KR20110000776A - Method for producing hydrogen using recombinant microorganism - Google Patents

Abstract

PURPOSE: A method for producing hydrogen using microorganism which is transformed with (nickel-iron)-hydrogenase gene is provided to enhance efficiency of biological hydrogenation and to apply as an electrocatalyst. CONSTITUTION: A (nickel-iron)-hydrogenase protein which is derived from Hydrogenovibrio marinus has an amino acid sequence of sequence number 2. A gene encoding the (nickel-iron)-hydrogenase protein has a base sequence of sequence number 1. A recombinant vector has the gene. A method for producing hydrogen from microorganism comprises: a step of transforming the microorganism with the recombinant vector; and a step of culturing the transformed microorganism in a culture medium containing Ni and Fe under the microaerobic condition. The microorganism is E.coli.

Description

Hydrogen production method using recombinant microorganism {Method for Producing Hydrogen Using Recombinant Microorganism}

The present invention relates to a method for producing hydrogen using recombinant microorganisms, and more specifically, Hydrogennovibrio marinus ) [Nickel-Iron] -hydrogenase protein derived from a strain, a gene encoding the protein, a recombinant vector comprising the gene, a microorganism transformed with the recombinant vector, and a hydrogen-producing hydrogen using the transformed microorganism. It is about a method.

At present, attention is focused on hydrogen as an energy source to overcome environmental problems and energy crisis caused by fossil fuels. Since hydrogen is produced from the constituents of water and organic substances, there is little concern about depletion of raw materials, clean energy generated only per unit weight, and there is a great possibility of practical use of technologies such as fuel cells. Photosynthetic microbes that use sunlight as energy are active environmental treatment technologies that not only generate hydrogen but also reduce carbon dioxide in the air. Research into hydrogen evolution by microorganisms has already begun since the late 1800s, and basic research is being carried out using algae and bacteria. This biological hydrogen production has many advantages. It is environmentally friendly, can be recycled and used for food waste and sewage sludge treatment.

Common algae receive light to fix carbon dioxide in the air by photosynthesis, and simultaneously decompose water to generate oxygen and hydrogen at the same time. The reason algae can generate hydrogen is because they have hydrogenase (hydrogenase) that helps produce hydrogen. Hydrogenases play a major role in biological hydrogen production. Hydrogenases catalyze the reversible oxidation of hydrogen molecules and play an important role in the energy metabolism of microorganisms. However, there are many challenges that must be solved with the current technology for commercialization. Among them, anhydrous conditions are essential for hydrogen production because the hydrogenase involved in the reaction is very sensitive to oxygen generated at the same time and inhibits hydrogen generation, which is an inefficient process. Therefore, in order to increase the efficiency of hydrogen production, many researchers are interested in hydrogenase and carry out improvement studies. For this purpose, it is necessary to develop a technology for detecting and searching for hydrogenases.

Hydrogenases are [iron] -hydrogenases ([Fe] -hydrogenases), [nickel-iron] -hydrogenases, and metal ions, depending on the composition of the hydrogen-activating site. -free hydrogenase). Among these, [nickel-iron] -hydrogenase has a merit of having high affinity for a substrate even though its activity is lower than that of [iron] -hydrogenase. Nevertheless, [nickel-iron] -hydrogenase is little known compared to [iron] -hydrogenase.

The present inventors recognize the usefulness of hydrogen energy, isolate the hydrogenase gene from H. marinus using Degenerate PCR, and express it in the form of recombinant protein in E. coli to produce hydrogen in vivo. To develop a recombinant strain.

In order to solve the above problems, the present invention is Hydrogenovibrio marinus [Nickel-iron] -hydrogenase protein from strains is provided.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a microorganism transformed with the recombinant vector.

The present invention also provides a method for producing hydrogen using the transformed microorganism.

According to the present invention, the hydrogenase obtained by the present invention may increase the efficiency of biological hydrogen production. It will also be applied as an electrocatalyst in the development of biofuel cells and biosensors. Instead of the chemical reactions commonly used in current fuel cells, the use of hydrases could lead to simpler processes and clean fuel cells without contamination. In addition, it may be used in biocorrosion induced by anaerobic microorganisms.

In order to achieve the object of the present invention, the present invention consists of an amino acid sequence of SEQ ID NO: 2, Hydrogennovibrio ( Hydrogenovibrio marinus ) [Nickel-iron] -hydrogenase protein from strains is provided.

[Nickel-iron] -hydrogenase has a lower affinity than the [iron] -hydrogenase, but has a high affinity for the substrate and has a three-dimensional structure including nickel as well as iron. [Nickel-iron] -hydrogenases consist of two subunits: a large subunit and a small subunit. In general, large subunits mainly contain conserved sequences of about 1800 bp in size, and small subunits mainly of variable sequences of about 1100 bp in size. Among these small subunits, there is a secretion signal sequence that signals the position between the outer membrane and the inner membrane, ie periplasm, and the small subunit and the large subunit bind before secretion occurs. The bound [nickel-iron] -hydrogenase is transported to the membrane via the twin-arginine translocation pathway.

The range of [nickel-iron] -hydrogenase protein according to the present invention is Hydrogennovibrio marinus ) Proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from strains and functional equivalents of such proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means having activity of [nickel-iron] -hydrogenase.

The present invention also provides a gene encoding the [nickel-iron] -hydrogenase protein of the present invention. The present inventors align the conserved segment sequence and secretion sequence of the carboxyl terminus of a large subunit among known genes of [nickel-iron] -hydrogenase and then prepare a (nickel-iron) -hydrogenase amplification primer. Hydrogennovibrio using Hydrogenovibrio marinus ) About 2900 bp [nickel-iron] -hydrogenase gene was isolated from the strain. The base sequence of the isolated gene was determined, and the determined base sequence is shown in SEQ ID NO: 1. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, of the present invention The gene may comprise a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1.

The "% sequence homology" for a polynucleotide is determined by comparing the two optimally arranged sequences with the comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

In addition, the present invention provides the [nickel-iron] -hydrogenase of the present invention. A recombinant vector comprising a gene is provided.

Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9 (Qiagen); pBS vector, PHAGESCRIPT vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia; pRSET-B from Invitrogen Inc .; And pET21b (Novagen). Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene); And pSVK3, pBPV, pMSG and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art. In the present invention, one of the E. coli expression vector pET21b vector was used.

The recombinant vector will preferably comprise one or more selectable markers. Such markers include dihydrofolate reductase, neomycin resistance, and the like for eukaryotic cell culture, and include tetracycline, ampicillin resistance genes, etc., for culture in E. coli and other bacteria.

Preferably, the [nickel-iron] -hydrogenase of the present invention The recombinant vector including the gene may be the recombinant vector described in FIG. 5, but is not limited thereto.

The present invention also provides a microorganism transformed with the recombinant vector of the present invention.

Recombinant constructs can be introduced into cultured host cells using well known techniques such as infection, transduction, transfection, transfection, electroporation, and transformation. Representative examples of hosts include bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; And fungal cells such as yeast cells. Suitable culture media and conditions for host cells are known in the art.

Preferably, the transformed microorganism is E. coli, more preferably E. coli BL21.

In addition, the present invention comprises the steps of transforming the microorganism with the recombinant vector of the present invention; And

It provides a method for producing hydrogen in a microorganism comprising culturing the transformed microorganism in a culture medium containing Ni and Fe in microaerobic conditions.

Hydrogen production method of the present invention first comprises the step of transforming the microorganism with the recombinant vector of the present invention, the method of transforming the microorganism with the recombinant vector is as described above. Next, the step of culturing the transformed microorganisms in a culture medium containing Ni and Fe in microaerobic conditions, the medium, culture temperature, incubation time, etc. required to culture the microorganisms Use known methods. In the present invention, in particular, microaerobic conditions close to anaerobic are used, which specifically refers to conditions that use only oxygen in the gas present in the interior of the container, in particular in the space inside the sealed container. In addition, the culture medium may include, in particular, Ni and Fe. Preferably, the microorganism is Escherichia coli.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example

Example 1: Isolation of [nickel-iron] -hydrogenase genes

Using this information (FIG. 1), almost all [nickel-iron] -hydrogenases have sequences that signal the cleavage by endopeptidase to become a complete enzyme at the C terminus of large subunits (FIG. 1). Degenerate primers were designed from the sequence of and the genes were obtained by degenerate PCR (see Korean Patent Publication No. 2007-0107299). Hydrogenovibrio marinus was analyzed by gene sequencing. The entire sequence of [nickel-iron] -hydrogenase derived from the strain was obtained.

In the obtained nucleotide sequence, 1781 bp corresponds to a large subunit (FIG. 2, SEQ ID NO: 1). As a result of comparing the homology with the hydrogenase of E. coli, the amino acid of the amino acid is different despite its biochemical characteristics are different. It showed high homology of 72% in sequence (FIG. 3).

Example 2: Expression and Hydrogen Production of Recombinant Hydrogenase in Escherichia Coli

Based on the obtained hydrogenase DNA sequence, an expression vector was prepared to express H. marinus hydrogenase in E. coli (FIG. 4), and inserted into E. coli to analyze protein expression and activity of the expressed enzyme. Primers for vector construction are as follows: hydS forward-CGC TGC AGA AGG AGA TAT ACA TAT GTC ATC TCA AGT TGA AAC GTT C (Pst I), hydS reverse-CCG GTA CCT TAT TTA TCT CCT TTC TTT TGA GCC GC ( Acc 65I), hydL forward-GCT AGC AAG AGG TAT ATA TTA ATG AGC GTA TTA AAC ACA CCA AAC C (Nhe I), and hydL reverse-CTC GAG TTA ACG CAC CTG CAC AGA GAT.

The BL21 strain transformed with the vector using the Trc promoter was prepared in 100 ml of M9CA medium (Na ₂ HPO ₄ 6 g, KH ₂ PO ₄ 3 g, NH ₄ Cl 1 g, NaCl 0.5 g, Casamino acid 5 g, Thiamine 1 mg). / l, supplemented with 2 mM MgSO ₄ , 0.1 mM CaCl ₂ ) in a 125 ml serum bottle. Three hours after inoculation, 1 mM IPTG was induced, and one BL21 / pTrc-hydL; S was placed in an air-vented cotton plug, two in a serum bottle, and then in a serum bottle. The flask and the rest of the serum bottles were filled with 30 μM / Fe of 20 μM and only 30 μM of Ni was added to the remaining serum bottle. And we examined the hydrogen production and protein expression for the three strains. In addition, the wild type BL21 was cultured under the same conditions to measure the hydrogen production.

Protein expression was determined by extracting whole cells and cell membranes from the culture sample 16 hours after induction by IPTG to transfer proteins to nitrocellulose membranes, and then to the large and small subunits of His antibodies and H. marinus , respectively. Detected and confirmed using the prepared rabbit polyclonal antibody (Fig. 5). As a result, it was confirmed that large subunits and small subunits of hydrogenase were expressed in whole cells and cell membrane parts.

In addition, the hydrogen produced in the strain-specific culture is gas collected by using a gas tight syringe (gas tight syringe) from the upper gas portion of the serum bottle sealed with a rubber stopper before extraction of protein samples using gas chromatography (GC) Analyzed. Quantification of the amount of hydrogen produced was converted from the area value of the hydrogen peak by GC for each sample after obtaining a standard curve for the peak area of the standard gas using 1% H ₂ standard gas (Scott). The results are shown in Table 1 below.

Comparison of Hydrogen Production According to H. marinus Hydrogenase Expression BL21
(Wild type) BL21 / pTrc-HydL, S 16 hours after induction Miho
(Ni 30μM /
Fe 20μM) Exhalation
(Ni 30μM /
Fe 20μM) Miho
(Ni 30μM /
Fe 20μM) Miho
(Ni 30μM /
Fe 0μM) H ₂ production
(ml H ₂ / L culture) 0 0 108 ± 0.25 0.2 ± 0.05 OD ₆₀₀ 2.5 4.7 1.83 1.44

In the case of hydrogen production, the wild type had 0 hydrogen production, and BL21 transformed with pTrc-HydL, S had no hydrogen production under aerobic conditions, but both Ni and Fe were added under microaerobic conditions near anaerobic conditions. Hydrogen of 108 ml H ₂ / L culture was produced. However, even under the same microaerobic conditions, when Fe was not added, almost no hydrogen was produced in 0.2 ml H ₂ / L culture (FIG. 6). These results show that H. marinus hydrogenase itself is resistant to oxygen but not intracellularly produced under aerobic conditions. Even though hydrogenase itself is resistant to oxygen, many proteins are involved in the maturation stage of hydrogenase. And because they are regulated by complex intracellular mechanisms including oxygen, it is considered that the function of hydrogenase is inhibited for this reason. In the case of protein expression without Fe, the bands corresponding to the size of the H. marinus hydrogenation enzyme large subunit and the small subunit were confirmed in all cell and membrane samples. In the sample of the cell membrane portion, only a small amount of protein was detected than the whole cell portion, but the hydrogenase was secreted into the cell membrane as originally characterized.

Example 3: H. marinus  Comparison of Hydrogen Production According to Dehydrogenase and Escherichia Coli Derived Hydrogenase Expression

Hydrogenovibrio When the hydrogenase derived from the marinus strain and the hydrogenase derived from E. coli were expressed in recombinant protein form, hydrogen production, ie, oxygen tolerance, was compared under the same exposure to oxygen.

As shown in FIG. 7, a small amount of oxygen is present in the serum bottle, which is relatively large, when the volume of the culture medium is 50 ml than when 60 ml and when 60 ml is 70 ml. It was confirmed in the graphs of FIGS. 8 and 9 that the difference in the amount of oxygen also had a great influence on the hydrogen production capacity by the hydrogenase. FIG. 8 shows the area values of peaks analyzed by GC for the amount of hydrogen produced in each culture medium (50, 60, 70, 80, 90 ml) (in the gas sample in 100 ul of culture vessel). will be. FIG. 9 compares values of hydrogen contained in the entire gas as a ratio of unit culture volume from the values of FIG. 8 because the volume of the gas part in each vessel is different and the amount of the culture medium is different. Although the tendency is slightly different, it can be confirmed that the hydrogen production capacity is changed by the hydrogenase according to the amount of oxygen in the culture vessel as a whole, and that H. marinus is less inhibited by the presence of oxygen than that of Escherichia coli. .

10 shows that gene expression of hydrogenase 1 is influenced by various external factors according to research papers published in the past, among which it is known that the expression is activated under the condition of anaerobic condition and formate. In the light of the experimental results shown in Figures 8 and 9 of the analysis of the effect of the addition of formate in the culture conditions in a 50 ml culture medium that is significantly inhibited hydrogen production by oxygen in the culture. As a result, the inhibitory effect of oxygen was significantly recovered when formate was added to both recombinant strains expressing E. coli and H. marinus hydrogenase, and formate when H. marinus was expressed. It reacted more quickly with the effect of increasing the hydrogen production.

This result also shows that H. marinus hydrogenase has relatively higher oxygen resistance than E. coli. In addition, it can be seen that the hydrogenated enzyme of H. marinus is also positively affected by formate.

1 shows an endopeptidase recognition sequence present in the C-terminal sequence of a large subunit of a hydrogenase gene.

Figure 2 shows the nucleotide sequence of a large subunit of the hydrogenase gene of the H. marinus strain.

Figure 3 compares the amino acid sequence homology of the hydrolase large subunit of E. coli and the hydrolase large subunit of H. marinus .

Figure 4 shows a vector schematic for expressing the hydrogenase of H. marinus in E. coli.

5 is a Western blot analysis to observe the expression of small and large subunits of H. marinus hydrogenase expressed by pTrc-HydL, S vector.

6 shows a culture medium in which bubbles are formed by hydrogen production. Left: WT. BL21, middle: BL21 / pET21b-HmH (Ni 30 μM / Fe 0 μM), right: BL21 / pET21b-HmH (Ni 30 μM / Fe 20 μM).

Figure 7 shows an experiment to see the change in the hydrogen production according to the difference in the degree of exposure to oxygen in culture by varying the volume of the culture. 60, 70, 80, 90 ml / 125 ml serum bottle from left.

FIG. 8 shows the area values of peaks analyzed by GC for the amount of hydrogen produced in each culture medium (50, 60, 70, 80, 90 ml) (in the gas sample in 100 ul of culture vessel). (EcH: E. coli hydratase, HmH: H. marinus hydrase) The peak area of hydrogen produced by GC analysis, measured for 50 ~ 90 ml culture volume).

FIG. 9 compares values of hydrogen contained in the gas as a ratio of unit culture volume from the values of FIG. 8.

Figure 10 shows the effect on hydrogen production when formate is added while culturing strains expressing small and large subunit genes of Escherichia coli and H. marinus hydrogenase in a pET vector.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Method for Producing Hydrogen Using Recombinant Microorganism <130> M07-6398-FD <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1781 <212> DNA <213> Hydrogenovibrio marinus <400> 1 atgagcgtat taaacacacc aaaccactat aagatggaca actccggtcg tcgagtagtc 60 attgatcctg ttactcgtat cgaaggccac atgcgttgtg aagtcaacgt tgatgaaaac 120 aatgtcatcc aaaatgccgt atccacaggc actatgtggc gaggccttga ggttattctt 180 cgcggacgcg accctcgtga cgcatgggca tttgtagaac gcatatgtgg tgtctgcact 240 ggctgccatg ctttggcatc agttcgagcg gttgaggatg ctctagatat caaaattccg 300 cacaatgcca ctttaattcg cgaaattatg gccaaaacac ttcaaattca tgaccatatt 360 gtgcatttct accatttaca tgctctagac tgggtcaatc cagtaaatgc tttgaaggca 420 gatcctcagg caacctctga actacaaaaa ctagtctcac cgcatcaccc aatgtctagc 480 cctggctact tcaaagacat tcaaatcaga atccaaaagt tcgtagattc tgggcaactc 540 ggaatcttca agaatggtta ttggagtaac cccgcctata aattatctcc cgaagcagat 600 cttatggctg ttacccacta tcttgaagct ctggacttcc aaaaagaaat cgtcaaaatc 660 catgcgatat tcggtggtaa aaaccctcac cctaactata tggttggagg tgtgccttgt 720 gcaataaaca tcgatggaga tatggccgcg ggtgccccta taaacatgga gcgtttaaac 780 tttgtcaaat cacttataga acaagggcga acctttaaca ccaatgtcta tgtacccgac 840 gttatagcta tcgcagcttt ctatcgcgac tggctatatg gtggagggct atccgcgaca 900 aacgttatgg attatggcgc ataccctaaa actccatatg ataaatctac agatcaacta 960 cctggaggtg caatcattaa tggagattgg gggaaaattc atccagttga ccctagagat 1020 cctgagcagg ttcaagaatt tgtcactcat tcttggtaca aatacccaga cgaaacaaaa 1080 ggacttcatc catgggatgg tattacagag ccaaactatg aactaggatc tagaacaaaa 1140 gggtcgagaa caaacatcat cgaaatcgac gaatctgcaa aatattcctg gatcaaatcc 1200 ccgcgctgga gggggcatgc tgtcgaagta gggccacttg cccgctatat acttgcctat 1260 gcccaaggcg ttgaatacgt taagactcag gttcacacaa gcctaaatag gtttaacgct 1320 gtatgtcgtt tgctagaccc aaaccataaa gacatcaccg acctgaaagc tttccttgga 1380 tcaacaatcg gccgaactct tgctcgtgct cttgaatcag agtattgcgg agacatgatg 1440 ctagatgact tcaaccaatt gatcagcaac attaaaaacg gtgatagcag tacggctaat 1500 acagacaagt gggatccttc aagctggcct gaacatgcca aaggtgtggg aacagttgca 1560 gctcctcgcg gtgctctggc gcactggatt gttattgaga aaggaaagat caaaaactac 1620 cagtgtgttg tgccaacaac ttggaatggc tctcctcgtg accctaaagg aaatatagga 1680 gcttttgaag catctcttat gggaactcta atggaacgcc cagatgaacc ggtcgaagta 1740 cttcgtaccc ttcacagttt tgatccctgc ctcgcctgct c 1781 <210> 2 <211> 593 <212> PRT <213> Hydrogenovibrio marinus <400> 2 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 Val Val Pro Asp Val Ile Ala Ile Ala Ala Phe Tyr         275 280 285 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 Arg 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 385 390 395 400 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         435 440 445 His Lys Asp Ile Thr Asp Leu Lys Ala Phe Leu Gly Ser Thr Ile Gly     450 455 460 Arg Thr Leu Ala Arg Ala Leu Glu Ser Glu Tyr Cys Gly Asp Met Met 465 470 475 480 Leu Asp Asp Phe Asn Gln Leu Ile Ser Asn Ile Lys Asn Gly Asp Ser                 485 490 495 Ser Thr Ala Asn Thr Asp Lys Trp Asp Pro Ser Ser Trp Pro Glu His             500 505 510 Ala Lys Gly Val Gly Thr Val Ala Ala Pro Arg Gly Ala Leu Ala His         515 520 525 Trp Ile Val Ile Glu Lys Gly Lys Ile Lys Asn Tyr Gln Cys Val Val     530 535 540 Pro Thr Thr Trp Asn Gly Ser Pro Arg Asp Pro Lys Gly Asn Ile Gly 545 550 555 560 Ala Phe Glu Ala Ser Leu Met Gly Thr Leu Met Glu Arg Pro Asp Glu                 565 570 575 Pro Val Glu Val Leu Arg Thr Leu His Ser Phe Asp Pro Cys Leu Ala             580 585 590 Cys      

Claims (8)

Hydrogenovibrio marinus consisting of the amino acid sequence of SEQ ID NO: 2 [Nickel-iron] -hydrogenase protein from strains. A gene encoding the [Nickel-Iron] -hydrogenase protein of claim 1. The method according to claim 2, A gene comprising the nucleotide sequence of SEQ ID NO: 1. Recombinant vector comprising the gene of claim 2. Microorganisms transformed with the recombinant vector of claim 4. The method according to claim 5, The microorganism is characterized in that E. coli. Transforming the microorganism with the recombinant vector of claim 4; And Culturing the transformed microorganism in a culture medium containing Ni and Fe in microaerobic conditions. The method of claim 7, The microorganism is E. coli.

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