4 Met Ala Arg Lys Thr Val Phe Ile Asp Phe Ser Lys Cys Ile Glu Cys 1 5 10 15 Arg Ala Cys Glu Val Ala Cys Glu Arg Glu His Asn Gly Arg Ser Phe 20 25 30 Ile Asn Val Phe Glu Trp Gln Glu Met Ala Ala Met Ala Leu Asn Cys 35 40 45 Arg His Cys Glu Lys Ala Pro Cys Leu Glu Val Cys P ro Thr Asn Ala 50 55 60 Leu Tyr Arg Asp Gly Asp Gly Ala Val Leu Leu Ala Pro Gln Lys Cys 65 70 75 80 Ile Gly Cys Leu Met Cys Gly Ile Val Cys Pro Phe Gly Ile Pro Glu 85 90 95 Leu Asp Ser Leu Asp Lys Ile Met Met Lys Cys Asp Leu Cys Ala His 100 105 110 Arg Arg Ala Glu Gly Lys Leu Pro Ala Cys Val Glu Thr Cys Pro Thr 115 120 125 Asp Ala Leu Leu Phe Gly Asp Phe Asn Asp Ile Gln Arg Met Arg Arg 130 135 140 Gln Lys Phe Thr Glu Lys Ala Ile Glu Ile Ala Lys Ser Gly Glu Arg 145 150 155 160 Ile Gln Phe Arg Gly Val 165

[Brief description of the drawings]

FIG. 1 shows the homology between various formate dehydrogenases.

FIG. 2 shows the construction of an expression vector for a thermostable formate dehydrogenase.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Satoshi Ezaki 64 Floral Kitayama, Kamigamo Ikehatacho, Kita-ku, Kyoto, Kyoto 108 F term (reference) 4B024 BA08 CA03 DA06 EA03 EA04 GA11 HA01 4B050 CC03 DD02 FF03E 4B065 AA01Y AA26X AB01 AC14 BA02 BD01 BD15 CA28

Claims (12)

[Claims] 1. A thermostable formate dehydrogenase comprising the amino acid sequence of SEQ ID NO: 2, or a formate dehydrogenase comprising one or more amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 A variant thereof having enzymatic activity. 2. A thermostable formate dehydrogenase comprising the amino acid sequence of SEQ ID NO: 4, or a formate dehydrogenase containing an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4. A variant thereof having enzymatic activity. 3. A heat-stable formate dehydrogenase comprising the amino acid sequences of SEQ ID NOs: 2 and 4, or an amino acid sequence comprising the amino acid sequences of SEQ ID NOs: 2 and 4 wherein one or several amino acids are deleted, substituted or added. And a variant thereof having formate dehydrogenase activity. 4. The thermostable formate dehydrogenase according to claim 1, which is derived from the hyperthermophilic archaeon KOD1 strain, or a variant thereof. 5. A DNA encoding the thermostable formate dehydrogenase according to claim 1 or a variant thereof. 6. A DNA encoding the thermostable formate dehydrogenase according to claim 2 or a variant thereof. 7. The DNA according to claim 5, comprising the nucleotide sequence of positions 1 to 2130 of SEQ ID NO: 1. 8. The DNA according to claim 6, comprising the nucleotide sequence of positions 1 to 498 of SEQ ID NO: 3. 9. The DNA according to claim 5, which is derived from the hyperthermophilic archaeon KOD1 strain. 10. The DNA according to any one of claims 5 to 9
Vector containing. 11. A recombinant host cell comprising the vector according to claim 10. A method for producing a thermostable formate dehydrogenase or a variant thereof, comprising a step of culturing the host cell according to claim 11.