JP2008043245A - Oxygen-resistant hydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a hydrogenase having resistance to reversible inactivation by oxygen. <P>SOLUTION: The oxygen-resistant hydrogenase has a hydrogenase surface modified with an antibody or a crosslinking agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an oxygen-resistant hydrogenase, and more particularly to a hydrogenase with improved oxygen tolerance by blocking the entry of oxygen molecules into the hydrogenase by modifying the surface with an antibody or a cross-linking agent.

Hydrogenases, nickel catalytic active site - has an iron complex or iron complex, the reaction to produce hydrogen molecules by reducing protons ^- and _{^{(2H + + e → H 2}} ), protons and electrons of hydrogen molecules It is an enzyme that catalyzes a reaction (H ₂ → 2H ⁺ + e ⁻ ) that decomposes into _two to produce a reducing power. Hydrogen can be produced by decomposing water, burns to generate energy, and then returns to water, so it is clean energy for the natural environment. Therefore, hydrogen is attracting the most attention as a clean next-generation energy carrier to replace fossil fuels, while the rapid expansion of the use of non-fossil fuel energy to save global resources and prevent global warming. ing. Therefore, it can be said that hydrogenases having the ability to generate hydrogen molecules and resolution have various possibilities. For example, a light-driven hydrogen synthesis device that combines hydrogenase and a photosynthetic reaction center such as cyanobacteria or green algae has been proposed (Patent Document 1). On the other hand, the hydrogen molecule resolution of hydrogenase can be used for a hydrogen sensor that detects hydrogen leakage in order to replace the platinum electrode of a fuel cell or to ensure the safety of the fuel cell (Non-patent Documents 1 and 2). Therefore, the importance of hydrogenase is expected to increase further in the future.

  However, hydrogenase has a problem that it is easily deactivated by oxygen molecules. Therefore, when hydrogenase is used, oxygen molecules must be sufficiently removed, which is disadvantageous in terms of cost. It is known that the hydrogenase deactivation process by oxygen can be divided into two stages. First, reversible inactivation occurs in the first stage, and then irreversible inactivation occurs in the second stage. In a reversible inactivated state, it is activated in a hydrogen atmosphere or in the presence of a reducing agent, but it is not activated in an irreversible inactivated state. Attempts to improve the resistance of hydrogenase to oxygen reported so far can be roughly divided into three. That is, (1) search for natural hydrogenase, (2) amino acid substitution of hydrogenase, and (3) immobilization of hydrogenase. Examples of (1) include hydrogenases derived from Ralstonia eutropha (Non-patent Documents 1 and 2), hydrogenases derived from Hydrogenovibrio marinus (Non-patent Document 3), and hydrogenases derived from Desulfovibrio vulgaris (Non-patent Document 4). . These hydrogenases are resistant to the second stage of irreversible inactivation, and it has been reported that irreversible inactivation does not occur for several days to several weeks when left in air at 4 ° C. Yes. This is in contrast to Clostridium-derived hydrogenase, which has been well studied so far, in an irreversible manner in a few minutes. However, the activity of all hydrogenases decreases in the presence of oxygen, suggesting that reversible inactivation occurs due to oxygen molecules, and resistance to reversible inactivation at the first stage has been obtained. I can say no. As an example of (2), the resistance to reversible inactivation of the first step is improved in the amino acid substitution product in which one of the cysteine residues coordinated to the iron-sulfur complex of Azotobacter vinelandii hydrogenase is substituted with serine. (Non-patent document 5). The natural Azotobacter vinelandii hydrogenase was almost completely inhibited in hydrogenolysis activity at an oxygen concentration of 76 μM, but the amino acid substitution product retained the same activity in the absence of oxygen even at the above oxygen concentration. This result suggests that the oxygen tolerance of the amino acid substitution product is improved. However, the hydrogenolysis rate of amino acid substitution products is 16 nmol / min / gram of cell, and this rate is dramatically 1500 times lower than that of natural type 24500 nmol / min / gram of cell in the absence of oxygen. Very low. Therefore, although this amino acid substitution provides oxygen tolerance, it has a problem that the original activity is greatly reduced because it greatly affects the electron transfer function of hydrogenase. As an example of (3), there can be mentioned a research example in which oxygen resistance is improved by immobilizing hydrogenase on a carrier such as silica gel or ion exchange resin (Non-patent Documents 6 and 7). However, the hydrogenase immobilized on the carrier has improved resistance to the second-stage irreversible inactivation, but there is no report of improved resistance to the first-stage reversible inactivation. Therefore, the problem of reversible inactivation of hydrogenase by oxygen remains a problem to be solved.

  On the other hand, there have been several reports on modification of the enzyme protein surface by intramolecular crosslinking and antibody binding. For example, regarding intramolecular crosslinking, α-galactosidase is cross-linked between lysines on the protein surface using hexamethylene diisocyanate (Non-patent Document 8), and chymotrypsin is 1-ethyl-3- (3-dimethylaminopropyl). ) Example of cross-linking between aspartic acid and glutamic acid on the protein surface using carodiimide (EDC) (Non-patent Document 9), glyceraldehyde-3-phosphate dehydrogenase using carboxylic acid between lysine on the protein surface There are cross-linked examples (Non-Patent Document 10), and all of them are aimed at improving heat resistance and protease resistance. On the other hand, regarding the enzyme stability by antibodies, α-galactosidase (Non-patent Document 8), RNase (Non-patent Document 11), papain (Non-patent Document 12), and glucose-6-phosphate dehydrogenase (Non-patent Document 13). It has been reported that the heat resistance, protease resistance and denaturant (urea) resistance have been improved by mixing each enzyme with serum containing an antibody against it. However, all of the above reported examples relate to enzyme proteins that are completely different from hydrogenases in terms of molecular weight, subunit structure, and function, and none have been studied for improving oxygen tolerance.

JP 2006-20614 Analytical Chemistry, 2005, Vol.77, p.4969-4975 Proc.Natl.Acad.Sci.USA, 2005, Vol.102, p.16951-16954 Biochemical and Biophysical Research Communications, 1997, Vol.232, p.766-770 FEBS letter, 1978, Vol.86, p.122-126 Journal of Bacteriology, 1995, Vol.177, p.3960-3964 Biochemical and Biophysical Research Communications, 1980, Vol.92, p.1091-1096 Proc.Natl.Acad.Sci.USA, 1978, Vol.75, p.3640-3643 Biochimica et. Biophysica Acta, 1974, Vol. 350, p.432-440 Biochimica et. Biophysica Acta. 1978, Vol.522, p.277-283 J. Mol. Catal., 1983, Vol. 19, p.291-301 Biochimica et Biophysica Acta, 2001, Vol.1548, p.114-120 Biotechnol. Appl. Biochem. 2000, Vol. 32, p.89-94 Biochimica et. Biophysica Acta, 1974, Vol.343, p.431-434

  An object of the present invention is to provide a hydrogenase that is resistant to reversible inactivation by oxygen.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the resistance to reversible inactivation by oxygen can be imparted by modifying the surface with an antibody or a crosslinking agent. It came to complete.

That is, the present invention includes the following inventions.
(1) An oxygen-resistant hydrogenase, wherein the hydrogenase surface is modified by means for blocking the entry of oxygen molecules into the hydrogenase.
(2) The oxygen-resistant hydrogenase according to (1), wherein the blocking means is an antibody.
(3) The oxygen-resistant hydrogenase according to (2), wherein the antibody is a polyclonal antibody or a monoclonal antibody.
(4) The oxygen-resistant hydrogenase according to (1), wherein the blocking means is a crosslinking agent.
(5) The oxygen-resistant hydrogenase according to (4), wherein the crosslinking agent is a bifunctional crosslinking agent.
(6) An electrode formed by immobilizing the oxygen-resistant hydrogenase according to any one of (1) to (5).

  According to the present invention, an oxygen-resistant hydrogenase is provided. In the oxygen-resistant hydrogenase of the present invention, reversible inactivation by oxygen, which has been a problem in the past, is remarkably suppressed as compared with natural hydrogenase, and the decrease in enzyme activity itself is small. Therefore, the oxygen-resistant hydrogenase of the present invention is very useful as an alternative to rare metals such as platinum and palladium used in the construction of biomass hydrogen production systems using photosynthetic microorganisms and fuel cells.

  Hereinafter, the present invention will be described in detail.

  The oxygen-resistant hydrogenase of the present invention is characterized in that the hydrogenase surface is modified by means for blocking the entry of oxygen molecules into the hydrogenase.

  In the present specification, “oxygen tolerance” refers to resistance to reversible inactivation at the first stage in a hydrogenase deactivation process by oxygen in two stages. In addition, if there is resistance to reversible inactivation at the first stage, it can be said that it has resistance against irreversible inactivation at the second stage.

  In the present invention, the hydrogenase that is subjected to surface modification for imparting oxygen resistance is not particularly limited, but examples thereof include those derived from microorganisms, animals, or plants, and preferably include microorganism-derived hydrogenases. Among microorganisms, hydrogenase derived from Desulfovibrio vulgaris is preferable. In addition, hydrogenases obtained by culturing and purifying these microorganisms and the like and hydrogenases obtained by recombinants can also be used.

In the present invention, an antibody is used as the first means for blocking the entry of oxygen molecules into the hydrogenase. The antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody. The antibody can be produced by a general method using purified hydrogenase or a part thereof as an antigen. The antibody may be a fragment, and for example, F (ab ′) ₂ and Fab ′ can also be used.

  For example, the polyclonal antibody is prepared as follows. First, animals are immunized with purified hydrogenase. Mammals (eg, rats, mice, rabbits, etc.) are used as animals. In the case of rabbits, the dose of antigen per animal is, for example, 100 to 500 μg using an adjuvant. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.

  The site of administration is intravenous, subcutaneous or intraperitoneal. Further, the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 3 times at intervals of several days to several weeks, preferably 2 to 3 weeks. Then, the antibody titer is measured 6 to 60 days after the final immunization day, and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. The antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), or the like.

  When purification of antibody from antiserum is required, it should be purified by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof. Can do.

  The monoclonal antibody is prepared as follows, for example. First, animals are immunized as described above. If necessary, in order to effectively immunize, an adjuvant (commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, etc.) may be mixed as described above. Similarly, mammals (eg, rats, mice, rabbits, etc.) may be used as the animals. For example, in the case of mice, the dose of antigen is about 50 μg per mouse. The administration site is mainly intravenous, subcutaneous and intraperitoneal. The interval between immunizations is not particularly limited, and is performed at least 2-3 times at intervals of several days to several weeks, preferably at intervals of 2-3 weeks. Then, antibody-producing cells are collected after the final immunization. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells being preferred.

  Next, in order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As myeloma cells to be fused with antibody-producing cells, cell lines derived from animals such as mice and generally available can be used. As a cell line to be used, it has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. What has is preferable. For example, specific examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1), P3 / NSI / 1-Ag4-1, Sp2 / 0-Ag14. Thereafter, the myeloma cell and the antibody-producing cell are fused. For cell fusion, antibody-producing cells and myeloma cells are mixed at a ratio of 15: 1 to 25: 1 in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and cells such as polyethylene glycol The fusion reaction is performed in the presence of a fusion promoter or by electric pulse treatment (for example, electroporation).

  The target hybridoma is selected from the cells after cell fusion treatment. For example, cells can be cultured using a medium containing hypoxanthine, aminopterin and thymidine, and the growing cells can be obtained as hybridomas. Next, it is screened whether the target antibody exists in the culture supernatant of the grown hybridoma. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme-linked immunosorbent assay (ELISA), RIA (radioimmuno assay), or the like. Cloning of the fused cells is performed by a limiting dilution method or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established.

As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method or the like can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium or MEM medium containing 10% fetal bovine serum under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration) for 3 to 10 days. Then, an antibody is obtained from the culture supernatant. In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, gel chromatography or the like is appropriately selected, or these It can be purified by a combination of methods.

  The antibody obtained as described above, for example, antiserum collected from an immunized animal and a hydrogenase solution (0.1 μM) are mixed at a ratio of 1: 1, preferably 10: 1, and a temperature of 4 to 30 ° C. for 5 to 30 minutes. By reacting, a hydrogenase-antibody complex in which the hydrogenase surface is coated with an antibody can be obtained.

  In the present invention, a crosslinking agent is used as the second means for blocking the entry of oxygen molecules into the hydrogenase. The cross-linking agent used in the present invention is not limited as long as it can be covalently bonded to an amino acid residue on hydrogenase, but it is not limited between ε-amino groups of lysine side chains, between sulfhydryl groups of cysteine side chains, or lysine. A bifunctional crosslinking agent capable of crosslinking between the ε-amino group of the side chain and the sulfhydryl group of the cysteine side chain is preferred, and a bifunctional crosslinking agent capable of crosslinking the ε-amino group of the lysine side chain is particularly preferred.

  As such bifunctional crosslinkers, homobifunctional crosslinkers containing amine-reactive N-hydroxysuccinimide (NHS) esters at both ends, homobifunctional crosslinks containing sulfhydryl-reactive maleimide groups at both ends And a heterobifunctional cross-linking agent each containing an NHS ester and a maleimide group at each end. The spacer arm length of the cross-linking agent is in the range of 11 to 35 ohm strong, preferably 14 to 22 ohm strong, depending on the type of cross-linking agent.

Specific examples of the above-mentioned bifunctional crosslinking agent include lysine-lysine crosslinking agents such as Bis (NHS) PEO ₅ : Bis (N-succinimidyl) -pentaethylene glycol ester (spacer arm length 22 mm), EGS: Ethylene glycol bis [succinimidyl succinate] (spacer arm length 16.1 mm), Sulfo-EGS: Ethylene glycol bis [sulfosuccinimidyl succinate] spacer arm length 16.1 mm), Bis (sulfosuccinimidyl) sebacate (spacer arm length 14 mm), BSOCOES: Bis [2 -(succinimidooxycarbonyloxy) ethyl] sulfone (spacer arm length 13 mm), DSP: Dithiobis [succinimidyl propionate] (spacer arm length 12 mm), DTSSP: 3,3'-Dithiobis [sulfosuccinimidyl propionate] (spacer arm length 12 mm), DSS: Disuccinimidyl suberate (spacer arm length 11.4 mm), BS3: Bis (sulfosuccinimidyl) suberate (spacer arm length 11.4 mm), DSG: Disuccinimidyl glutarate (spacer arm length 7.7 mm), DST: Disuccinimidyl tartar ate (spacer arm length 6.4 mm), sulfo-DST: Disulfosuccinimidyl tartrate (spacer arm length 6.4 mm), Sulfo-SANPAH: (N-Sulfosuccinimidyl-6- [4´-azido-2´-nitrophenylamino] hexanoate) Specific photo-activated cross-linking, spacer arm length 18.2 mm), etc., and examples of cysteine-cysteine cross-linking agents include BM [PEO] ₂ : (1,8-Bis-Maleimidodiethyleneglycol (spacer arm length 14.7 mm) , BM [PEO] ₃ : (1,11-Bis-Maleimidodiethyleneglycol (spacer arm length 17.8 mm) and the like, and as a lysine-cysteine cross-linking agent, for example, Sulfo-KMUS: N- [κ-Maleimidoundecanoyloxy] sulfosuccinimide ester (Spacer arm length 16.3 mm), NHS-PEO ₂ -Mal: (succinimidyl-[(N-maleimidopropionamido) -diethyleneglycol] ester (spacer arm length 17.6 mm), NHS-PEO ₄ -Mal: (succinimidyl-[(N- maleimidopropionamido) -tetraethyleneglycol] ester (spacer arm length 24.6Å _{, NHS-PEO 8 -Mal: (} succinimidyl - [(N-maleimidopropionamido) -octaethyleneglycol] ester ( spacer arm length 39.3A) but the like, but are not limited to. Moreover, a commercial item (for example, product made by PIERCE) can be used for each of the above-mentioned crosslinking agents.

  Intramolecular cross-linking of hydrogenase with the above cross-linking agent, dissolve the hydrogenase in a suitable solvent, add the cross-linking agent and react at a temperature of 4 to 30 ° C. for 60 to 600 minutes, and then remove the unreacted cross-linking agent. Can be done. The reaction is preferably performed at a hydrogenase concentration in the range of 1 to 100 μM. If the hydrogenase concentration is too high, the enzyme's proper conformation is not maintained, so that the enzyme activity decreases, or intermolecular cross-linking occurs to cause the enzyme protein to aggregate, resulting in a significant decrease in enzyme activity. The crosslinking agent is dissolved in a water-miscible solvent such as DMSO or ethanol, and is added in an amount of 0.01% to 10%, preferably 0.01 to 0.1% with respect to the hydrogenase solution. Furthermore, if the unreacted crosslinking agent remains in the solution, the enzyme activity may be lowered. Therefore, it is preferable to remove the unreacted crosslinking agent by a method such as dialysis or column chromatography.

  In the present invention, oxygen resistance of hydrogenase, that is, resistance to reversible inactivation by oxygen can be evaluated as follows. First, an active hydrogenase is exposed to air. This inactivates part of the hydrogenase. When a benzyl viologen solution saturated with hydrogen gas is added thereto, the active hydrogenase decomposes hydrogen to generate a reducing power, and this reducing power is utilized for reactivation of the inactive hydrogenase. When almost everything is activated, the reducing power is then used to reduce benzyl viologen. The time (lag time) until the inactive hydrogenase is sufficiently reactivated and the reduction of benzyl viologen begins is measured. Since this lag time is considered to be proportional to the amount of inactivated hydrogenase, it can be evaluated that if the lag time is short, the resistance to reversible inactivation by oxygen is high.

  The present invention also provides an electrode formed by immobilizing the above oxygen-resistant hydrogenase. The electrode can be used for a fuel cell, for example. As the electrode, an electrode made of gold, platinum, carbon, graphite or the like can be used. Methods for immobilizing enzymes on electrodes include methods using cross-linking reagents (such as polyfunctional acylating agents), methods of encapsulating in polymer matrices, methods of coating with dialysis membranes, methods of adsorbing to conductive polymers These may be used in combination. Typically, the oxygen-resistant hydrogenase of the present invention is mixed with a carrier protein and then immobilized on a carbon electrode by treatment with glutaraldehyde. Preferably, glutaraldehyde is then blocked by treatment with a reagent having an amine group. The carrier protein is not particularly limited as long as the immobilized enzyme is kept stable for a long time, has good substrate permeability, and can be formed into a thin film. For example, bovine serum albumin can be used.

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

(Example 1) Preparation of complex of hydrogenase and anti-hydrogenase / polyclonal antibody
Hydrogenase derived from Desulfovibrio vulgaris Miyazaki F was dissolved in a PBS aqueous solution to a concentration of 1 mg / ml, and about 2 ml of the hydrogenase solution was injected 5 times into 5 mice over about 2 months. After confirming that the antibody titer was improved, serum was collected. An equal volume of 0.1 μM hydrogenase solution (PBS aqueous solution) was mixed with 100 μl of serum to prepare a complex of hydrogenase and anti-hydrogenase antibody.

Example 2 Preparation of Intramolecular Crosslinking Hydrogenase Bis (N-succinimidyl) -pentaethylene glycol ester (spacer length 22 mm), Bis (sulfosuccinimidyl) sebacate (spacer length 14 mm), Bis (sulfosuccinimidyl) suberate (spacer length) 11Å) was used. About 1 mg of each of the above cross-linking reagents was added to 500 μl of a hydrogenase solution (borate buffer aqueous solution, pH 7.3, hydrogenase concentration derived from Desulfovibrio vulgaris Miyazaki F), and the mixture was stirred at 15 ° C. overnight. After the reaction, the unreacted crosslinking reagent was removed by a gel filtration column, and the hydrogenase fraction was collected as an intramolecular crosslinking hydrogenase. FIG. 1 shows a schematic diagram of the structure of the crosslinking reagent and the structure of the intramolecular crosslinking hydrogenase.

(Example 3) Oxygen tolerance evaluation of hydrogenase The oxygen tolerance evaluation test of hydrogenase was performed as follows according to the operation procedure shown in FIG. The antibody-bound hydrogenase prepared in Example 1, the three intramolecular cross-linked hydrogenases prepared in Example 2, and the natural hydrogenase were each added to a 3 mM benzyl viologen solution (Tris buffer solution) so that the final concentration was about 0.05 μM. , pH 7.3). Hydrogenase was activated by blowing hydrogen gas to about 1 ml of this hydrogenase solution for 15 minutes. The activation of hydrogenase was confirmed by a purple color due to the reduction of benzyl viologen. The activated hydrogenase solution was then stirred in air. Within a few seconds after stirring, it was confirmed that the purple benzyl viologen was oxidized and became colorless, and oxygen molecules began to dissolve in the hydrogenase solution. After stirring for 5 minutes in air, 20 μl of the hydrogenase solution was added to 3 ml of 3 mM benzyl viologen solution (Tris buffer, pH 7.3) saturated with hydrogen gas. When the absorbance (540 nm) of the hydrogenase solution was measured over time, the hydrogenase that had been inactivated by stirring in the air was reactivated, so that the reduction of benzyl viologen did not occur in the first few minutes. Did not rise. Thereafter, hydrogenase reactivation sufficiently progressed, benzyl viologen was reduced, the solution turned purple, and the absorbance increased. The time until the absorbance began to rise was defined as the time (lag time) until hydrogenase that had been inactivated by stirring in air was reactivated. The activity of hydrogenase was determined by measuring the reduction rate of benzyl viologen (the slope of the absorbance curve).

  Each hydrogenase solution [1: Intramolecular crosslinking hydrogenase (crosslinking reagent; Bis (N-succinimidyl) -pentaethylene glycol ester), 2: Intramolecular crosslinking hydrogenase (crosslinking reagent; (Bis (sulfosuccinimidyl) sebacate)), 3: Intramolecular crosslinking hydrogenase (Crosslinking reagent; Bis (sulfosuccinimidyl) suberate), 4: natural hydrogenase, 5: hydrogenase-polyclonal antibody complex] is shown in Fig. 3. Also, the lag time measured for each hydrogenase solution and the reduction of benzyl viologen. The speed is shown in Table 1 below.

  As a result, the time required for hydrogenase reactivation (lag time) is about 20 minutes for natural hydrogenase, but is reduced to about 5 minutes by antibody binding and to about 1 minute by intramolecular cross-linking. It was revealed that resistance to activation was improved.

The schematic diagram of the structure of a crosslinking reagent and the structure of molecular bridge | crosslinking hydrogenase is shown. The scheme of the oxygen tolerance evaluation test of hydrogenase is shown. It shows the change in absorbance of each hydrogenase solution [1: Intramolecular crosslinking hydrogenase (crosslinking reagent; Bis (N-succinimidyl) -pentaethylene glycol ester), 2: Intramolecular crosslinking hydrogenase (crosslinking reagent; (Bis (sulfosuccinimidyl) sebacate), 3 : Intramolecular cross-linking hydrogenase (cross-linking reagent; Bis (sulfosuccinimidyl) suberate), 4: natural hydrogenase, 5: hydrogenase-polyclonal antibody complex].

Claims (6)

  An oxygen-resistant hydrogenase, wherein the hydrogenase surface is modified by means for blocking the entry of oxygen molecules into the hydrogenase.   2. The oxygen tolerant hydrogenase of claim 1 wherein the blocking means is an antibody.   The oxygen-resistant hydrogenase according to claim 2, wherein the antibody is a polyclonal antibody or a monoclonal antibody.   The oxygen tolerant hydrogenase according to claim 1, wherein the blocking means is a cross-linking agent.   The oxygen-resistant hydrogenase according to claim 4, wherein the cross-linking agent is a bifunctional cross-linking agent.   An electrode formed by immobilizing the oxygen-resistant hydrogenase according to any one of claims 1 to 5.

Images