JP2000350585A - Heat-resistant and oxygen-resistant hydrogenase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new heat-resistant and oxygen-resistant hydrogenase gene which contains a specific amino acid sequence, has a hydrogenase activity, and can be used for producing hydrogen useful for the clean energy system and so on. SOLUTION: This is a new heat-resistant and oxygen-resistant hydrogenase gene which contains an amino acid sequence represented by formula I and/or II, or an amino acid sequence modified by deletion, substitution, or addition of one or more amino acid(s) from, in, or to the amino acid sequence, has a hydrogenase activity, is excellent as an energy medium, and can be used for producing hydrogen useful for constructing the clean energy system and so on. This protein is obtained by preparing a genome DNA library derived from chromosomal DNA of a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus, screening the obtained library using its partial sequence as a probe, followed by expressing the obtained gene in an expression system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a heat- and oxygen-resistant hydrogenase protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, a method for producing a hydrogenase protein, and a hydrogenase. The present invention relates to a method for making proteins heat resistant and / or oxygen resistant.

[0002]

2. Description of the Related Art At present, fossil fuels such as petroleum and coal are mainly used as fuels. These fuels cause environmental problems such as air pollution and global warming due to the rise of carbon dioxide concentration.In particular, global warming due to carbon dioxide is an unavoidable problem that is always present as long as fossil fuels are used. is there. Under such circumstances, development of a new energy source to replace fossil fuels is required, and hydrogen is considered as one of the promising candidates.
Hydrogen has a large exothermic energy per unit weight,
It is excellent not only as an energy medium, but also excellent in constructing a clean energy system, for example, it can be efficiently converted to heat energy by burning and to electric energy if a fuel cell is used.

As a method for producing hydrogen, physicochemical techniques such as steam reforming of natural gas and electrolysis of water are known. However, biological methods using green algae, cyanobacteria, photosynthetic bacteria, anaerobic bacteria and the like are known. Hydrogen can also be produced by a technique. For example, if blue-green algae or green algae is used, hydrogen can be produced from water, carbon dioxide, and light energy, and an ultimate environment-friendly energy production system can be constructed.

[0004] However, there are some problems to be solved for practical use. One of the problems is stabilization of an enzyme involved in hydrogen generation. Hydrogenase and nitrogenase are known as enzymes directly involved in the generation of hydrogen in hydrogen-producing organisms. Hydrogenase is an enzyme that catalyzes the reduction of an electron carrier by hydrogen and the generation of hydrogen by the reverse reaction, and there are various types depending on the types of electron carriers that can react. Hydrogenases are generally unstable to heat and oxygen and are easily inactivated.

[0005]

SUMMARY OF THE INVENTION The present invention relates to a heat- and oxygen-resistant hydrogenase protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, and production of the hydrogenase protein. The aim is to provide a method.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus (Hydrogenovibrio m.
arinus) and succeeded in isolating a novel hydrogenase gene from the chromosomal DNA of the present invention, thereby completing the present invention.

That is, the present invention relates to the following protein (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 (b) at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 Having a modified amino acid sequence and having hydrogenase activity

Further, the present invention is a hydrogenase gene encoding the following protein (a) or (b). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 (b) at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 Having a modified amino acid sequence and having hydrogenase activity

Further, the present invention relates to the following (a) or (b):
It is a gene containing A. (a) DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3 (b) Hybridizing with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3 under stringent conditions DNA encoding soybean and protein having hydrogenase activity

[0010] Further, the present invention is a recombinant vector containing the above gene. Furthermore, the present invention is a transformant containing the above-mentioned recombinant vector. Furthermore, the present invention is a method for producing a hydrogenase protein, which comprises culturing the above transformant and collecting a hydrogenase protein from the resulting culture.

Furthermore, the present invention provides the following steps: (a) comparing the amino acid sequence of a thermostable and / or oxygen-resistant hydrogenase with the amino acid sequence of a non-thermostable and / or non-oxygen-resistant hydrogenase; (b) (C) specifying an amino acid residue specifically found in a thermostable and / or oxygen-resistant hydrogenase based on the comparison result obtained in the step (a); In the amino acid sequence, the amino acid residue at the position corresponding to the amino acid residue specified in step (b) (for example, represented by SEQ ID NO: 56 or 110, or represented by SEQ ID NO: 4 The 21st, 39th, 116th, 169th and 169th amino acids of the amino acid sequence
171st, 213th, 254th, 282nd, 326th, 333th, 349th, 356th and 368th at least one selected from the group consisting of) Substituting with an amino acid residue found in (1) above. Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The hydrogenase gene of the present inventor is a gene encoding a hydrogenase having heat resistance and oxygen resistance, unlike the conventional hydrogenase gene. The hydrogenase gene of the present invention can be cloned from Hydrogenobrio marinus by the following procedure.

1. Cloning of the hydrogenase gene of the present invention (1) Culture of hydrogenovibrio marinus As a source of the hydrogenase of the present invention, bacteria belonging to the genus hydrogenovibrio (e.g., hydrogenovibrio
Marinas MH-110). This bacterium is a comma-shaped gram-negative bacterium with polar flagella, and is cultivated in liquid or solid for 1 to 24 hours in a medium consisting of only inorganic compounds under a gas phase consisting of hydrogen, oxygen and carbon dioxide. Can be proliferated. Hydrogenobibrio marinus MH-110 used in the present invention has been deposited with the RIKEN Microorganism Strain Conservation Facility under the Application No. 7688, and is easily available.

(2) Preparation of Chromosomal DNA Preparation of chromosomal DNA from bacteria can be performed by a commonly used technique. For example, the cells obtained in the above (1), suspended in a suitable buffer such as TE buffer,
Next, the cells are destroyed by a chemical treatment method using a surfactant (eg, SDS, CTAB) or the like, and nucleic acids are extracted. Then, chromosomal DNA can be prepared by decomposing the RNA by ribonuclease treatment and then performing alcohol precipitation.

(3) Isolation and purification of hydrogenase and determination of N-terminal amino acid sequence On the other hand, in order to prepare a probe for cloning the hydrogenase gene of the present invention, hydrogenase is isolated and purified and its N-terminal amino acid sequence is determined. I do. First,
After centrifugation of the cells obtained in the above (1), suspended in a suitable buffer such as phosphate buffer or PIPES buffer, mechanical disruption method (e.g., ultrasonic crushing, carborundum milling,
(Alumina grinding). Next, a membrane fraction is prepared from the obtained disrupted cells by ultracentrifugation or the like, and the membrane protein is solubilized with a surfactant such as sucrose monodecanoate and octylthioglucoside (OTG). Then, a hydrogenase-active fraction is collected and contaminating proteins are removed by ion exchange chromatography such as DEAE-Toyopearl, hydrophobic chromatography, gel filtration chromatography, or the like.

Next, the purified hydrogenase fraction thus obtained is subjected to denaturation treatment and then subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins in the gel are transferred to a blotting membrane such as polyvinylidene fluoride (PVDF) membrane, and amide black or Coomassie brilliant blue G-25 is transferred.
After staining with 0 or the like, the band of the hydrogenase protein is cut out based on the molecular weight. Finally, the cut membrane section was subjected to an amino acid sequencer (e.g., ABI, model
470A) to determine the N-terminal amino acid sequence.

(4) Cloning of the hydrogenase gene Based on the N-terminal amino acid sequence determined in the above (3), a PCR primer is synthesized, and using this primer, the present hydrogenase is synthesized from the chromosomal DNA obtained in the above (2). Amplify part of the gene. The hydrogenase gene is cloned using the amplified DNA fragment as a probe. The hydrogenases of the present invention are class I (membrane-bound,
(A type that holds a nickel-iron cluster in the molecule). Generally, it is known that a gene encoding a class I hydrogenase is adjacent to a small subunit gene and a large subunit gene in the order of chromosomal DNA [L.-F.
nd MAMandrand, FEMS Microbiology Reviews 104: 243
-270 (1993)]. Therefore, after the N-terminal amino acid sequence of the small subunit and the N-terminal amino acid sequence of the large subunit are determined, PCR is performed using chromosomal DNA as a template, using primers synthesized based on these amino acid sequences. The full length gene of the small subunit can be amplified.

For example, the PCR primers that can be used here include the N subtype of the hydrogenase small subunit.
Terminal amino acid sequence Met Glu Thr Lys Pro Arg Thr Pro Va
l 5'-ATGGAAAC synthesized based on Ile Trp (SEQ ID NO: 6)
TAA (A / G) CC (A / T) CGTAC (A / G / T) CC (A / T) GT (A / T) AT (A / C /
T) TGG-3 '(SEQ ID NO: 8) and N-terminal amino acid sequence of large subunit Met Asp Asn Ser Gly Arg Arg Val Val Ile As
5 '-(A / G) TCGAT (A / T) A synthesized based on p (SEQ ID NO: 7)
C (A / T) ACACGACGACC (A / T) GA (A / G) TT (A / G) TCCAT-3 ′ (SEQ ID NO: 9) can be used. However, the present invention is not limited to these primers. The synthesis can be performed with a DNA synthesizer (for example, model A391, model 391).

Then, the nucleotide sequence of the amplified DNA fragment is determined, and it is confirmed that the deduced amino acid sequence has homology with the partial amino acid sequence encoded by the small subunit gene of class I hydrogenase. If confirmed, after labeling the PCR product, this is used as a probe, and a restriction enzyme digest of chromosomal DNA (e.g., HindIII, Kpn
I, EcoRI digest, etc.).
Thus, it is possible to measure the size of the fragment containing the target hydrogenase gene on the gel after electrophoresis of the digested product of the restriction enzyme of chromosomal DNA. Based on the determined size, from the gel after electrophoresis of the digested product of the restriction enzyme of chromosomal DNA, cut out a gel near that size and extract the DNA, and then use an appropriate vector (e.g.,
(pBluescriptIIKS (+), pUC19, pBR322, pFOS1, M13, etc.)
Connect to Then, the ligated product is transformed into E. coli (for example, XL-1).
(blue, C600, JM109, S17-1, etc.) to prepare a chromosomal DNA library containing chromosome-derived DNA fragments of the desired size.

Next, colony PCR is performed using the obtained chromosomal DNA library and the above primers (for example, SEQ ID NO: 8 and SEQ ID NO: 9) to select colonies in which a DNA fragment of the desired size has been amplified. Finally, a plasmid is extracted from the colony, the nucleotide sequence of the inserted DNA fragment is determined, and finally a positive clone of the hydrogenase gene is determined.

The nucleotide sequence is determined by subjecting the target DNA fragment to pGEM-T
(Promega) and pBluescriptIIKS (+) (Stratagene)
After ligation to an appropriate vector such as maxam-Gilbert, it can be performed by a known method such as a chemical modification method of Gilbert, or a dideoxynucleotide chain termination method using M13 phage, but is usually performed by an automatic nucleotide sequencer (for example, manufactured by ABI) , Model 377A). From the determined base sequence, a DNA analysis software such as DNASIS (manufactured by Hitachi Software Engineering Co.) was used to search for an open reading frame, and the homology between the deduced amino acid sequence and the amino acid sequence of a known hydrogenase was determined. Find out.

SEQ ID NO: 1 is the nucleotide sequence of the small subunit gene of the present invention, SEQ ID NO: 2 is the amino acid sequence of the small subunit protein of the present invention, SEQ ID NO: 3 is the nucleotide sequence of the large subunit gene of the present invention, SEQ ID NO: 4 illustrates the amino acid sequence of the large subunit protein of the present invention, the small subunit gene of the present invention and the large subunit gene of the present invention adjacent to SEQ ID NO: 5, and the protein containing these amino acid sequences is At least one amino acid in the amino acid sequence may be mutated such as deletion, substitution or addition as long as it has hydrogenase activity.

For example, at least one, preferably about 1 to 20, of the amino acid sequence represented by SEQ ID NO: 2 or 4,
More preferably, 1 to 10 amino acids may be deleted, or at least one, preferably about 1 to 20, more preferably 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 2 or 4. Amino acids may be added, or at least one, preferably about 1 to 30, and more preferably 1 to 10 amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4 are substituted with other amino acids. You may.

The gene of the present invention also includes a DNA capable of hybridizing with the above gene under stringent conditions. The stringent condition refers to, for example, a condition at a temperature of 52 to 70 ° C, preferably 62 to 70 ° C. To introduce mutations into genes, the Kunkel method, G
For example, a mutagenesis kit (for example, Mutan-K (manufactured by TAKARA), Mutan-G) using a site-directed mutagenesis method by a known method such as the apped duplex method or a method analogous thereto.
(TAKARA)) or TAKARA LA
PCR can be performed using the in vitro Mutagenesis series kit.

Once the nucleotide sequence of the gene of the present invention has been determined, it is then subjected to chemical synthesis or cDNA or genomic analysis of the gene.
The gene of the present invention can be prepared by PCR using DNA as a template. For example, a gene encoding the full-length amino acid sequence of the small subunit of the hydrogenase of the present invention is
When amplifying by PCR, hydrogenovibrio
5'-ATGTCATCTCAAG using chromosomal DNA of Marinas as a template
TTGAAACGTTCT-3 '(SEQ ID NO: 10) and 5'-TTTATCTCCTTTCTTT
It can be performed by using a primer having the nucleotide sequence of TGAGCCGCT-3 ′ (SEQ ID NO: 11). However, in the present invention, the primer is not limited to these. In addition, 5 'upstream or 3' downstream DNA of the gene of the present invention
When a region is desired to be prepared, it can be performed by a technique such as gene walking. Gene walking is screening from a gene library using a fragment of the 5 'or 3' end of a clone already obtained as a probe, and isolating a clone in which a part of the sequence partially overlaps the sequence of the clone, This is a method in which the analysis is advanced in the 5 ′ or 3 ′ direction by repeating this method. For example, when DNA of the 3 ′ downstream region of the large subunit of the hydrogenase of the present invention is obtained by gene walking, a 300 bp region of the 3 ′ terminal fragment of the large subunit of Hydrogenobibrio marinus (SEQ ID NO: 12) ) Can be used as a probe. However, in the present invention, the probe is not limited to these.

2. Preparation of Recombinant Vector and Transformant (1) Preparation of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating (inserting) the gene of the present invention into an appropriate vector.
The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. Since the hydrogenase gene of the present invention is composed of a gene encoding a small subunit and a gene encoding a large subunit, when constructing a recombinant vector for expressing a hydrogenase, the two subunits are combined into one. The vectors may be tandemly linked or the small and large subunit genes may be linked to separate vectors.

As the plasmid DNA, plasmids derived from E. coli (for example, pBR322, pBR325, pUC118, pUC119, pUC
18, pUC19, pCBD-C, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pHY300PLK DNA, etc.), and yeast-derived plasmids (eg, YEp13, YEp24, YCp50, YIp30, pAUR101D)
NA, pAUR123 DNA, etc.), a plasmid derived from cyanobacteria (e.g., pECA
N8 [RHLau and NAStraus, FEMS Microbiology Lette
rs 27: 253-256 (1985)]), and phage DNA includes λ phage.

To insert the gene of the present invention into a vector, first, the purified DNA is cut with an appropriate restriction enzyme,
For example, a method of inserting into an appropriate restriction enzyme site or a multiple cloning site of vector DNA and ligating to the vector is employed. The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exerted. Therefore, the vector of the present invention includes, in addition to the promoter and the gene of the present invention, cis elements such as enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), if desired.
And the like can be linked. In addition, as a selection marker, for example, an ampicillin resistance gene,
Neomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene and the like.

(2) Preparation of Transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host of the present invention
There is no particular limitation as long as it can express DNA. For example, the genus Escherichia such as Escherichia coli and Bacillus subtilis
s), Pseudomonas putida (Pseudomonas
as putida), bacteria belonging to the genus Rhizobium such as Rhizobium meliloti (Rhizobium meliloti), and Rhodobacter sphaeroides (Rhodoba
ctersphaeroides) and Synechococcus (S
ynechococcus) Cyanobacteria such as PCC7942 strain, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pomb)
e) and the like.

The hydrogenase gene of the present invention comprises
Because it consists of a gene encoding a small subunit and a gene encoding a large subunit, in the above (1),
When the small subunit gene and the large subunit gene are ligated to different vectors, they may be transformed into the same host. When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and at the same time, a promoter, a ribosome binding sequence,
It is preferable that the gene of the present invention is composed of a transcription termination sequence. Further, a gene controlling a promoter may be included.

As Escherichia coli, for example, Escherichia
Coli (Escherichia coli) JM109, DH5α and the like,
As Bacillus subtilis, for example, Bacillus subtilis (Bacillu
s subtilis) MI114, 207-21 and the like. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example trp promoter, lac promoter, P _L promoter, and P _R promoter may be used from Escherichia coli or phage. An artificially designed and modified promoter such as a tac promoter may be used.

The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions
[SNCohen et al., Proc. Natl. Acad. Sci., USA 69: 2
110 (1972)], electroporation, bonding [R. Sim
on et al., BIO / TECHNOLOGY 784-791 (Nov. 1983)], a natural transformation method [JGK Williams et al., Methods.
ol. 167: 766-778 (1988)].

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe
e), Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast.For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, CMV promoter, AOX1 promoter and the like.

The method for introducing the recombinant vector into yeast is not particularly limited, as long as it is a method for introducing DNA into yeast. For example, an electroporation method [DMBecker et al.
l., Methods. Enzymol. 194: 182 (1990)], spheroplast method [A. Hinnen et al., Proc. Natl. Acad. Sci., U.
SA 75: 1929 (1978)], lithium acetate method [H. Itoh et al.,
J. Bacteriol. 153: 163 (1983)].

3. Production of the protein of the present invention The protein of the present invention has an amino acid sequence encoded by the hydrogenase gene of the present invention, or has an amino acid sequence in which the mutation is introduced into at least one amino acid in the amino acid sequence. Say.
The protein of the present invention can be produced from the culture of the transformant obtained in the above 2 (2) as follows. Here, “culture” refers to the culture supernatant after culturing,
It refers to either cells obtained by culture or crushed cells. The method for culturing the transformant obtained in the above 2 (2) is performed according to a usual method used for culturing those cells.

The medium may be any of natural medium and synthetic medium as long as the medium contains a carbon source, a nitrogen source, inorganic salts and the like which can be used by the cells and can efficiently culture the cells. May be used. Here, as the carbon source,
Carbohydrates such as glucose, fructose, sucrose, and mannose; organic acids such as acetic acid and lactic acid; alcohols such as methanol; animal oils; Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; nitrates such as potassium nitrate and sodium nitrate; peptone, meat extract, yeast extract, malt extract, and corn steep liquor. Is used. Furthermore, compounds containing trace elements such as nickel, potassium, sodium, calcium, magnesium, manganese, cobalt, zinc, iron, molybdenum, copper, and sulfuric acid, phosphoric acid,
An anionic salt such as hydrochloric acid is used.

In the culture of the transformant, if the transformant has been transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. . For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, culturing a microorganism transformed with an expression vector using a trp promoter such as isopropyl-β-D-thiogalactopyranoside (IPTG). In this case, indoleacrylic acid (IAA) or the like may be added to the medium.

After the culture, when the hydrogenase protein of the present invention is produced in the cells, the cells are crushed to extract the hydrogenase protein. The disruption of the cells can be performed using ultrasonic waves, a French press, a homogenizer using glass beads, or the like, but can also be performed in combination with lysozyme or a freeze-thaw method.
When the hydrogenase protein of the present invention is produced outside the cells, the culture solution is used as it is, or the cells are removed by centrifugation or the like. Thereafter, by using a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or the like, alone or in appropriate combination, The protein of the present invention can be isolated and purified from the culture.

4. Measurement of hydrogenase activity Since hydrogenase catalyzes the hydrogen oxidation reaction (H ₂ → 2H ⁺ + 2e ⁻ ) and the hydrogen generation reaction (2H ⁺ + 2e ⁻ → H ₂ ), its activity is determined by examining the oxidation of hydrogen or the generation of hydrogen. Can be measured. For example, in a method of measuring hydrogenase activity by hydrogen oxidation, the gas phase of the reaction system is filled with hydrogen, and the amount of oxidized methyl viologen reduced in a unit time by the hydrogenase is determined by the spectrophotometry. By measuring with a meter, the hydrogenase activity can be calculated based on the measured value. On the other hand, in the hydrogenase activity measurement method by the hydrogen generation reaction, the reaction system is replaced with an inert gas such as argon, a reducing agent such as dithionite is added thereto, and an electron carrier such as methyl viologen present in the reaction system is removed. By using the reduced form, the amount of hydrogen generated per unit time can be measured using gas chromatography or the like, and the hydrogenase activity can be measured based on the measured value.

5. The hydrogenase of the present invention has heat resistance and oxygen resistance despite being derived from a normal-temperature bacterium. Therefore,
By modifying the amino acid sequence of the non-thermostable and / or non-oxygen-resistant hydrogenase based on the structure of the hydrogenase of the present invention, the hydrogenase can be made thermotolerant and oxygen-resistant.

In general, the deactivation of an enzyme by heat or oxygen
It is because the three-dimensional structure of the protein is destroyed. Normal,
The three-dimensional structure of the protein is maintained by α-helix structure, β-sheet structure, hydrogen bond, hydrophobic interaction, SS bond, electrostatic interaction, metal ion coordination, etc., and strengthens these interactions By performing an amino acid mutation, it is possible to make the hydrogenase protein heat- and oxygen-resistant. When comparing the hydrogenase which is unstable to heat and oxygen with the amino acid of the hydrogenase of the present invention, a plurality of characteristic amino acids as shown in Table 1 are found in the hydrogenase of the present invention. In Table 1, the position means the position of the amino acid located from the N-terminal methionine in the large subunit or the small subunit. Further, in the present specification, when a number is given to an amino acid symbol such as, for example, Ile21, it means that it is the 21st isoleucine from the N-terminal methionine.

[0042]

[Table 1]

Accordingly, by substituting the amino acid at the corresponding position with the same amino acid as the hydrogenase of the present invention, the non-thermostable and / or non-oxygen-resistant hydrogenase can be rendered thermotolerant and / or oxygen-resistant. Amino acid substitution can be performed by introducing point mutations by PCR [Kaoru Saigo,
Translated by Yuko Sano, Molecular Biology Experiment Protocol I, p.249-
270, Maruzen, 1997].

6. Hydrogen production by biological techniques (1) Creation of hydrogen-producing cells having thermostable and oxygen-tolerant hydrogenase The mechanism of hydrogen generation by algae, photosynthetic bacteria, and anaerobic bacteria is shown in Fig. 1. Has not been put to practical use due to problems such as heat and oxygen instability of the hydrogenase directly involved in the enzyme. Therefore, the heat-resistant and oxygen-tolerant hydrogenase gene of the present invention is introduced into the above-mentioned biological cells, or the hydrogenase is modified by the method for increasing the temperature and oxygen-tolerance of the hydrogenase described in 5 above, thereby breeding a hydrogen-producing cell having a heat- and oxygen-tolerant hydrogenase. be able to. For example, the expression of the heat- and oxygen-tolerant hydrogenase gene of the present invention introduced into the cell is as follows:
In the case of algae belonging to the genus Synechocystis, JGKWillia
ms et al. [JGKWilliams et al., Methods. Enzymo
l. 167: 766-778 (1988)], the method of R. Simon et al. [R. Simon et a
l., BIO / TECHNOLOGY 784-791 (Nov. 1983)].

(2) Hydrogen Production by Bioreactor A highly stable hydrogen production system can be constructed by the bioreactor using the hydrogen producing cell obtained in the above (1). Bioreactors are those in which hydrogen-producing cells are suspended in a culture tank containing an appropriate medium,
A culture tank or column filled with immobilized hydrogen-producing cells may, for example, be mentioned. Here, the production of immobilized hydrogen-producing cells can be performed by immobilizing the cells on a gel of alginic acid, carrageenan, agarose or the like according to a conventional method. When using photosynthetic bacteria or anaerobic bacteria,
Since an organic substance such as an organic acid or glucose is required, an appropriate medium or suspension is replenished with time. In addition, when cyanobacteria or green algae is used, hydrogen can be produced from water and light energy. Therefore, in hydrogen production by algae, cells are irradiated with light suitable for the organism, such as sunlight, light from a fluorescent lamp, or a light emitting diode.

[0046]

The present invention will be described below in more detail with reference to examples. However, the present invention is not limited to these examples. [Example 1] Culture of Hydrogenovibrio marinus and preparation of chromosomal DNA (1) Culture of Hydrogenovibrio marinus Marine hydrogen oxidizing bacterium Hydrogenovibrio marinus (Hyd
rogenovibrio marinus) MH-110 (RIKEN Microbial Strain Preservation Strain) was added to 1 liter of growth medium (per liter: 2.0 g (NH ₄ ) ₂ SO ₄ , 2.5 g K ₂ HPO ₄ , 0.5 g KH ₂ P
O ₄ , 0.2g MgSO ₄・ 7H ₂ O, 29.3g NaCl, 10.0mg CaCl ₂ , 1
0.0mg FeSO ₄ · 7H ₂ O, were inoculated into a medium containing trace elements solution (pH 7.0)) of _{0.6mg NiSO 4 · 7H 2 O,} 2.0ml Hirsch (Hirsh). Here, the composition of the trace element solution 1 liter _{Hirsch, 1.0mg MoO 3, 7.0mg ZnSO 4} · 7H 2 O, 0.5mg CuSO 4 · 5H
_{2 O, 1.0mg H 3 BO 3} , 1.0mg MnSO 4 · 5H 2 O, 1.0mg CoCl 2 · 6H 2
O. Next, the gas phase was replaced with a gas having a composition of H ₂ : O ₂ : CO ₂ = 7: 2: 1, and the cells were cultured at 37 ° C. for 14 hours. After completion of the culture, the culture broth was collected by centrifugation at 8,000 × g for 20 minutes at 4 ° C.

(2) Preparation of chromosomal DNA From the cultured cells obtained in the above (1), the method of Ausubel et al. [FMAusubel et al., Current protocols in mol
ecular biology. Wiley, New York. (1987)]. That is, 10
After collecting 0 ml of the cell suspension, washing with 0.8% NaCl,
0 ml STE buffer (500 mM sucrose, 25 mM Tris, 10 mM ED
TA). After adding 40 mg of lysozyme and reacting at 37 ° C for 10 minutes, add 20 μl of 25 mg / ml proteinase K and
The reaction was performed for 90 minutes. Add 500 μl of 10% SDS and 20 μl of 25 mg / ml proteinase K and react at 37 ° C. for 30 minutes.
After adding 1.5 ml of SDS, a phenol-chloroform solution (T
E-saturated phenol / chloroform = 1: 1 solution) was added in an equal amount, and phenol extraction was performed three times. After adding twice the volume of cold ethanol to the supernatant and performing ethanol precipitation at -20 ° C overnight, the precipitate was washed with 70% ethanol and dried under vacuum.
This was suspended in 1 ml of TE (pH 8.0), and 1 μl RNase (25 mg / m
l) and react at 37 ° C for 30 minutes.
Dialysis was performed overnight. Further, after ethanol precipitation, washing with 70% ethanol, and vacuum drying, 400 μl of TE
(PH 8.0) to give a chromosomal DNA solution.

Example 2 Enzymological Properties of Hydrogenase from Hydrogenobrio marinus (1) Preparation of Enzyme Solution An enzyme solution was prepared from the cells obtained in Example 1 (1). That is, 1 g of wet cells were suspended in 5 ml of a 50 mM phosphate buffer (pH 7.0), and an ultrasonic crusher (manufactured by BRANSON, S
Using ONIFIER-450), crushing was performed three times for 1 minute at an output of 20 kHz. The homogenate was centrifuged at 8,000 × g for 20 minutes at 4 ° C. Then, the supernatant after centrifugation was further added at 4 ° C. and 128,000 ×
Ultracentrifuged at g for 1 hour. The obtained precipitate was used as a membrane fraction, and this was suspended in a 50 mM phosphate buffer (pH 7.0) and subjected to the following experiment as an enzyme solution.

(2) Method for measuring enzyme activity Hydrogenase activity was measured by the following two methods. Measurement of hydrogenase activity by hydrogen absorption activity measurement method The hydrogenase activity was determined from the rate of increase in the absorbance of methyl viologen, which exhibits a blue color when reduced. That is, first, 2.5 ml of a 20 mM phosphate buffer (pH 7.0) containing 1 mM methyl viologen was placed in a glass cell, and the vessel was sealed and replaced with hydrogen gas for 3 minutes. Then, at any reaction temperature (30 ° C.-9
(0 ° C.) for 5 minutes, 10 μl of the enzyme solution was injected to start the reaction, and the change in absorbance at 600 nm was measured with a self-recording spectrophotometer to measure the activity value.

Measurement of Hydrogenase Activity by Hydrogen Evolving Activity Measurement Method First, methyl vialogen and 20 mM phosphate buffer (pH 7.0) were added to a vial bottle to a final concentration of 2.5 mM, and the vial was sealed with argon gas for 5 minutes. Was replaced. Next, 10 μl of the enzyme solution was added, and the reaction was performed at any reaction temperature (30 ° C. to 90
C) for 5 minutes. 200 replaced with argon gas
100 μl of mM dithionite was added to the vial to start the reaction. Hydrogen generated in the gas phase was determined by gas chromatography.

(3) Measurement of Hydrogenase Oxygen Tolerance The hydrogenase solution in which the gas phase was replaced with air or oxygen was stored at 30 ° C., and the remaining enzyme activity was measured over time. H. marinus hydrogenase ("-●-" and "-■" in FIG. 2)
-") Was shown to have higher oxygen tolerance than those of the same hydrogen bacterium, Alcaligenes eutrophus (Al. Eutrophus) (" -o- "and"-□-"in Fig. 2).

(4) Measurement of Thermal Stability of Hydrogenase Hydrogenase solution was placed in air or hydrogen gas for 70 minutes.
The temperature was kept at 90 ° C. or 90 ° C., and the enzyme activity remaining over time was measured. Approximately 90 even when heated at 70 ° C for 50 minutes in air
% Activity. When the gas phase was replaced with hydrogen gas, the thermal stability further increased, and even after heating at 90 ° C. for 50 minutes, almost the same activity as before heating was maintained (FIG. 3).

The hydrogenase of the present invention and a known hydrogenase derived from Pyrodictium brokii [TDPhil and RJMaier, J. Bacteriol. 173: 18]
39-1844 (1991) (Table 2, reference 1)], a hydrogenase derived from Thermotoga maritima [A. Jusz
czak, S. Aono and MWWAdams, J. Biol. Chem. 266: 138
34-13841 (1991) (Table 2, Reference 2)], hydrogenase derived from Bacillus schlegelli [M. Pi
nkwart, K. Schneider and HG Schlegel, FEMS Microbio
l. Letter 17: 137-141 (1983) (Table 3, Reference 3)], Clostridium thermos
aceticum) -derived hydrogenase [HL Drake, J. Bacteri
ol. 150: 702-709 (1982) (Table 2, Reference 4)], and the results of comparing thermal stability and oxygen tolerance are shown in Table 2. As is clear from the results, the hydrogenovibrio of the present invention
The hydrogenase derived from marinus has both thermostability and oxygen tolerance, and was found to be superior in stability to known hydrogenases.

[0054]

[Table 2]

Example 3 Isolation and Purification of Hydrogenase from Hydrogenobrio marinus and Analysis of N-terminal Amino Acid Sequence (1) Purification of Hydrogenase Hydrogenase was solubilized from the membrane fraction obtained in Example 2 (1). did. That is, after adjusting the protein concentration of the membrane fraction to 2 mg / ml, a nonionic surfactant octylthioglucoside was added to 20 mM, and the temperature was adjusted to 50 ° C, 20 ° C.
Solubilization was performed by a heat treatment for 5 minutes. The obtained solubilized hydrogenase protein was previously 10% glycerol,
50 mM Tris-HCl buffer containing 0.5% Triton X-100 (pH
7.7) was added to the DEAE-Toyopearl column (manufactured by Tosoh Corporation) equilibrated in the above, the column was washed with the above buffer solution, NaC of 0 to 500 mM
The protein adsorbed on the column was eluted with a gradient of l. When the hydrogenase activity of each fraction was measured by a hydrogen absorption activity measurement method, it was found that the target hydrogenase was eluted with 25 mM NaCl.

(2) SDS-polyacrylamide gel electrophoresis An equal volume of denaturing buffer (60 mM Tris-HCl (pH 6.8), 2% SDS, 20%) was applied to the hydrogenase eluate obtained in (1) above.
Glycerol, 10% 2-mercaptoethanol, 0.01%
Bromophenol blue), mix and add
Denaturation for SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). SDS-PAGE is based on Laemmli's method [UKLa
emmli, Nature 227: 680-685 (1970)]. Separation gel concentration is 10%, running buffer is 25mM Tris, 0.192M
Glycine, 0.1% SDS was used. From the results of SDS-PAGE,
The hydrogenase of the present invention was estimated to consist of a small subunit having a molecular weight of about 38,000 and a large subunit having a molecular weight of 74,000.

(3) Determination of N-terminal amino acid sequence The protein in the gel after SDS-PAGE separation obtained in (2) above was applied to a clear blot P membrane (manufactured by ATTO) at 2 mA /
Electroblotting was performed at a current of cm ² for 55 minutes. The clear blot P membrane was then coated with Coomassie Brilliant Blue.
The target band was cut out by staining with G-250, and the membrane section was applied to a protein sequencer (ABI, model 470A) to decode the amino acid sequence. As a result, the N-terminal amino acid sequence of the hydrogenase of the present invention has a small subunit containing Met G
lu Thr Lys Pro Arg Thr Pro Val Ile Trp (SEQ ID NO: 6), large subunit of which is Met Asp Asn Ser Gly Arg Arg V
al Val Ile Asp (SEQ ID NO: 7).

Example 4 Cloning of Hydrogenase Gene (1) Synthesis of Primer A degenerate primer was synthesized based on the amino acid sequence determined in Example 3 (3). That is, as the 5 ′ degenerate sense primer sequence, the N-terminal amino acid sequence of the small subunit Met Glu Thr Lys Pro Arg Thr Pro Val Ile
5′-ATGGAAACTAA (A / G) CC (A / G
T) CGTAC (A / G / T) CC (A / T) GT (A / T) AT (A / C / T) TGG-3 ′ (SEQ ID NO: 8) as a 3 ′ degenerate antisense primer, N-terminal amino acid sequence of large subunit Met Asp Asn Ser Gly
5 '-(A based on Arg Arg Val Val Ile Asp (SEQ ID NO: 7)
/ G) TCGAT (A / T) AC (A / T) ACACGACGACC (A / T) GA (A / G) TT (A /
G) TCCAT-3 '(SEQ ID NO: 9) was synthesized. Note that the synthetic oligonucleotide was a fully automatic DNA synthesizer (ABI, model 39).
It was chemically synthesized using 1).

(2) Amplification of the hydrogenase small subunit gene by PCR Using the hydrogenovibrio marinus chromosomal DNA obtained in Example 1 as a template and performing the PCR using the primer of (1) above, the hydrogenase small subunit is amplified. The unit gene was obtained. The composition of the PCR reaction solution is as follows.

1.3 mg / ml chromosomal DNA solution 0.5 μl 10 × PCR buffer (TAKARA) 10 μl 20 μM primer (sense) 2 μl 20 μM primer (antisense) 2 μl 1 mM dNTP mix 1 μl 50 mM MgCl ₂ 5 μl 5 U / μl Taq polymerase ( 1 μl sterile water 78.5 μl total volume 100 μl

The PCR was performed under the following conditions: heat denaturation at 96 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension reaction at 72 ° C. for 2 minutes.
As a cycle, 30 cycles were performed. After the completion of the reaction, the PCR product was recovered according to a conventional method, and then subjected to 1% agarose gel electrophoresis, and about 1 kbp of the small subunit gene was tested.
The DNA fragment was confirmed. Next, the PCR product was TA Cloning
E. coli (TOP10)
F 'strain). A single colony is cultured in LB medium, the plasmid is purified, and Dye Terminator Cycle Sequenci
ng FS Ready Reaction Kit (PE Biosystems) and a DNA sequencer (ABI, Model 377A)
The nucleotide sequence of the PCR product was determined (SEQ ID NO: 13).

(3) Cloning of the full length sequence of the hydrogenase gene and determination of the nucleotide sequence Using the PCR product obtained in the above (2) as a probe, screening of the full length hydrogenase sequence from the chromosomal DNA of Hydrogenobrio marinus was attempted. Was. First, probe the DNA Labeling and Detection Kit, Nonr
Digoxigenin labeling was performed using adio active (Boehringer Mannheim Biochemica). Next, using this labeled probe, a HindIII digest of the chromosomal DNA of Hydrogenobibrio marinus obtained in Example 1 was subjected to Southern hybridization. Here, the hybridization solution is a QuikHyb Hybridization Solution (Stratage
ne Cloning System). as a result,
A strong signal was obtained around 6 kbp.

Next, a HindIII digest of the genomic DNA of Hydrogenobrio marinus was separated by 0.8% agarose gel electrophoresis, and a fragment of about 6 kbp was cut out. The obtained DNA fragment was ligated to the HindIII site of the multicloning site of the pBluescript II KS (+) plasmid, and transformed into Escherichia coli XL-1 Blue (Stratagene).

Next, using the primers synthesized in (1) above for each of the obtained transformants, colony PCR was performed.
As shown in FIG. 4, a 1-kbp fragment of the small subunit was amplified to examine whether a positive clone was screened. As a result, two positive clones were obtained from 532 colonies. PCR
For 30 cycles, the conditions of heat denaturation at 96 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, and extension reaction at 72 ° C. for 2 minutes are defined as one cycle. The composition of the reaction solution used for the colony PCR is shown below.

Transformant (colony) Small amount 10 × PCR buffer (TAKARA) 2 μl 20 μM primer (sense) 0.4 μl 20 μM primer (antisense) 0.4 μl 1 mM NTP mix 0.2 μl 50 mM MgCl ₂ 0.4 μl Taq polymerase 0.2 μl (5U) H ₂ 0 16.4μl Total volume 20μl

Next, a recombinant plasmid pMH518 was prepared from the obtained positive clones, subjected to subcloning with a restriction enzyme and production of a deletion mutant using an Erase-a-Base System (manufactured by Promega), and then to a DNA sequencer (ABI). 377A), the nucleotide sequence was determined. As a result, the adjacent small subunit gene and large subunit gene were found (SEQ ID NO: 5). That is, 36
A small subunit gene (SEQ ID NO: 1) encoding a protein consisting of one amino acid (SEQ ID NO: 2) and a large subunit gene (SEQ ID NO: 3) encoding a protein consisting of 457 amino acids (SEQ ID NO: 4) I found it.

(4) Homology search For the amino acid sequence determined in (3) above, GenBank
A homology search was performed using the database. As a result, as shown in Table 3, both the small subunit and the large subunit were alkaligenes (Alcaligenes), Azotobacter (Azotobacter), Rhodobacter (Rhodobacter), thiocapsa (Thiocapsa), Pseudomonas (Pseudomonas), Bradyrhizobium (Bradyrhizobium), and oligotropha. (Oli
gotropha), Rubrivivax, and Escherichia derived from class I membrane-bound NiFe-hydrogenase. In addition, homology was observed with a thermostable hydrogenase derived from the hydrogen-oxidizing bacterium Aquifex aeolicus having an optimum growth temperature of about 70 ° C.

[0068]

[Table 3]

Example 5 Identification of Amino Acids Involved in the Thermostability of Hydrogenobibrio marinus-derived Hydrogenase The amino acids involved in the thermostability of Hydrogenobibrio marinus (H. marinus) hydrogenase are identified. Therefore, the amino acid sequence of the hydrogenase of the present invention and a known non-thermostable hydrogenase (Alcaligenes eutrophus (Alcaligeneseutrophus; Al.
Azotobacter vinelandi
i; Desulfovibrio giga (Desulfovibrio) derived from Az.
gigas; hydrogenase derived from D. gigas), a thermostable hydrogenase (Aqifex eoricus)
And a hydrogenase derived from Pyrococcus furiosus (P. furiosus). Figure 5 shows the alignment comparison results of the large subunit.
FIG. 6 shows the alignment comparison results of the small subunits. In the figure, the amino acids found only in the hydrogenase of Hydrogenobrio marinus are boxed, and the amino acids that are different from normal non-thermostable class I hydrogenases but are common to the three thermostable hydrogenases are underlined.

Looking at the underlined and squared amino acids in the large subunit (FIG. 5), the amino acids Val and Leu are replaced by Ile in hydrogenovibrio marinus and other thermostable hydrogenases in normal NiFe-hydrogenase. It was found that there were many cases (nine places).
This is a substitution that increases the hydrophobicity of amino acids, but Val
It is thought that the substitution of Leu and Ile from the compound increases the hydrophobicity due to the addition of a methylene group and does not significantly affect the structure of the protein itself. Although Phe213 is not a substitution for Ile, it is also a substitution of Leu for normal hydrogenase,
A substitution that increases hydrophobicity. Ile282 is Phe in Pyrococcus furiosus, and Ile333 is Phe in Axifex aeoricus. Both of them increase hydrophobicity similarly to the substitution with Ile, and are therefore underlined in the figure. Thr349 Se
Substitution from r is also found in Axifex aeolicus, which is also a substitution whose hydrophobicity is increased by the addition of a methylene group. The substitution from Ala found in Pro356 is Ile in Pyrococcus furiosus, which is also a substitution that increases hydrophobicity. As described above, hydrogenovibrio marinus and other thermostable hydrogenases showed many amino acid substitutions with increased hydrophobicity. Therefore,
It is considered that the heat resistance and oxygen resistance are increased by performing substitution of the amino acid site of a hydrogenase having low thermostability and oxygen resistance, such as Alcaligenes eutrophas, which increases hydrophobicity like Hydrogenobibrio marinus.

Furthermore, there is a report that formation of ionic bonds due to the presence of an electric charge on an amino acid present on the surface of a protein contributes to heat resistance. A substitution for a charged amino acid commonly found in Hydrogenobrio marinus and other thermostable hydrogenases was found in Glu39. In Pyrococcus furiosus, it is Asp, but both are substitutions for negatively charged amino acids.

In the small subunit (FIG. 6), there are not as many amino acid substitutions characteristic of the thermostable enzyme as in the large subunit, but the substitution from Leu to Ile was observed in Ile56 as in the case of the large subunit. . Substitution with a charged amino acid is L
Found on ys110, which was common with Pyrococcus furiosas.

Table 4 summarizes the characteristic amino acid substitutions found in these thermostable hydrogenases. When the position of this enzyme in the three-dimensional structure was assigned based on the result of alignment of Desulfovibrio gigas with hydrogenase, all amino acids were present evenly on the molecular surface. As a result, it has been clarified that the amino acids in Table 4 may be involved in thermostability especially in the hydrogenase of the present invention.

[0074]

[Table 4]

[0075]

According to the present invention, there are provided a heat- and oxygen-resistant hydrogenase protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, and a method for producing the hydrogenase protein. You.

[0076]

[Sequence List] SEQUENCE LISTING <110> TOKYO GAS CO., LTD. <120> Thermostable and Oxygen-stable Hydrogenase <160> 13 <210> 1 <211> 1355 <212> DNA <213> Hydrogenovibrio marinus <220> < 221> CDS <222> (270) .. (1352) <400> 1 aaaatcaagg gtgtgtttaa gtaatgtgag atatgacttg ttattgtaat attcttttta 60 tgctggaata tgctatgatt cattaaagac aaactttaat gtaattcaat gtgcggtcaa 120 aatttcttac cattaagaaa tgagcaaaca ttaatgtaaa aattaataaa atatagtctc 180 aaaatgaaca aaaataaatg tagggttaca cttacgccct ttttagtact taatctattt 240 caagttaaaa aataaaaacg aggattgct atg tca tct caa gtt gaa acg ttc 293 Met Ser Ser Gln Val Glu Thr Phe 1 5 tat gaa gtc atg cgg cgc caa gga att acg cgt cgc agc ttt ttg aaa 341 Tyr Glu Val Met Arg Arg Gln Gly Ile Thr Arg Arg Ser Phe Lys 10 15 20 tac tgt agc ctt act gcg gct gca tta ggg ctt agt cct gct tat gcg 389 Tyr Cys Ser Leu Thr Ala Ala Ala Leu Gly Leu Ser Pro Ala Tyr Ala 25 30 35 40 aac aaa att gct cat gcg atg gaa aca aag cct cgc acg cct gtt atc 437 Asn Lys Ile Ala His Ala Met Glu Thr Lys Pro Arg Thr Pro Val Ile 45 50 55 tgg ttg cat ggt ttg gaa tgt acg tgt tgt tca gaa tcc ttt att cgg 485 Trp Leu His Gly Leu Glu Cys Thr Cys Cys Ser Glu Ser Phe Ile Arg 60 65 70 tct gct cac cca cta gca aaa gac gta gtg tta tcc atg atc tct ctg 533 Ser Ala His Pro Leu Ala Lys Asp Val Val Leu Ser Met Ile Ser Leu 75 80 85 gat tat gac gac acc ctc atg gca gcc tct gga cat gca gcg gaa gcg 581 Asp Tyr Asp Asp Thr Leu Met Ala Ala Ser Gly His Ala Ala Glu Ala 90 95 100 att ctt gat gaa att aaa gaa aaa tat aaa gga aac tat att ctt gcg 629 Ile Leu Asp Glu Ile Lys Glu Lys Tyr Lys Gly Asn Tyr Ile Leu Ala 105 110 115 120 gta gaa ggg aac cct ccg ctt aat cag gat gga atg tcc tgc att atc 677 Val Glu Gly Asn Pro Pro Leu Asn Gln Asp Gly Met Ser Cys Ile Ile 125 130 135 ggt ggc cgc cct ttt tcc gaa caa tta cgc atg gcc gat gat gca 725 Gly Gly Arg Pro Phe Ser Glu Gln Leu Lys Arg Met Ala Asp Asp Ala 140 145 150 aaa gct atc att tca tgg ggt tct tgc gcg tca tgg gga tgc gta cag 773 Lys Ala Ile Ile Ser Trp Gly Cys Ala Ser Trp Gly Cys Val Gln 155 160 165 gca gca aaa cca aac cct acg cag gcc acg cca gta cat aaa ttt ctt 821 Ala Ala Lys Pro Asn Pro Thr Gln Ala Thr Pro Val His Lys Phe Leu 170 175 180 ggc ggc ggg tat gac aaa ccg att atc aaa gtg cct ggg tgt cct cca 869 Gly Gly Gly Tyr Asp Lys Pro Ile Ile Lys Val Pro Gly Cys Pro Pro 185 190 195 200 att gct gaa gtc atg act ggc gtc att acc tat atg cta acc ttt gat 917 Ile Ala Glu Val Met Thr Gly Val Ile Thr Tyr Met Leu Thr Phe Asp 205 210 215 cgc atc cct gag ctg gac cga caa ggt cgt cct aaa atg ttc tac tca 965 Arg Ile Pro Glu Leu Asp Arg Gln Gly Arg Pro Lys Met Phe Tyr Ser 220 225 230 caa cgt att cat gat aaa tgc tac cgc cgc cct cac ttt gat gcc ggt 1013 Gln Arg Ile His Asp Lys Cys Tyr Arg Arg Pro His Phe Asp Ala Gly 235 240 245 caa ttt gta gaa gag tgg gat gac gag ggt cgt aaa ggt tac tgt 1061 Gln Phe Val Glu Glu Trp Asp Asp Glu Gly Ala Arg Lys Gly Tyr Cys 250 255 260 tta tac aaa gtt gga tgc aaa ggc cca aca act tat aac gcc tgc tca 1109 Leu Tyr Lys Val Gly Cys Lys Gly Pro Thr Thr Tyr Asn Ala Cys Ser 265 270 275 280 280 acc gtc cgt tgg aat gga gga act tcc ttc ccg att caa tct ggt cat 1157 Thr Val Arg Trp Asn Gly Gly Thr Ser Phe Pro Ile Gln Ser Gly His 285 290 295 295 ggc tgt atc ggc tgt tct gaa gat ggg ttc tgg gac aaa gga tcc ttc 1205 Gly Cys Ile Gly Cys Ser Glu Asp Gly Phe Trp Asp Lys Gly Ser Phe 300 305 310 tac tca cgc gat aca gag atg aac gca ttt ggc atg gca 1253 Tyr Ser Arg Asp Thr Glu Met Asn Ala Phe Gly Ile Glu Ala Thr Ala 315 320 325 gac gat att ggt aaa acc gcc atc ggt gtt gtc ggt gca gca gta gtc 1301 Asp Asp Asp Ile Gly Lys Thr Ala Ile Gly Val Val Gly Ala Ala Val Val 330 335 340 gct cat gca gcg ata agt gct gta aaa gcg gct caa aag aaa gga gat 1349 Ala His Ala Ala Ile Ser Ala Val Lys Ala Ala Gln Lys Lys Gly Asp 345 350 355 360 aaa taa 1355 Lys <210 > 2 <211> 361 <212> PRT <213> Hydrogenovibrio marinus <400> 2 Met Ser Ser Gln Val Glu Thr Phe Tyr Glu Val Met Arg Arg Gln Gly 1 5 10 15 Ile Thr Arg Arg Ser Phe Leu Lys Tyr Cys Ser Leu Thr Ala Ala Al a 20 25 30 Leu Gly Leu Ser Pro Ala Tyr Ala Asn Lys Ile Ala His Ala Met Glu 35 40 45 Thr Lys Pro Arg Thr Pro Val Ile Trp Leu His Gly Leu Glu Cys Thr 50 55 60 Cys Cys Ser Glu Ser Phe Ile Arg Ser Ala His Pro Leu Ala Lys Asp 65 70 75 80 Val Val Leu Ser Met Ile Ser Leu Asp Tyr Asp Asp Thr Leu Met Ala 85 90 95 Ala Ser Gly His Ala Ala Glu Ala Ile Leu Asp Glu Ile Lys Glu Lys 100 105 110 Tyr Lys Gly Asn Tyr Ile Leu Ala Val Glu Gly Asn Pro Pro Leu Asn 115 120 125 Gln Asp Gly Met Ser Cys Ile Ile Gly Gly Arg Pro Phe Ser Glu Gln 130 135 140 Leu Lys Arg Met Ala Asp Asp Ala Lys Ala Ile Ile Ser Trp Gly Ser 145 150 155 160 Cys Ala Ser Trp Gly Cys Val Gln Ala Ala Lys Pro Asn Pro Thr Gln 165 170 175 Ala Thr Pro Val His Lys Phe Leu Gly Gly Gly Gly Tyr Asp Lys Pro Ile 180 185 190 Ile Lys Val Pro Gly Cys Pro Pro Ile Ala Glu Val Met Thr Gly Val 195 200 205 Ile Thr Tyr Met Leu Thr Phe Asp Arg Ile Pro Glu Leu Asp Arg Gln 210 215 220 Gly Arg Pro Lys Met Phe Tyr Ser Gln Arg Ile His Asp Lys Cys Tyr 225 230 235 240 Ar g Arg Pro His Phe Asp Ala Gly Gln Phe Val Glu Glu Trp Asp Asp 245 250 255 Glu Gly Ala Arg Lys Gly Tyr Cys Leu Tyr Lys Val Gly Cys Lys Gly 260 265 270 Pro Thr Thr Tyr Asn Ala Cys Ser Thr Val Arg Trp Asn Gly Gly Thr 275 280 285 Ser Phe Pro Ile Gln Ser Gly His Gly Cys Ile Gly Cys Ser Glu Asp 290 295 300 Gly Phe Trp Asp Lys Gly Ser Phe Tyr Ser Arg Asp Thr Glu Met Asn 305 310 315 320 Ala Phe Gly Ile Glu Ala Thr Ala Asp Asp Ile Gly Lys Thr Ala Ile 325 330 335 Gly Val Val Gly Ala Ala Val Val Ala His Ala Ala Ile Ser Ala Val 340 345 350 Lys Ala Ala Ala Gln Lys Lys Gly Asp Lys 355 360 <210> 3 < 211> 1373 <212> DNA <213> Hydrogenovibrio marinus <220> <221> CDS <222> (3) .. (1373) <400> 3 ta atg agc gta tta aac aca cca aac cac tat aag atg gac aac tcc 47 Met Ser Val Leu Asn Thr Pro Asn His Tyr Lys Met Asp Asn Ser 1 5 10 15 ggt cgt cga gta gtc att gat cct gtt act cgt atc gaa ggc cac atg 95 Gly Arg Arg Val Val Ile Asp Pro Val Thr Arg Ile Glu Gly His Met 20 25 30 cgt tgt gaa gtc aac gtt gat gaa aac aat gtc atc caa aat gcc gta 143 Arg Cys Glu Val Asn Val Asp Glu Asn Asn Val Ile Gln Asn Ala Val 35 40 45 tcc aca ggc act atg tgg cga ggc ctt gag gtt att ctt cgc gga cgc 191 Ser Thr Gly Thr Met Trp Arg Gly Leu Glu Val Ile Leu Arg Gly Arg 50 55 60 gac cct cgt gac gca tgg gca ttt gta gaa cgc ata tgt ggt gtc tgc 239 Asp Pro Arg Asp Ala Trp Ala Phe Val Glu Arg Ile Cys Gly Val Cys 65 70 75 act ggc tgc cat gct ttg gca tca gtt cga gcg gtt gag gat gct cta 287 Thr Gly Cys His Ala Leu Ala Ser Val Arg Ala Val Glu Asp Ala Leu 80 85 90 95 gat atc aaa att ccg cac aat gcc act tta att cgc gaa att atg gcc 335 Asp Ile Lys Ile Pro His Asn Ala Thr Leu Ile Arg Glu Ile Met Ala 100 105 110 aaa aca ctt caa att cat gac cat att gtg cat ttc tac cat tta cat 383 Lys Thr Leu Gln Ile His Asp His Ile Val His Phe Tyr His Leu His 115 120 125 gct cta gac tgg gtc aat cca gta aat gct ttg aag gca gat cct cag 431 Ala Leu Asp Trp Val Asn Pro Val Asn Ala Leu Lys Ala Asp Pro Gln 130 135 140 gca acc tct gaa cta caa aaa cta gtc tca ccg cat cac cca atg tct 479 Ala Thr Ser Glu Leu Gln Lys Leu Val Ser Pro His His Pro Met Ser 145 150 155 agc cct ggc tac ttc aaa gac att caa atc aga atc caa aag ttc gta 527 Ser Pro Gly Tyr Phe Lys Asp Ile Gln Ile Arg Ile Gln Lys Phe Val 160 165 170 175 gat tct ggg caa ctc gga atc ttc aag aat ggt tat tgg agt aac ccc 575 Asp Ser Gly Gln Leu Gly Ile Phe Lys Asn Gly Tyr Trp Ser Asn Pro 180 185 190 gcc tat aaa tta tct ccc gaa gca gat ctt atg gct gtt acc cac tat 623 Ala Tyr Lys Leu Ser Pro Glu Ala Asp Leu Met Ala Val Thr His Tyr 195 200 205 ctt gaa gct ctg gac ttc caa aaa gaa atc gtc aaa atc cat gc ata 671 Leu Glu Ala Leu Asp Phe Gln Lys Glu Ile Val Lys Ile His Ala Ile 210 215 220 ttc ggt ggt aaa aac cct cac cct aac tat atg gtt gga ggt gtg cct 719 Phe Gly Gly Lys Asn Pro His Pro Asn Tyr Met Val Gly Gly Val Pro 225 230 235 tgt gca ata aac atc gat gga gat atg gcc gcg ggt gcc cct ata aac 767 Cys Ala Ile Asn Ile Asp Gly Asp Met Ala Ala Gly Ala Pro Ile Asn 240 245 250 255 atg gag cgt tta aac ttt gtcaaa tca ctt ata gaa caa ggg cga acc 815 Met Glu Arg Leu Asn Phe Val Lys Ser Leu Ile Glu Gln Gly Arg Thr 260 265 270 ttt aac acc aat gtc tat gta ccc gac gtt ata gct atc gca gct ttc 863 Phe Asn Thr Asn Val Tyr Val Pro Asp Val Ile Ala Ile Ala Ala Phe 275 280 285 tat cgc gac tgg cta tat ggt gga ggg cta tcc gcg aca aac gtt atg 911 Tyr Arg Asp Trp Leu Tyr Gly Gly Gly Leu Ser Ala Thr Asn Val Met 290 295 300 gat tat ggc gca tac cct aaa act cca tat gat aaa tct aca gat caa 959 Asp Tyr Gly Ala Tyr Pro Lys Thr Pro Tyr Asp Lys Ser Thr Asp Gln 305 310 315 cta cct gga ggt gca atc att aat gga gat tgg ggg aaa att cat cca 1007 Leu Pro Gly Gly Ala Ile Ile Asn Gly Asp Trp Gly Lys Ile His Pro 320 325 330 335 gtt gac cct aga gat cct gag cag gtt caa gaa ttt gtc act cat tct 1055 Val Asp Pro Arg Asp Pro Glu Gln Val Gln Glu Phe Val Thr His Ser 340 345 350 tgg tac aaa tac cca gac gaa aca aaa gga ctt cat cca tgg gat ggt 1103 Trp Tyr Lys Tyr Pro Asp Glu Thr Lys Gly Leu His Pro Trp Asp Gly 355 360 365 att aca gag c ca aac tat gaa cta gga tct aaa aca aaa ggg tcg aga 1151 Ile Thr Glu Pro Asn Tyr Glu Leu Gly Ser Lys Thr Lys Gly Ser Arg 370 375 380 aca aac atc atc gaa atc gac gaa tct gca aaa tat tcc tgg atc aaa 1199 Thr Asn Ile Ile Glu Ile Asp Glu Ser Ala Lys Tyr Ser Trp Ile Lys 385 390 395 tcc ccg cgc tgg agg ggg cat gct gtc gaa gta ggg cca ctt gca cgc 1247 Ser Pro Arg Trp Arg Gly His Ala Val Glu Val Gly Pro Leu Ala Arg 400 405 410 415 tat ata ctt gcc tat gcc caa ggc gtt gaa tac gtt aag act cag gtt 1295 Tyr Ile Leu Ala Tyr Ala Gln Gly Val Glu Tyr Val Lys Thr Gln Val 420 425 430 cac aca agc cta aat agg ttt aac gct gta tgt cgt ttg cta gac cca 1343 His Thr Ser Leu Asn Arg Phe Asn Ala Val Cys Arg Leu Leu Asp Pro 435 440 445 aac cat aaa gac atc acc gac ctg aaa gct 1373 Asn His Lys Asp Ile Thr Asp Leu Lys Ala 450 455 <210> 4 <211> 457 <212> PRT <213> Hydrogenovibrio marinus <400> 4 Met Ser Val Leu Asn Thr Pro Asn His Tyr Lys Met Asp Asn Ser Gly 1 5 10 15 Arg Arg Val Val Ile Asp Pro Val Thr Arg Ile Glu Gly His Met Arg 20 25 30 Cys Glu Val Asn Val Asp Glu Asn Asn Val Ile Gln Asn Ala Val Ser 35 40 45 Thr Gly Thr Met Trp Arg Gly Leu Glu Val Ile Leu Arg Gly Arg Asp 50 55 60 Pro Arg Asp Ala Trp Ala Phe Val Glu Arg Ile Cys Gly Val Cys Thr 65 70 75 80 Gly Cys His Ala Leu Ala Ser Val Arg Ala Val Glu Asp Ala Leu Asp 85 90 95 Ile Lys Ile Pro His Asn Ala Thr Leu Ile Arg Glu Ile Met Ala Lys 100 105 110 Thr Leu Gln Ile His Asp His Ile Val His Phe Tyr His Leu His Ala 115 120 125 Leu Asp Trp Val Asn Pro Val Asn Ala Leu Lys Ala Asp Pro Gln Ala 130 135 140 Thr Ser Glu Leu Gln Lys Leu Val Ser Pro His His Pro Met Ser Ser 145 150 155 160 Pro Gly Tyr Phe Lys Asp Ile Gln Ile Arg Ile Gln Lys Phe Val Asp 165 170 175 Ser Gly Gln Leu Gly Ile Phe Lys Asn Gly Tyr Trp Ser Asn Pro Ala 180 185 190 Tyr Lys Leu Ser Pro Glu Ala Asp Leu Met Ala Val Thr His Tyr Leu 195 200 205 Glu Ala Leu Asp Phe Gln Lys Glu Ile Val Lys Ile His Ala Ile Phe 210 215 220 Gly Gly Lys Asn Pro His Pro Asn Tyr Met Val Gly Gly Val Pro Cys 225 230 235 240 Ala Ile Asn Ile Asp Gly Asp Met Ala Ala Gly Ala Pro Ile Asn Met 245 250 255 Glu Arg Leu Asn Phe Val Lys Ser Leu Ile Glu Gln Gly Arg Thr Phe 260 265 270 Asn Thr Asn Val Tyr Val Pro Asp Val Ile Ala Ile Ala Ala Phe Tyr 275 280 285 arg Arg Asp Trp Leu Tyr Gly Gly Gly Leu Ser Ala Thr Asn Val Met Asp 290 295 300 Tyr Gly Ala Tyr Pro Lys Thr Pro Tyr Asp Lys Ser Thr Asp Gln Leu 305 310 315 320 Pro Gly Gly Ala Ile Ile Asn Gly Asp Trp Gly Lys Ile His Pro Val 325 330 335 Asp Pro Arg Asp Pro Glu Gln Val Gln Glu Phe Val Thr His Ser Trp 340 345 350 Tyr Lys Tyr Pro Asp Glu Thr Lys Gly Leu His Pro Trp Asp Gly Ile 355 360 365 Thr Glu Pro Asn Tyr Glu Leu Gly Ser Lys Thr Lys Gly Ser Arg Thr 370 375 380 Asn Ile Ile Glu Ile Asp Glu Ser Ala Lys Tyr Ser Trp Ile Lys Ser 385 390 395 395 400 Pro Arg Trp Arg Gly His Ala Val Glu Val Gly Pro Leu Ala Arg Tyr 405 410 415 Ile Leu Ala Tyr Ala Gln Gly Val Glu Tyr Val Lys Thr Gln Val His 420 425 430 Thr Ser Leu Asn Arg Phe Asn Ala Val Cys Arg Leu Leu Asp Pro Asn 435 44 0 445 His Lys Asp Ile Thr Asp Leu Lys Ala 450 455 <210> 5 <211> 2728 <212> DNA <213> Hydrogenovibrio marinus <220> <221> CDS <222> (270) .. (1352) <220 > <221> CDS <222> (1358) .. (2728) <400> 5 aaaatcaagg gtgtgtttaa gtaatgtgag atatgacttg ttattgtaat attcttttta 60 tgctggaata tgctatgatt cattaaagac aaactttaat gtaattcaat gtgcggtcaa 120 aatttcttac cattaagaaa tgagcaaaca ttaatgtaaa aattaataaa atatagtctc 180 aaaatgaaca aaaataaatg tagggttaca cttacgccct ttttagtact taatctattt 240 caagttaaaa aataaaaacg aggattgct atg tca tct caa gtt gaa acg ttc 293 Met Ser Ser Gln Val Glu Thr Phe 1 5 tat gaa gtc atg cgg cgc caa gga att acg cgt cgc agc ttt ttg aaa 341 Tyr Glu Val Met Arg Arg Gln Gly Ile Thr Phe Leu Lys 10 15 20 tac tgt agc ctt act gcg gct gca tta ggg ctt agt cct gct tat gcg 389 Tyr Cys Ser Leu Thr Ala Ala Ala Ala Leu Gly Leu Ser Pro Ala Tyr Ala 25 30 35 40 aac aaa att gct cat gcg atg gaa aca aag cct cgc acg cct gtt atc 437 Asn Lys Ile Ala His Ala Met Glu Thr Lys Pro Arg Thr Pro Val Ile 45 50 55 tgg ttg cat ggt ttg gaa tgt acg tgt tgt tca gaa tcc ttt att cgg 485 Trp Leu His Gly Leu Glu Cys Thr Cys Cys Ser Glu Ser Phe Ile Arg 60 65 70 tct gct cac cca cta gca aaa gac gta gtg tta tcc atg atc tct ctg 533 Ser Ala His Pro Leu Ala Lys Asp Val Val Leu Ser Met Ile Ser Leu 75 80 85 gat tat gac gac acc ctc atg gca gcc tct gga cat gca gcg gaa gcg 581 Asp Tyr Asp Asp Thr Leu Met Ala Ala Ser Gly His Ala Ala Glu Ala 90 95 100 att ctt gat gaa att aaa gaa aaa tat aaa gga aac tat att ctt gcg 629 Ile Leu Asp Glu Ile Lys Glu Lys Tyr Lys Gly Asn Tyr Ile Leu Ala 105 110 115 120 gta gaa ggg aac cct ccg ctt aat cag gat gga atg tcc tgc att atc 677 Val Glu Gly Asn Pro Pro Leu Asn Gln Asp Gly Met Ser Cys Ile Ile 125 130 135 ggt ggc cgc cct ttt tcc gaa caa tta aaa cgc atg gcc gat gat gca 725 Gly Arg Pro Phe Ser Glu Gln Leu Lys Arg Met Ala Asp Asp Ala 140 145 150 aaa gct atc att tca tgg ggt tct tgc gcg tca tgg gga tgc gta cag 773 Lys Ala Ile Ile Ser Trp Gly Ser Cys Ala Ser Trp Gly Cys Val Gln 155 160 165 gca gca aaa cca aac cct acg cag gcc acg cca gta cat aaa ttt ctt 821 Ala Ala Lys Pro Asn Pro Thr Gln Ala Thr Pro Val His Lys Phe Leu 170 175 180 ggc ggc ggg tat gac aaa ccg att atc aaa gtg cct ggg tgt cct cca 869 Gly Gly Gly Tyr Asp Lys Pro Ile Ile Lys Val Pro Gly Cys Pro Pro 185 190 195 200 att gct gaa gtc atg act ggc gtc att acc tat atg cta acc ttt gat 917 Ile Ala Glu Val Met Thr Gly Val Ile Thr Tyr Met Leu Thr Phe Asp 205 210 215 cgc atc cct gag ctg gac cga caa ggt cgt cct aaa atg ttc tac tca 965 Arg Ile Pro Glu Leu Asp Arg Gln Gly Arg Pro Lys Met Phe Tyr Ser 220 225 230 caa cgt att cat gat aaa tgc tac cgc cgc cct cac ttt gat gcc ggt 1013 Gln Arg Ile His Asp Lys Cys Tyr Arg Arg Pro His Phe Asp Ala Gly 235 240 245 caa ttt gta gaa gag tgg gat gac gaa ggt gct cgt aaa ggt tac t Phe Val Glu Glu Trp Asp Asp Glu Gly Ala Arg Lys Gly Tyr Cys 250 255 260 tta tac aaa gtt gga tgc aaa ggc cca aca act tat aac gcc tgc tca 1109 Leu Tyr Lys Val Gly Cys Lys Gly Pro Thr Thr Tyr Asn Ala C ys Ser 265 270 275 280 acc gtc cgt tgg aat gga gga act tcc ttc ccg att caa tct ggt cat 1157 Thr Val Arg Trp Asn Gly Gly Thr Ser Phe Pro Ile Gln Ser Gly His 285 290 295 ggc tgt atc ggc tgt tct gaa gat ggg ttc tgg gac aaa gga tcc ttc 1205 Gly Cys Ile Gly Cys Ser Glu Asp Gly Phe Trp Asp Lys Gly Ser Phe 300 305 310 tac tca cgc gat aca gag atg aac gca ttt ggc att gaa gca acg gca 1253 Tyr Ser Arg Asp Glu Met Asn Ala Phe Gly Ile Glu Ala Thr Ala 315 320 325 gac gat att ggt aaa acc gcc atc ggt gtt gtc ggt gca gca gta gtc 1301 Asp Asp Ile Gly Lys Thr Ala Ile Gly Val Val Gly Ala Ala Val Val 330 335 340 gct cat gca gcg ata agt gct gta aaa gcg gct caa aag aaa gga gat 1349 Ala His Ala Ala Ile Ser Ala Val Lys Ala Ala Gln Lys Lys Gly Asp 345 350 355 360 aaa taata atg agc gta tta aac aca cca aac cac tat aag atg gac aac 1399 Lys Met Ser Val Leu Asn Thr Pro Asn His Tyr Lys Met Asp Asn 365 370 375 tcc ggt cgt cga gta gtc att gat cct gtt act cgt atc gaa ggc cac 1447 Ser Gly Arg Arg Val Val Ile Asp Pro Val Thr Arg Ile Glu Gly His 380 385 390 atg cgt tgt gaa gtc aac gtt gat gaa aac aat gtc atc caa aat gcc 1495 Met Arg Cys Glu Val Asn Val Asp Glu Asn Asn Val Ile Gln Asn Ala 395 400 405 gta tcc aca gg act atg tgg cga ggc ctt gag gtt att ctt cgc gga 1543 Val Ser Thr Gly Thr Met Trp Arg Gly Leu Glu Val Ile Leu Arg Gly 410 415 420 cgc gac cct cgt gac gca tgg gca ttt gta gaa cgc ata tgt ggt gtc 1591 Ar Asp Pro Arg Asp Ala Trp Ala Phe Val Glu Arg Ile Cys Gly Val 425 430 435 tgc act ggc tgc cat gct ttg gca tca gtt cga gcg gtt gag gat gct 1639 Cys Thr Gly Cys His Ala Leu Ala Ser Val Arg Ala Val Glu Asp Ala 440 445 450 455 cta gat atc aaa att ccg cac aat gcc act tta att cgc gaa att atg 1687 Leu Asp Ile Lys Ile Pro His Asn Ala Thr Leu Ile Arg Glu Ile Met 460 465 470 gcc aaa aca ctt caa att cat gac cat att gtg cat ttc tac cat tta 1735 Ala Lys Thr Leu Gln Ile His Asp His Ile Val His Phe Tyr His Leu 475 480 485 cat gct cta gac tgg gtc aat cca gta aat gct ttg aag gca gat cct 1783 His Ala Leu Asp T rp Val Asn Pro Val Asn Ala Leu Lys Ala Asp Pro 490 495 500 cag gca acc tct gaa cta caa aaa cta gtc tca ccg cat cac cca atg 1831 Gln Ala Thr Ser Glu Leu Gln Lys Leu Val Ser Pro His His Pro Met 505 510 515 tct agc cct ggc tac ttc aaa gac att caa atc aga atc caa aag ttc 1879 Ser Ser Pro Gly Tyr Phe Lys Asp Ile Gln Ile Arg Ile Gln Lys Phe 520 525 530 535 gta gat tct ggg caa ctc gga atc gtc tat tgg agt aac 1927 Val Asp Ser Gly Gln Leu Gly Ile Phe Lys Asn Gly Tyr Trp Ser Asn 540 545 550 ccc gcc tat aaa tta tct ccc gaa gca gat ctt atg gct gtt acc cac 1975 Pro Ala Tyr Lys Leu Ser Pro Glu Ala Asp Leu Met Ala Val Thr His 555 560 565 tat tat ctt gaa gct ctg gac ttc caa aaa gaa atc gtc aaa atc cat gcg 2023 Tyr Leu Glu Ala Leu Asp Phe Gln Lys Glu Ile Val Lys Ile His Ala 570 575 gt gttc aaa aac cct cac cct aac tat atg gtt gga ggt gtg 2071 Ile Phe Gly Gly Lys Asn Pro His Pro Asn Tyr Met Val Gly Gly Val 585 590 595 cct tgt gca ata aac atc gat gga gat atg gcc gcg ggt gcc cct ata 211 9 Pro Cys Ala Ile Asn Ile Asp Gly Asp Met Ala Ala Gly Ala Pro Ile 600 605 610 615 aac atg gag cgt tta aac ttt gtc aaa tca ctt ata gaa caa ggg cga 2167 Asn Met Glu Arg Leu Asn Phe Val Lys Ser Leu Ile Glu Gln Gly Arg 620 625 630 acc ttt aac acc aat gtc tat gta ccc gac gtt ata gct atc gca gct 2215 Thr Phe Asn Thr Asn Val Tyr Val Pro Asp Val Ile Ala Ile Ala Ala 635 640 645 ttc tat cgc gac tgg cta tat ggt gga ggg cta tcc gcg aca aac gtt 2263 Phe Tyr Arg Asp Trp Leu Tyr Gly Gly Gly Leu Ser Ala Thr Asn Val 650 655 660 atg gat tat ggc gca tac cct aaa act cca tat gat aaa tct aca gat 2311 Met Asp Tyr Gly Ala Tyr Pro Lys Thr Pro Tyr Asp Lys Ser Thr Asp 665 670 675 caa cta cct gga ggt gca atc att aat gga gat tgg ggg aaa att cat 2359 Gln Leu Pro Gly Gly Ala Ile Ile Asn Gly Asp Trp Gly Lys Ile His 680 685 690 695 cca gtt gac cct aga gat cct gag cag gtt caa gaa ttt gtc act cat 2407 Pro Val Asp Pro Arg Asp Pro Glu Gln Val Gln Glu Phe Val Thr His 700 705 710 tct tgg tac aaa tac cca gac gaa aca aaa gga ctt cat cca tgg gat 2455 Ser Trp Tyr Lys Tyr Pro Asp Glu Thr Lys Gly Leu His Pro Trp Asp 715 720 725 ggt att aca gag cca aac tat gaa cta gga tct aaa aca aaa ggg tcg 2503 Gly Ile Thr Glu Pro Asn Tyr Glu Leu Gly Ser Lys Thr Lys Gly Ser 730 735 740 aga aca aac atc atc gaa atc gac gaa tct gca aaa tat tcc tgg atc 2551 Arg Thr Asn Ile Ile Glu Ile Asp Glu Ser Ala Lys Tyr Ser Trp Ile 745 750 755 755 taaa ccg cgc tgg agg ggg cat gct gtc gaa gta ggg cca ctt gca 2599 Lys Ser Pro Arg Trp Arg Gly His Ala Val Glu Val Gly Pro Leu Ala 760 765 770 775 cgc tat ata ctt gcc tat gcc caa ggc gtt gaa tac gtt aag act ca 2647 Arg Tyr Ile Leu Ala Tyr Ala Gln Gly Val Glu Tyr Val Lys Thr Gln 780 785 790 gtt cac aca agc cta aat agg ttt aac gct gta tgt cgt ttg cta gac 2695 Val His Thr Ser Leu Asn Arg Phe Asn Ala Val Cys Arg Leu Leu Asp 795 800 805 cca aac cat aaa gac atc acc gac ctg aaa gct 2728 Pro Asn His Lys Asp Ile Thr Asp Leu Lys Ala 810 815 <210> 6 <211> 11 <212> PRT <213> Hydrogenovibrio marinus <400 > 6 Met Glu Thr Lys Pro Arg Thr Pro Val Ile Trp 1 5 10 <210> 7 <211> 11 <212> PRT <213> Hydrogenovibrio marinus <400> 7 Met Asp Asn Ser Gly Arg Arg Val Val Ile Asp 1 5 10 <210 > 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based on the N terminal sequence of small subunit of hydrogenase. <400> 8 atggaaacta arccwcgtac dccwgtwath tgg 33 <210> 9 <211 > <212> DNA <213> Artificial Sequence <220> 33 <223> Designed oligonucleotide based on the N terminal sequence of large subunit of hydrogenase. <400> 9 rtcgatwacw acacgacgac cwgarttrtc cat 33 <210> 10 <211> 25 <212 > DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based on 5 'terminal sequence of small subunit gene of hydrogenase. <400> 10 atgtcatctc aagttgaaac gttct 25 <210> 11 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> Designed oligonucleotide based on 3 'terminal sequence of small subunit gene of hydrogenase. <400> 11 tttatctcct ttcttttgag ccgct 25 <210> 12 <211> 300 <212> DNA <213> Hydrogenovibrio marinus <400> 12 gaaacaaaag gacttcatcc atgggatggt attacagagc caaactatga actaggatct 60 aaaacaaaag ggtcgagaac aaacatcatc gaaatcgacg aatctgcaaa atattcctgg 120 atcaaatccc cgcgctggag ggggcatgct gtcgaagtag ggccacttgc acgctatata 180 cttgcctatg cccaaggcgt tgaatacgtt aagactcagg ttcacacaag cctaaatagg 240 tttaacgctg tatgtcgttt gctagaccca aaccataaag acatcaccga cctgaaagct 300 <210> 13 <211 > 1121 <212> DNA <213> Hydrogenovibrio marinus <400> 13 atgtcatctc aagttgaaac gttctatgaa gtcatgcggc gccaaggaat tacgcgtcgc 60 agctttttga aatactgtag ccttactgcg gctgcattag ggcttagtcc tgcttatgcg 120 aacaaaattg ctcatgcgat ggaaacaaag cctcgcacgc ctgttatctg gttgcatggt 180 ttggaatgta cgtgttgttc agaatccttt attcggtctg ctcacccact agcaaaagac 240 gtagtgttat ccatgatctc tctggattat gacgacaccc tcatggcagc ctctggacat 300 gcagcggaag cgattcttga tgaaattaaa gaaaaatata aaggaaacta tattcttgcg 360 gtagaaggga accctccgct taatcaggat ggaatgtcct gcattatcgg tggccgccct 420 ttttccgaac aattaaaacg catggccgat gatgcaaaag ctatcattt c atggggttct 480 tgcgcgtcat ggggatgcgt acaggcagca aaaccaaacc ctacgcaggc cacgccagta 540 cataaatttc ttggcggcgg gtatgacaaa ccgattatca aagtgcctgg gtgtcctcca 600 attgctgaag tcatgactgg cgtcattacc tatatgctaa cctttgatcg catccctgag 660 ctggaccgac aaggtcgtcc taaaatgttc tactcacaac gtattcatga taaatgctac 720 cgccgccctc actttgatgc cggtcaattt gtagaagagt gggatgacga aggtgctcgt 780 aaaggttact gtttatacaa agttggatgc aaaggcccaa caacttataa cgcctgctca 840 accgtccgtt ggaatggagg aacttccttc ccgattcaat ctggtcatgg ctgtatcggc 900 tgttctgaag atgggttctg ggacaaagga tccttctact cacgcgatac agagatgaac 960 gcatttggca ttgaagcaac ggcagacgat attggtaaaa ccgccatcag tgttgtcggatagag tctgccatagag gag tctgccatagag tctgccatagag gag tagctcagag tctgccatagag agg

[0077]

[Sequence List Free Text] SEQ ID NO: 8: oligonucleotide designed based on N-terminal sequence of small subunit of hydrogenase SEQ ID NO: 9: Oligonucleotide designed based on N-terminal sequence of large subunit of hydrogenase SEQ ID NO: 10: Oligonucleotide designed based on 5 'terminal sequence of hydrogenase small subunit gene SEQ ID NO: 11: Oligonucleotide designed based on 3' terminal sequence of hydrogenase small subunit gene

[Brief description of the drawings]

FIG. 1 is a diagram showing the mechanism of hydrogen generation by algae, photosynthetic bacteria, and anaerobic bacteria.

FIG. 2 is a diagram showing the degree of oxygen tolerance of hydrogenase derived from Hydrogenobrio marinus and Alcaligenes eutrophus.

FIG. 3 is a graph showing the degree of thermostability of a hydrogenase derived from Hydrogenobrio marinus.

FIG. 4 is a diagram showing the structure of a hydrogenase gene derived from Hydrogenobrio marinus and the positions of primers used for amplifying the gene.

FIG. 5 is a view showing an amino acid sequence alignment of a large subunit of a hydrogenase derived from various microorganisms.

FIG. 6 shows an amino acid sequence alignment of small subunits of hydrogenases derived from various microorganisms.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/02 C12N 5/00 A // (C12N 15/09 ZNA C12R 1:01) (72) Inventor Hiroshi Nishihara 6-23-4 Minami, Ushiku-shi, Ibaraki F-term (reference) 4B024 AA03 BA08 CA03 CA04 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL05 4B065 AA01Y AA26X AB01 AC14 BA02 BA22 CA28

Claims (8)

[Claims] 1. A protein of the following (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 (b) at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 Having a modified amino acid sequence and having hydrogenase activity 2. A hydrogenase gene encoding the following protein (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 (b) at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 4 Having a modified amino acid sequence and having hydrogenase activity 3. A gene comprising the following DNA (a) or (b): (a) DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3 (b) Hybridizing with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3 under stringent conditions DNA encoding soybean and protein having hydrogenase activity 4. A recombinant vector containing the gene according to claim 2 or 3. A transformant comprising the recombinant vector according to claim 4. 6. A method for producing a hydrogenase protein, comprising culturing the transformant according to claim 5, and collecting a hydrogenase protein from the resulting culture. 7. The following steps: (a) comparing the amino acid sequence of a thermostable and / or oxygen-resistant hydrogenase with the amino acid sequence of a non-thermostable and / or non-oxygen-resistant hydrogenase; (b) step (a) Specifying the amino acid residue specifically found in the thermostable and / or oxygen-resistant hydrogenase based on the comparison result obtained in (c), among the amino acid sequences of the non-thermostable and / or non-oxygen-resistant hydrogenase An amino acid residue at a position corresponding to the amino acid residue specified in step (b),
Substituting the amino acid residue specifically found,
A method for producing a thermostable and / or oxygen-resistant hydrogenase, comprising: 8. The position corresponding to the amino acid residue is at position 56 or 110 of the amino acid sequence represented by SEQ ID NO: 2, or at position 21 corresponding to the amino acid sequence represented by SEQ ID NO: 4.
Th, 39th, 116th, 169th, 171st,
213st, 254th, 282nd, 326th, 333th
The manufacturing method according to claim 7, wherein the method is at least one selected from the group consisting of a 349th, a 356th, and a 368th.