Reduction of nitrogen-containing groups in target compounds using biocatalysts

Abstract

Provided herein is a method of producing functional groups in a target compound using biocatalysts described in more detail herein. Related systems and compositions are also provided.

Description

Technical Field

[0001] The present invention relates to a method for reducing functional groups in a target compound using a biocatalyst. In particular, the present invention relates to a method for reducing nitrogen-containing functional groups in a target compound using a biocatalyst supported on a carrier material. The present invention also relates to a system and an apparatus for implementing this method. Background Art

[0002] Chemical manufacturing processes are often associated with a number of environmental issues. The reagents used (e.g., catalysts) are often non-renewable and / or toxic. Extreme operating conditions, such as elevated temperatures and pressures, are often required, providing such conditions is energy inefficient. Toxic solvents are often required to obtain satisfactory yields. Furthermore, the reagents used are often non-selective, requiring complex synthetic strategies to selectively treat only the desired functional groups within the molecule.

[0003] The non-selectivity of conventional methods is particularly problematic in the field of fine chemical manufacturing, including in pharmaceutical synthesis, where it is particularly important to reduce the generation of impurities as much as possible. Such impurities usually need to be removed by purification strategies, but such purification methods are costly and generally inefficient. Therefore, the generation of unwanted by-products in chemical reactions increases economic costs and causes environmental damage, as the chemicals used in the purification process are also usually harmful.

[0004] A key class of chemical reactions that is particularly important in synthesis is the reaction of nitrogen-containing functional groups (such as nitro (-NO 2 ), azido (-N 3 ), nitrile (-CN)). Although the desired chemical outcome of this reaction is usually the formation of an amine (-NH 3 ) or quaternary ammonium (-NH 4 + ) groups, but known methods for carrying out this reaction are generally inefficient, produce large amounts of by-products, and / or use expensive, non-renewable reagents and environmentally damaging reaction conditions.

[0005] For example, some strategies for industrial reduction of nitro groups to amine groups rely on the use of noble metal catalysts, such as palladium (Pd). However, this catalyst is extremely expensive, non-renewable, and is associated with significant environmental impacts in its production process, and in the presence of common pollutants in the raw material, its activity is usually reduced. For example, carbon monoxide (CO) is a common pollutant in the hydrogen source obtained from natural gas, and it is usually used as a reducing agent in this reaction, which means that high-purity hydrogen (with its own economic and environmental impact) is usually required in this reaction. In addition, when using this catalyst, necessary reaction conditions are usually harsh, using high temperature and / or harmful organic solvents. In addition, this reaction often produces a large amount of by-products, such as by-products produced by non-specific reduction, incomplete reduction and catalysis unwanted side reactions (such as bond rearrangement).

[0006] In order to avoid the problems associated with the use of noble metal catalysts for nitrogen-based reduction, some recent efforts have focused on the use of biocatalysts, such as enzymes. Compared with traditional chemical treatment methods, enzyme treatment of chemical reagents provides advantages. Enzymes are renewable and biodegradable, thus overcoming environmental problems associated with the production and handling of chemical catalysts. Enzymes are generally harmless and non-toxic, thus solving safety issues associated with chemical catalysts. Enzymes are generally operated at moderate temperatures and atmospheric pressures, thus reducing the energy requirements associated with conventional chemical treatments. Enzymes are also generally highly selective for their chemical substrates, and methods such as quantitative enzyme engineering and directed mutagenesis continue to expand the scope of reactions that can be performed. Therefore, compared with conventional chemical methods, enzyme catalysis provides many advantages.

[0007] These advantages have led to the search for industrial applications of enzymes capable of catalyzing nitro reduction, such as nitroreductases. Such enzymes have active sites capable of catalyzing nitro reduction. However, while the use of such enzymes does solve at least some of the problems caused by the above-mentioned noble metal catalysts, significant problems still exist.

[0008] For example, enzyme catalysts such as nitroreductase are often used as homogeneous catalysts in solution with reactant (and often also product) molecules. However, this creates problems associated with the need to purify the enzyme from the final product mixture. The need to separate the liquid phase enzyme from the final product increases costs and processing time, may involve separation techniques using harsh chemical conditions, and may even cause the product to undergo further undesirable reactions in the purification workflow.

[0009] Furthermore, nitroreductases that have been studied for the reduction of nitrogen-containing functional groups generally require auxiliary cofactors to enable their catalytic cycle to operate. As described below, a particular difficulty in using such enzymes is the need to recycle the cofactors required in their catalytic cycle.

[0010] Cofactors are non-protein compounds that play an important role in many enzyme-catalyzed biochemical reactions, and they are often used to transfer chemical groups between enzymes. Cofactors are sometimes also called "cosubstrates", reflecting the processing of them by the enzyme in the process of catalyzing its primary reaction. For example, an oxidoreductase that catalyzes the oxidation of a reagent to produce a product can couple this oxidation reaction with a reduction reaction of the cofactor that acts as an electron hole. In this case, the entire reaction catalyzed by the enzyme can be expressed as:

[0011] Reducing agent + oxidizing cofactor → oxidizing product + reducing cofactor

[0012] Similarly, enzymes that catalyze the reduction of a reagent to produce a product typically couple this reduction reaction to an oxidation reaction of a cofactor that serves as a source of electrons or reducing equivalents such as hydride ions:

[0013] Oxidizing agent + reducing cofactor → reducing product + oxidizing cofactor

[0014] The biological applications of cofactors are not limited to the simple redox reactions mentioned above, but also include more complex reactions such as atom insertion reactions, rearrangement reactions, etc.

[0015] Key biological cofactors include: nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and flavins such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin.

[0016] Although enzymes that rely on cofactor regeneration are well known, their application in industry is limited by the difficulty in providing enough cofactors for enzyme use. One option is to provide a superstoichiometric amount of cofactor relative to the reagent in question. However, the high cost and typical low stability of the reduced cofactor molecule mean that this is not a feasible method. Other methods that have been considered include using a system that regenerates the cofactor molecule in the desired form (i.e., recycling the cofactor molecule). However, traditional cofactor regeneration systems are problematic. For example, current industrial practices for enzymatic NAD (P) H recycling rely on superstoichiometric amounts of carbon-based (organic) sacrificial reductants, such as glucose or isopropanol. However, this leads to additional costs, waste generation, and requires additional downstream processing steps for the desired product, and the atomic efficiency is low. Due to the lack of a suitable way to recycle flavin cofactors, the industrial development of enzymes using flavins is almost completely hindered. Electrochemical reduction of oxidized flavin cofactors has been proposed, but such systems are difficult to incorporate into industrially relevant environments, involving the use of expensive materials (such as precious metals) and highly processed carbon materials as required electrode materials, and are prone to producing undesirable side reaction products.

[0017] Thus, there is a pressing need for improved methods of catalyzing the reduction of reducible functional groups, particularly nitrogen-containing functional groups. In particular, there is a need for methods that avoid the need for expensive or hazardous reactive chemical reagents; that are atom efficient; that avoid the difficulties associated with electrochemical handling of reagents; that do not rely on the use of expensive sacrificial organic reducing agents; that are selective; that result in complete reduction rather than just partial reduction; that avoid the production of byproducts; and / or that do not rely on complex cofactor regeneration methods. The present invention is intended to address some or all of these problems. Summary of the invention

[0018] In the process of seeking to solve the above problems, the inventors surprisingly found that target compounds containing reducible nitrogen-containing functional groups can be cleanly and efficiently reduced in the presence of molecular reducing agents (such as hydrogen) by using a biocatalyst comprising an oxidoreductase (such as hydrogenase) supported on a support material (such as carbon). Surprisingly, the inventors have found that by contacting the target compound with such a catalyst, the molecular reducing agent can be oxidized by the oxidoreductase and the nitrogen-containing functional groups can be reduced at the support material.

[0019] As explained in more detail herein, the reduction reaction is clean and atom efficient. The reaction is carried out under mild conditions and avoids or minimizes the use of toxic reagents (such as organic solvents), which are associated with the use of noble metal catalysts as described above. Side reactions, such as rearrangements, can be avoided, and expensive sacrificial organic reducing agents are not required. The enzymes used are generally independent of cofactors that require regeneration (although these are not necessarily excluded). The catalysts are heterogeneous, which makes it easier to purify the product from the reaction mixture. In particular, the reaction generally results in a more complete reduction (up to 100% complete reduction of nitrogen-containing functional groups) than using similar enzymes in solution.

[0020] Therefore, the present invention provides a method for reducing nitrogen-containing functional groups in a target compound, comprising: contacting the target compound with a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material in the presence of a molecular reducing agent for oxidation by an oxidoreductase or a functional fragment or derivative thereof, under the conditions that:

[0021] - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and

[0022] - The nitrogen-containing functional groups are reduced at the support material.

[0023] Preferably, the method comprises contacting the target compound with the support material.

[0024] Preferably, the reduction of the nitrogen-containing functional groups comprises direct electron transfer from the support material to the target compound. Preferably, the method comprises reducing the nitrogen-containing functional groups to form amine groups or quaternary ammonium groups.

[0025] Preferably, the oxidoreductase or functional fragment or derivative thereof is in electrical contact with the support material. Preferably, the oxidoreductase or functional fragment or derivative thereof transfers electrons to the support material via an intramolecular electron conduction pathway. Preferably, the intramolecular electron conduction pathway comprises a series of [FeS] clusters.

[0026] Preferably, the molecular reducing agent is selected from hydrogen, carbon monoxide, formate, isotopes thereof and mixtures thereof. Preferably, the molecular reducing agent comprises hydrogen or its isotopes, or consists of hydrogen or its isotopes.

[0027] Preferably, in one embodiment, the supported biocatalyst comprises a hydrogenase or a functional fragment or derivative thereof. Preferably, the hydrogenase is selected from or comprises an amino acid sequence of any one or more of SEQ ID NOs: 1-46, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0028] Preferably, in another embodiment, the loaded biocatalyst comprises carbon monoxide dehydrogenase or a functional fragment or derivative thereof. Preferably, the carbon monoxide dehydrogenase is selected from or comprises any one or more of SEQ ID NOs: 47-67, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0029] Preferably, in another embodiment, the supported biocatalyst comprises formate dehydrogenase or a functional fragment or derivative thereof. Preferably, the formate dehydrogenase is selected from or comprises any one or more of SEQ ID NOs: 68-78, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0030] Preferably, the oxidoreductase or a functional fragment or derivative thereof is immobilized on the carrier material. Preferably, the carrier material is conductive or semiconductive.

[0031] Preferably, the support material comprises: carbon, metal or metal alloy, metal oxide or mixed metal oxide, metal hydroxide, metal chalcogenide, semiconductor material or conductive polymer, or a mixture thereof.

[0032] Preferably, the support material comprises or consists of a carbon material.

[0033] Preferably, the carbon material comprises graphite, carbon nanotubes, carbon black, activated carbon, carbon nanopowder, glassy carbon, carbon fiber, carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite, doped or surface-modified carbon or doped diamond. Preferably, the carbon material comprises:

[0034] - doped graphene, wherein the graphene is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals;

[0035] - doped carbon nanotubes, wherein the carbon nanotubes are doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals;

[0036] - doped diamond, wherein the diamond is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen and silicon;

[0037] - doped carbon black, wherein the carbon black is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals; and / or

[0038] - doped activated carbon, wherein the activated carbon is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals;

[0039] And / or wherein the carbon material comprises carboxylic acid surface groups or is modified to comprise carboxylic acid surface groups.

[0040] Preferably, the nitrogen-containing functional group is a nitro group, an azido group, a hydroxylamine group, a nitroso group, a nitrile group, a diazo group, a diazonium group, an isocyano group, an isothiocyanate group, an isocyanate group, a hydrazone group, a hydrazine group, an amidine group, an azo group or a guanidine group.

[0041] Preferably, the target compound is a nitroaromatic compound. Preferably, the nitroaromatic compound comprises a hydrocarbyl aryl or heteroaryl group substituted by a nitro group, wherein the hydrocarbyl aryl or heteroaryl group is optionally further substituted.

[0042] Preferably, the biocatalyst does not comprise an oxidoreductase or a functional fragment or derivative thereof comprising an active site capable of catalyzing the enzymatic reduction of a nitrogen-containing functional group. Preferably, the biocatalyst does not comprise a nitroreductase. Preferably, the method does not comprise transferring electrons to the target compound via one or more cofactors.

[0043] The present invention also provides a system, comprising:

[0044] i) a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material;

[0045] ii) a molecular reducing agent; and

[0046] iii) a target compound comprising a reducible nitrogen-containing functional group;

[0047] Wherein, the system is configured such that, in use, (a) the molecular reductant is oxidized by an oxidoreductase or a functional fragment or derivative thereof; and (b) the nitrogen-containing functional group is reduced at the support material.

[0048] Preferably, in the system, the oxidoreductase or functional fragment or derivative transfers electrons to the support material, and the reduction of the nitrogen-containing functional group involves direct electron transfer from the support material to the target compound.

[0049] Preferably, in said system, said oxidoreductase or a functional fragment or derivative thereof is as defined herein; and / or said carrier material is as defined herein; and / or said molecular reductant is as defined herein; and / or said target compound and / or said nitrogen-containing functional group is as defined herein; and / or said biocatalyst is as defined herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : When the rotation speed is 0 rpm, the scanning rate is 100 mV s -1 Cyclic voltammograms of a PGE electrode in 50 mM tris-HCl (pH 8) (black in the original color image) and in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO and 1 mM nitrobenzene (red in the original color image) obtained at 40 °C. The reductive wave in the presence of nitrobenzene starts at a potential more positive than the hydrogen couple, confirming the potential for reducing nitrobenzene using hydrogen as a molecular reductant according to the disclosed method.

[0051] Figure 2 : When the rotation speed is 0 rpm (red in the original color image) and 2000 rpm (blue in the original color image), the scanning rate is 100 mV s -1 Figure 2 shows the cyclic voltammogram of the BP-modified PGE electrode in 50 mM tris-HCl pH 8 containing 2 vol% DMSO and 1 mM nitrobenzene obtained at 4 % t. The reductive wave in the presence of nitrobenzene starts at a potential more positive than the hydrogen couple, confirming the potential of reducing nitrobenzene using hydrogen as a molecular reductant according to the disclosed method.

[0052] Figure 3: Infrared difference spectrum of 2 mM nitrobenzene in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO minus the spectrum of pure 50 mM tris-HCl (pH 8) at open circuit potential. The dotted line indicates the characteristic peak of the nitro group.

[0053] Figure 4 : IR difference spectrum of 2 mM nitrobenzene in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO at -0.47 V (vs SHE, applied to BP-modified activated carbon paper working electrode) minus the spectrum at open circuit potential. It can be seen that the dotted line indicates that the characteristic peak of the nitro group is decreasing, indicating a decrease in hydrogen potential.

[0054] Figure 5 :When the rotation speed is 0rpm, the scanning rate is (A) 100mV s -1 or (B) 10mV s -1 Cyclic voltammograms of the BP-modified PGE electrode in 100 mM sodium phosphate (pH 6) (black in the original color image) and 1 mM nitrophenol (blue in the original color image) obtained at 4 ℃. The reductive wave in the presence of nitrophenol starts at a potential more positive than that of the hydrogen pair, confirming the possibility of using hydrogen as a molecular reducing agent to reduce nitrophenol according to the disclosed method.

[0055] Figure 6 : In a spectroelectrochemical cell at 100 mV s -1 Cyclic voltammograms of BP-modified activated carbon paper electrode in 50 mM tris-HCl (pH 8) (light gray) and 5 mM 2-azido-1-phenylethanone (black) containing 1 vol% DMSO obtained at a scan rate of . The reductive wave in the presence of azide compounds starts at a potential more positive than that of hydrogen pairs, confirming the possibility of using hydrogen as a molecular reducing agent to reduce azides according to the disclosed method.

[0056] Figure 7 : Infrared difference spectrum of 5 mM 2-azido-1-phenylethanone in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO at open circuit potential. The dotted line indicates the characteristic peak of the azido group.

[0057] Figure 8 : IR difference spectra of 5 mM 2-azido-1-phenylethanone in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO at different potentials (vs SHE) applied to the BP-modified activated carbon paper working electrode. It can be seen that the dotted line indicates that the characteristic peak of the azido group is decreasing, indicating a decrease in the hydrogen potential.

[0058] Fig. 9 : When the rotation speed is 0 rpm, the scanning rate is 10 mV s -1 Cyclic voltammogram of a PGE electrode modified with BP200 in 100 mM sodium phosphate (pH 6) containing 1 vol% DMSO and 1 mM tetracyanobenzene obtained at 4 ℃. The reductive wave in the presence of cyano compounds starts at a potential more positive than that of hydrogen pairs, confirming the possibility of using hydrogen as a molecular reducing agent to reduce cyano groups according to the disclosed method.

[0059] Fig.10 : Corresponding to Table 1 1 H NMR spectrum (see Example 1). 2 O / D 2 Compound standards (A, B, C) and reaction mixtures in O were run under water suppression. The conditions reported were used. Ref 4 Reduction of compound A to C gives in this case a mixture of B and C. A: Extended spectrum. B: Enlarged spectrum.

[0060] Fig.11 :A: corresponds to scheme 1(i) 1 H NMR spectrum (see Example 2). This example was taken at room temperature under 1 bar H 2 The reaction was carried out using phosphate buffer (50 mM, pH 7.0) under 4% CO. Hyd1 / C was formed by immobilizing 19 μg Hyd1 on 25 μg BP2000. 2 O / D 2 The compound standards (commercially available) and the reaction mixture in O were run under water peak suppression. B: 1H NMR spectrum corresponding to Scheme 1(ii) (see Example 2). The upper panel shows the spectrum of the authentic product standard (commercially available), while the lower panel shows the spectrum obtained from the reaction mixture. Both spectra were obtained under H 2 O / D 2 O, and water peak suppression was used.

[0061] Fig.12 : HPLC trace detected at 220 nm showing compounds of 2-azido-1-phenylethanone and 2-amino-1-phenylethanone that had been subjected to Boc derivatization at room temperature for 12 hours Standards: Amine (1 eq.), Boc in 1:1 methanol / water (1 mL). 2 O (1.5 equivalents) and NaHCO 3(2.5 equivalents). A 0.2 mL portion of the mixture was extracted into hexane (0.6 mL) containing 2 mM toluene as an internal standard. The solution was filtered using a 22 μm syringe filter (Fischer Scientific, nylon membrane, 13 nm diameter). The peak area was determined by integrating the peaks shown in the chromatogram, and a calibration curve was used to determine the correlation between compound concentration and peak area. The conversion rates listed in Table 2 (Example 4) were determined based on the concentrations of azide and amine calculated using this method.

[0062] Fig.13 : Corresponding to Scheme 3 1 H NMR spectrum (Example 6). Cinnamoyl azide (bottom spectrum) was synthesized from cinnamic alcohol using published procedures. 5 The middle spectrum uses 10 wt % Pd / C instead of Hyd1 / C.

[0063] Fig.14 : Cyclic voltammogram of BP-modified PGE electrode in 100 mM sodium phosphate (pH 6) and 1 mM nitrohexane obtained at a scan rate of 10 mV s-1 at a rotation speed of 0 rpm. The thermodynamic potential of the hydrogen pair is shown as a red dashed line in the original color image, and the overpotential when Hyd1 is running is shown as a blue dashed line in the original color image. Data discussed in Example 10. DETAILED DESCRIPTION

[0064] Method of the present invention

[0065] As described above, the present invention provides a method for reducing functional groups in a target compound, comprising: contacting the target compound with a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material in the presence of a molecular reducing agent for oxidation by an oxidoreductase or a functional fragment or derivative thereof, under the conditions that:

[0066] - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and

[0067] - The functional groups are reduced at the support material.

[0068] The functional group is a reducible functional group. Typically, the functional group is a nitrogen-containing functional group. Suitable nitrogen-containing functional groups are discussed below.

[0069] Therefore, the present invention provides a method for reducing nitrogen-containing functional groups in a target compound, comprising: contacting the target compound with a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material in the presence of a molecular reducing agent for oxidation by an oxidoreductase or a functional fragment or derivative thereof, under the conditions that:

[0070] - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and

[0071] - The nitrogen-containing functional groups are reduced at the support material.

[0072] The methods provided herein include reducing the functional groups of the target compound at the support material. Therefore, these methods generally include contacting the target compound with the support material. Suitable support materials are described in more detail herein.

[0073] Typically, the methods provided herein involve the direct transfer of electrons and / or hydride ions from a carrier material to a target compound. More typically, the methods provided herein involve the direct transfer of electrons from a carrier material to a target compound. Typically, these methods include transferring electrons directly from a carrier material to a reducible functional group of a target compound. In some embodiments, the direct transfer of electrons includes electron tunneling from a carrier material to an atom of a functional group of a target compound. In some embodiments, the reducible functional group of a target compound contacts the surface of a carrier material. Therefore, the direct electron transfer from a carrier material to a functional group of a target compound occurs on a solvent accessible surface of a carrier material, i.e., at the interface between a carrier material and a solvent (e.g., an aqueous reaction solvent). The direct electron transfer from a carrier material to a functional group of a target compound generally does not include contacting the target compound with a reductase (e.g., a nitroreductase) on the carrier material. In other words, the disclosed methods generally do not include contacting the target compound or its reducible functional group with an enzyme fixed on a carrier material. Therefore, it can be considered that the carrier material provides an "active site" (using a term similar to enzymology) for the reduction of reducible functional groups.

[0074] The electron transfer from the support material to the functional group is generally not mediated by an enzyme or its cofactors. The cofactors are as described above. In other words, the disclosed methods generally do not include the transfer of electrons to the target compound via one or more cofactors.

[0075] In particular, electron transfer from the support material to the functional group is not typically used for enzymatic catalysis of the reduction of the functional group of the target compound. Thus, for example, the supported biocatalyst typically does not include a reductase capable of catalyzing the reduction (e.g., complete reduction) of the functional group. In other words, the supported biocatalyst typically does not include an oxidoreductase or a functional fragment or derivative thereof that includes an active site (e.g., a substrate binding active site) capable of catalyzing (e.g., naturally catalyzing) the enzymatic reduction of a nitrogen-containing functional group, such as a nitroreductase.

[0076] Thus, it is clear that, as used herein, direct electron transfer from a support material to a functional group does not include enzymatic reduction of the functional group, such as using a nitroreductase, a cytochrome P450 monooxygenase, a nitrile reductase, an azoreductase, etc. Thus, typically the biocatalyst does not comprise a nitroreductase. Similarly, the biocatalyst typically does not comprise an azoreductase or a nitrile reductase or other enzymes comprising an active site (e.g., a substrate binding active site) capable of reducing a reducible functional group. In some embodiments, the supported biocatalyst consists of an oxidoreductase as described herein supported on a support.

[0077] Therefore, it is important to distinguish the oxidoreductase for catalyzing the oxidation of molecular reductants from the reductase having an active site (such as an active site for binding substrate) for the reduction of the reducible functional group of the target compound. As used herein, the oxidoreductase for catalyzing the oxidation of molecular reductants is not an example of a reductase for reducing the reducible functional group of the target compound. For example, the oxidoreductase for catalyzing the oxidation of molecular reductants does not generally include an active site (such as an active site for binding substrate) that can (such as naturally) accommodate the target compound or its reducible functional group. The oxidoreductase for catalyzing the oxidation of molecular reductants may only operate under potentials or reaction conditions that are incompatible with the enzymatic reduction of the reducible functional group of the target compound by reductases (such as nitroreductases, azoreductases or nitrile reductases). For example, the reductase for reducing reactive functional groups generally requires cofactors as described herein (such as NADH, NAD (P) H or flavin (such as FAD, FMN or riboflavin)) to mediate electron transfer to its oxidized substrate. Typically, in the method herein, there is no such cofactor. Thus, typically, the disclosed methods do not involve the use of cofactors in the reaction medium (eg, in the reaction solution).

[0078] As described above, the target compound contains a reducible functional group. Typically, the reducible functional group is a nitrogen-containing functional group. Examples of suitable nitrogen-containing functional groups are described in detail herein. It will be apparent from the discussion herein that these groups include: nitro (R-NO 2 ), azido (RN 3 ), hydroxylamine (R-NR'OH), nitroso (R-NO), nitrile (R-CN), diazo (R-CR'=N 2 ), diazonium (RN 2 + ), isonitrile (R-NC); isothiocyanate (R-NCS), isocyanate (RN=C=O), hydrazone (R-CR'=N-NR'), hydrazine (R-NR'-NR' 2 ), amidino (RC(NR')NR' 2), azo (RN = N = R'), and guanidine (R-NR'-C(NR')-NR' 2 ) etc.; wherein R represents the remaining part of the target compound, and each R' independently represents a group, such as H or a hydrocarbon group, such as an alkyl group, such as C 1-6 or C 1-4 Alkyl, such as methyl or ethyl; typically H. More typically, the nitrogen-containing functional group is a nitro group (such as an aromatic nitro group); an azide group (such as an aromatic azide group) or a nitrile group (such as an aromatic nitrile group). Still more typically, the nitrogen-containing functional group is a nitro group (such as an aromatic nitro group) or an azide group (such as an aromatic azide group); most typically, the nitrogen-containing functional group is a nitro group (such as an aromatic nitro group).

[0079] Generally, the methods disclosed herein include substantially complete or complete reduction of a reducible functional group. As used herein, the term "complete reduction" means that a reducible functional group (e.g., a nitrogen atom in a reducible functional group) is reduced to the lowest stable oxidation state. For example, complete reduction of a nitro group corresponds to a nitro group (R-NO 2 ) is reduced to an amine group (R-NH 2 ) or quaternary ammonium group (R-NH 3 + ), while partial reduction of the nitro group can lead to the formation of, for example, hydroxylamine (R-NHOH). Thus, for example, when the reducible functional group is a nitro group (R-NO 2 ) (e.g., an aromatic nitro group), the method generally comprises reducing the nitro group to an amine group or a quaternary ammonium group (R-NH 3 + )(wherein R is the remainder of the target compound). When the reducible functional group is an azido group (RN 3 ) (e.g., an aromatic nitrile group), the method generally comprises reducing the azide group to an amine group or a quaternary ammonium group (R—NH 3 + ) (wherein R is the remainder of the target compound). When the reducible functional group is a nitrile group (R—CN) (e.g., an aromatic nitrile group), the method generally includes reducing the nitrile group to an amine group or a quaternary ammonium group (e.g., R—CR′ 2 NH 3 + )(wherein R is the remainder of the target compound).

[0080] As defined in more detail herein, the method may comprise contacting a sample comprising a plurality of target compounds with a biocatalyst in the presence of a molecular reducing agent. Typically, at least 50%, such as at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.9% or at least 99.99% of the target compounds in the sample may be completely reduced at the nitrogen-containing functional groups.

[0081] The methods disclosed herein are generally highly selective. Generally, the methods disclosed herein are selective for the reduction of nitrogen-containing functional groups, even in the presence of a C=C double bond. For example, the method may include contacting a target compound comprising a nitrogen-containing reducible functional group and a C=C double bond with a biocatalyst as described herein. In such embodiments, generally the nitrogen-containing reducible functional group is reduced and the C=C double bond is not reduced. This is described in more detail in the Examples. This may represent a particular advantage of the disclosed methods, since such selective reduction is generally not possible with noble metal catalysts such as palladium.

[0082] reducing agent

[0083] It is apparent from the above discussion that the disclosed methods involve oxidation of a molecular reducing agent by an oxidoreductase or a functional fragment or derivative thereof. Suitable enzymes are described in more detail herein.

[0084] Typically, the molecular reducing agent is selected from hydrogen (H 2 ), carbon monoxide (CO), formate (deprotonated to CH(O)O - or protonated HC(O)OH, also known as formic acid), isotopes thereof, and mixtures thereof. The use of such reducing agents can produce particular benefits because their oxidation products can be easily removed from the reaction mixture and / or do not need to be removed. Thus, the protons formed by the oxidation of molecular hydrogen generally do not need to be removed from the reaction mixture. The carbon dioxide produced by the oxidation of CO or formate can be easily removed, for example, as a gas. Thus, reactions using such reducing agents can be clean and do not require extensive purification. Typically, the molecular reducing agent is selected from hydrogen and carbon monoxide, isotopes thereof, and mixtures thereof. More typically, the molecular reducing agent is hydrogen (i.e., molecular hydrogen) or an isotope thereof.

[0085] In some embodiments, the reducing agent used in the disclosed methods is determined by the oxidoreductase contained in the biocatalyst. In other embodiments, the oxidoreductase contained in the biocatalyst is determined by the reducing agent used. In view of the present disclosure, it will be straightforward for those skilled in the art to select compatible oxidoreductase / reductant pairs. Exemplary combinations of oxidoreductases and reducing agents are shown in the following table:

[0086] Oxidoreductase Molecular reducing agent Hydrogenase or its functional fragment or derivative Molecular hydrogen or its isotopes Carbon monoxide dehydrogenase or its functional fragment or derivative Carbon monoxide or its isotopes Formate dehydrogenase or its functional fragment or derivative Formate or its isotopes

[0087] Mixtures of oxidoreductases and corresponding molecular reductants can be used. For example, the biocatalyst may comprise a mixture of one or more hydrogenases (or functional fragments or derivatives thereof) and one or more carbon monoxide dehydrogenases (or functional fragments or derivatives thereof), and the molecular reductant may comprise a mixture of molecular hydrogen (or its isotope) and carbon monoxide (or its isotope). The biocatalyst may comprise a mixture of one or more hydrogenases (or functional fragments or derivatives thereof) and one or more formate dehydrogenases (or functional fragments or derivatives thereof), and the molecular reductant may comprise a mixture of molecular hydrogen (or its isotope) and formate (or its isotope). The biocatalyst may comprise a mixture of one or more carbon monoxide dehydrogenases (or functional fragments or derivatives thereof) and one or more formate dehydrogenases (or functional fragments or derivatives thereof), and the molecular reductant may comprise a mixture of carbon monoxide (or its isotope) and formate (or its isotope).

[0088] When the molecular reducing agent is hydrogen, suitable isotopes thereof include 1 H 2 , 2 H 2 and 3 H 2 Mixed isotopes (e.g. 1 H 2 H and 1 H 3 H) is also included. Preferably, hydrogen is 1 H 2 It will be apparent that, as used herein, organic molecules such as glucose, formate, and ethanol, isopropanol, etc. are not sources of molecular hydrogen, although in some embodiments, these compounds may be used as molecular reducing agents, as described in more detail herein.

[0089] Typically, when the reducing agent is molecular hydrogen, the molecular hydrogen is provided in gaseous form. The gas may be mixed with an aqueous solution in which the biocatalyst and the target compound and optionally other reaction components are present. 2 Under pressure, H 2 The solubility in water is 0.8 mM. In other words, when the reductant is molecular hydrogen provided in the form of molecular hydrogen gas, the concentration of hydrogen in the solution (i.e., the concentration at which the oxidoreductase works) is 0.8 mM hydrogen. Other pressures may also be used. For example, the gas pressure in the reaction vessel may be 0.01 to about 100 bar, such as 0.1 to 10 bar, such as about 0.2 to about 5 bar, such as 0.5 to 2 bar, such as about 1 bar. Increasing the gas pressure will increase the concentration of hydrogen in the reaction solution. Reducing the gas pressure will reduce the concentration of hydrogen in the reaction solution.

[0090] For the avoidance of doubt, molecular hydrogen may also be provided in the form of a solution in which the molecular hydrogen is dissolved (eg an aqueous solution, eg comprising a buffer salt as described in more detail herein).

[0091] When the reducing agent is molecular hydrogen, hydrogen can be reacted with other gases (such as CO, CO 2 , air, O 2 、N 2 When provided as a mixture, the mixture may contain about 0.1% to about 99% hydrogen, such as 1% to about 95%, for example about 2% to about 80% H 2 , such as about 5% to about 50% H 2 An exemplary mixture may include, for example, 50% hydrogen and 50% N 2 .

[0092] When the reducing agent is molecular hydrogen, the hydrogen can be of any suitable purity. For example, when it is important to control the level of impurities in the final product mixture, hydrogen with a purity of 99% or more (e.g., 99.9%, 99.99% or 99.999%) can be used. In other aspects, when it is not very important to control the level of impurities in the final product mixture, hydrogen of lower purity can be used. For example, relatively low-purity hydrogen can be provided in the form of "synthesis gas". Synthesis gas produced by coal gasification is typically a mixture of 30%-60% carbon monoxide, 25%-30% hydrogen, 5%-15% carbon dioxide and 0%-5% methane, and may also optionally contain smaller amounts of other gases. Waste hydrogen streams from other industrial processes can also be used generally. Therefore, the method of the present disclosure is ideally suited for use as part of a larger reaction sequence involving a reaction step containing hydrogen as a reactant or product, wherein the waste hydrogen from the reaction step is used as a molecular reducing agent in the disclosed method.

[0093] When the reducing agent is molecular hydrogen or an isotope thereof, the molecular hydrogen or an isotope thereof may be provided by any suitable source, such as a gas cylinder. Alternatively, the molecular hydrogen or an isotope thereof may be generated in-situ, for example by electrolysis of water.

[0094] When the molecular reducing agent is carbon monoxide, suitable isotopes thereof include 12 C 16 O. 13 C 16 O. 14 C 16 O. 12 C 17 O. 13 C 17 O. 14 C 17 O. 12C 18 O. 13 C 18 O and 14 C 18 O. Preferably, carbon monoxide is 12 C 16 O.

[0095] Usually, when the reductant is carbon monoxide, carbon monoxide is provided in gaseous form. The gas can be mixed with an aqueous solution in which there are biocatalysts and target compounds and optional other reaction components. At 1 bar CO pressure, the solubility of CO in water is 0.95 mM. In other words, when the reductant is carbon monoxide provided in the form of molecular carbon monoxide gas, the concentration of carbon monoxide in the solution (i.e., the concentration at which the oxidoreductase works) is 0.95 mM carbon monoxide. Other pressures can also be used. For example, the gas pressure in the reaction vessel can be 0.01 to about 100 bar, such as 0.1 to 10 bar, for example, about 0.2 to about 5 bar, for example, 0.5 to 2 bar, for example, about 1 bar. Increasing the gas pressure will increase the concentration of carbon monoxide in the reaction solution. Reducing the gas pressure will reduce the concentration of carbon monoxide in the reaction solution.

[0096] For the avoidance of doubt, the carbon monoxide may also be provided in the form of a solution in which the carbon monoxide is dissolved (eg an aqueous solution, eg comprising a buffer salt as described in more detail herein).

[0097] When the reducing agent is carbon monoxide, the carbon monoxide can be in the form of carbon monoxide and other gases (such as H 2 , CO 2 , air, O 2 、N 2 When provided as a mixture, the mixture may contain about 0.1% to about 99% CO, such as 1% to about 95%, for example about 2% to about 80% H 2 , such as about 5% to about 50% CO. An exemplary mixture may include, for example, 50% CO and 50% N 2 .

[0098] When the reducing agent is CO, the CO can be of any suitable purity. For example, when it is important to control the level of impurities in the final product mixture, CO having a purity of 99% or higher (e.g., 99.9%, 99.99%, or 99.999%) can be used. In other aspects, when it is not important to control the level of impurities in the final product mixture, CO of lower purity can be used. For example, as described above, relatively low purity CO can be provided in the form of "syngas." Waste CO streams from other industrial processes can also generally be used. Therefore, the methods of the present disclosure are ideally suited for utilization as part of a larger reaction sequence that includes a reaction step that includes CO as a reactant or product, wherein the waste CO from the reaction step is used as a molecular reducing agent in the disclosed methods.

[0099] When the reducing agent is CO or an isotope thereof, the CO or an isotope thereof may be provided from any suitable source, such as a gas cylinder.

[0100] When the molecular reducing agent is formate, suitable isotopes thereof include carbon isotopes such as 12 C-formate, 13 C-Formate and 14 C-formate; oxygen isotopes, such as 16 O-Formate, 17 O-Formate and 18 O-formate; and hydrogen isotopes, such as 1 H formate, 2 H formate and 3 Preferably, the formate is 12 C. 16 O and 1 H composition.

[0101] Typically, when the reducing agent is a formate, the formate is provided in the form of a metal formate or formic acid. Examples of formate include lithium formate, sodium formate, potassium formate, magnesium formate, calcium formate, ammonium formate, and the like. The formic acid may be provided as an aqueous solution comprising from about 1% to about 99% formic acid, or may be provided in a substantially pure form (e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.9% purity).

[0102] When the reducing agent is formate (or formic acid), formate (or formic acid) may be present in the reaction solution at a concentration of about 1 μM to about 1 M, such as about 10 μM to about 100 mM, such as about 100 μM to about 10 mM, such as about 1 mM. Other components may also be present, such as buffer salts, etc.

[0103] Oxidoreductase

[0104] As discussed in more detail herein, in the disclosed methods, the target compound is contacted with an oxidoreductase or a functional fragment or derivative thereof supported on a support material. The support material is typically conductive or semiconductive. Suitable support materials are described in more detail herein.

[0105] The oxidoreductase is usually in electrical contact with the support material. Typically, the active site of the oxidoreductase is in electronic communication with the support material. In other words, the biocatalyst is configured so that electrons can be transferred from the active site of the oxidoreductase (i.e., the active site that catalyzes the oxidation reaction of the molecular reductant) to the support material.

[0106] Typically, electrons can be transferred from an oxidoreductase (e.g., from the active site of an oxidoreductase) to a support material via an intramolecular electron conduction pathway. In other words, the electron transfer from a molecular reducing agent to a support material by an oxidoreductase is typically a direct electron transfer. Although the present invention does include the use of an electron mediator (e.g., a redox-active dye such as methyl or benzyl viologen) to mediate the electron transfer from a molecular reducing agent to a support material, the electron transfer is typically not mediated by an electron transfer agent (e.g., a mediator), e.g., typically not mediated by a redox-active dye (e.g., methyl or benzyl viologen).

[0107] Typically, electron transfer from a molecular reductant to a support material includes direct electron transfer by an oxidoreductase. Typically, direct electron transfer is carried out by an intramolecular electron conduction pathway. Typically, the intramolecular electron conduction pathway includes a series of [FeS] clusters. As will be appreciated by those skilled in the art, [FeS]-clusters include [3Fe4S] and [4Fe4S] clusters. [FeS] clusters can be described as distal clusters, proximal clusters, or intermediate clusters. The symbols "distal" and "proximal" in the context are conventional in the art. For proteins comprising an active site and a series or series of [FeS] clusters, the proximal cluster is the [FeS] cluster closest to the active site. The distal cluster is the [FeS] cluster closest to the solvent accessible surface of the protein, and is therefore the farthest from the active site. The [FeS] cluster between the proximal cluster and the distal cluster is referred to as an intermediate cluster. The distal cluster is typically solvent accessible. Thus, in some embodiments, electron transfer is carried out through a series of [FeS] clusters from the active site to the proximal cluster, from the proximal cluster to the distal cluster (optionally through one or more intermediate clusters), and from the distal cluster to the support material. Without being bound by theory, each step in the electron transfer can be an electron tunneling step.

[0108] In some embodiments, the oxidoreductase comprises a natural electron transfer partner, such as a cytochrome (such as the cytochrome of SEQ ID NO: 22, or a functional fragment, derivative or variant thereof). The cytochrome may be included in the oxidoreductase at a position for electron transfer to a distal [FeS] cluster. In other words, the cytochrome may be located at the end of a series of [FeS] clusters.

[0109] Typically, the oxidoreductase is a hydrogenase, a carbon monoxide dehydrogenase and / or a formate dehydrogenase, or a functional fragment or derivative thereof.

[0110] Preferably, the oxidoreductase is selected or modified to catalyze a reaction close to the thermodynamic reduction potential E of the reducing agent. o For example, when the reducing agent is molecular hydrogen, the oxidoreductase is preferably a hydrogenase and is preferably selected or modified to catalyze the oxidation of a molecular reducing agent close to 2H under the experimental conditions. + / H 2 Thermodynamic reduction potential E o (“E o (2H + / H 2 (Those skilled in the art will appreciate that at 25°C, pH 7.0 and 1 bar H 2 Next, E o (2H + / H 2 )=-0.413V, and varies according to the Nernst equation). Preferably, the oxidoreductase is selected or modified to o (2H + / H 2 ) is less than 100 mV, more preferably at an applied potential greater than E o (2H + / H 2 ) corrected for catalytic H at an applied potential less than 50 mV 2 or x H 2 Oxidation. Under the experimental conditions discussed, the enzyme catalyzed o (2H + / H 2 ) 2 The method for measuring the oxidation capacity is a conventional method for those skilled in the art, for example, as described in the literature by Vincent et al. (J. Am. Chem. Soc. (2005) 127, 18179-18189).

[0111] When the reductant is CO, the oxidoreductase is preferably a carbon monoxide dehydrogenase (CODH) and is preferably selected or modified to catalyze the reaction of CO with CO under the experimental conditions. 2 Thermodynamic reduction potential E of the / CO pairo (“E o (CO 2 / CO)”). (Those skilled in the art will appreciate that at 25°C, E o (CO 2 Preferably, the oxidoreductase is selected or modified to react at a ratio of E o (CO 2 / CO) is less than 100 mV, more preferably at an applied potential greater than E o (CO 2 Similarly, when the reductant is formate, the oxidoreductase is preferably a formate dehydrogenase and is preferably selected or modified to catalyze the oxidation of CO at an applied potential of less than 50 mV corrected for CO. 2 / Thermodynamic reduction potential E of formate o (“E o (CO 2 / formate)”). (Those skilled in the art will appreciate that at 25°C, E o (CO 2 / formate) = -0.42 V). Preferably, the oxidoreductase is selected or modified to react at a ratio of E o (CO 2 / formate) at an applied potential less than 100 mV, more preferably at a potential greater than E o (CO 2 catalyzes formate oxidation at an applied potential less than 50 mV corrected for formate.

[0112] In some embodiments, the oxidoreductase is selected or modified to oxidize the reducing agent at a potential that is more negative than the reduction potential of the target compound.

[0113] Oxidoreductases are often selected or modified to be highly active. For example, oxidoreductases can be selected or modified to have efficient substrate conversion.

[0114] The enzyme turnover rate can be calculated in a variety of ways. The Total Turnover Number (TTN, also referred to as TON) is a measure of the number of moles of product per mole of enzyme. As will be appreciated by those skilled in the art, TTN therefore indicates the number of times the enzyme has turned over (i.e. has oxidized molecules of a molecular reducing agent). Preferably, the TTN of the oxidoreductase is at least 10, such as at least 100, more preferably at least 1000, for example at least 10,000 or at least 100,000, such as at least 1,000,000, preferably at least 10 7 , such as at least 10 8 , for example at least 10 9 .

[0115] Turnover Frequency (TOF) is a measure of the number of moles of product produced per second per mole of enzyme present. Thus, in the methods provided herein, TOF represents the number of moles of oxidized reducing agent produced per second per mole of oxidoreductase. Thus, TOF can be determined by the number of catalytic cycles performed per enzyme molecule per second. Preferably, in the methods of the present invention, the first polypeptide has a 0.1s -1 Up to 1000s -1 TOF, preferably 1s -1 Up to 100s -1 , such as about 10s -1 To about 50s -1 .

[0116] When the supported biocatalyst comprises a hydrogenase, any suitable hydrogenase may be used. The hydrogenase may comprise an active site comprising an iron atom (such as in [FeFe]-hydrogenase) or a nickel and iron atom (such as in [NiFe]- and [NiFeSe]-hydrogenase). Preferably, the hydrogenase comprises an active site comprising a nickel and an iron atom. Suitable proteins are described below.

[0117] When the supported biocatalyst comprises a hydrogenase, the hydrogenase is typically an uptake hydrogenase or a hydrogen-sensitive hydrogenase. Uptake hydrogenases are used in vivo by organisms to generate energy by oxidizing molecular hydrogen in their environment. In vivo, they convert H 2 Oxidation of anaerobic receptors (such as nitrate and sulfate or O 2 ). Typically, hydrogen uptake enzymes contain a signal peptide (usually about 30 to about 60 amino acid residues in length) at the N-terminus of the small subunit. Typically, the signal peptide contains a [DENST]RRxFxK motif. Hydrogen-sensitive hydrogenases (also called regulatory hydrogenases) are used by organisms in vivo to sense hydrogen levels in order to control the response to H 2 The biosynthesis of hydrogenases. Regulatory hydrogenases usually do not contain signal peptides that are unique to hydrogenases. Regulatory hydrogenases usually 2 Insensitive.

[0118] Typically, hydrogenases used in the disclosed methods are selected or modified to be oxygen-tolerant. Oxygen-tolerant hydrogenases are capable of oxidizing H in the presence of oxygen. 2 or x H 2 , such as at least 0.01% O 2 , preferably at least 0.1% O 2 , more preferably at least 1% O 2 , such as at least 5% O 2 , for example at least 10% O 2 , such as at least 20% O2 or more 2 while retaining at least 1%, preferably at least 5%, such as at least 10%, preferably at least 20%, more preferably at least 50%, such as at least 80%, such as at least 90%, preferably at least 95%, such as at least 99% of their H 2 Oxidative Activity. Various oxygen-tolerant hydrogenases are known to those skilled in the art.

[0119] Typically, the hydrogenases used in the disclosed methods do not contain a catalytic enzyme for NAD(P) + Reduced natural flavin active sites (also called prosthetic groups), although such hydrogenases are not excluded. Some known hydrogenases do contain such active sites, such as soluble hydrogenases (SH) from R. eutropha, Rhodococcus opacus, Hydrogenophilus thermoluteolus, and Pyrococcus furiosus. However, without being bound by theory, it is believed that hydrogenases lacking such prosthetic groups generally have increased stability compared to hydrogenases containing such prosthetic groups. Examples of hydrogenases lacking a flavin prosthetic group include: Escherichia coli hydrogenase 1 (SEQ ID NOs: 1-2), Escherichia coli hydrogenase 2 (SEQ ID NOs: 3-4), Ralstonia eutropha membrane-bound hydrogenase (SEQ ID NOs: 5-7), Ralstonia eutropha regulatory hydrogenase (SEQ ID NOs: 8-9), Aquifex aeolicus hydrogenase 1 (SEQ ID NOs: 10-11), and Hydrogenovibrio marinus membrane-bound hydrogenase (SEQ ID NOs: 12-13).

[0120] When the supported biocatalyst comprises a hydrogenase, the hydrogenase is typically a class 1 or class 2b hydrogenase. Reference to a class of hydrogenases such as class 1 or class 2b refers to the validated Vignais classification scheme described by Vignais and Billoud (Chem. Rev. 2007, 107, 4206-4272), which is known to those skilled in the art. The hydrogenase may be any of the class 1 or class 2b hydrogenases listed in Vignais and Billoud (Chem. Rev. 2007, 107, 4206-4272), the contents of which are incorporated herein by reference.

[0121] Preferably, when the supported biocatalyst comprises a hydrogenase, the hydrogenase is selected from or includes:

[0122] i) the amino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2) or an amino acid sequence having at least 60% homology thereto;

[0123] ii) the amino acid sequence of Escherichia coli hydrogenase 2 (SEQ ID NO: 3 and / or 4) or an amino acid sequence having at least 60% homology thereto;

[0124] iii) an amino acid sequence of a portion of the membrane-bound hydrogenase of Ralstonia eutropha (SEQ ID NO: 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto;

[0125] iv) an amino acid sequence of a portion of the regulatory hydrogenase of Ralstonia eutropha (SEQ ID NO: 8 and / or 9) or an amino acid sequence having at least 60% homology thereto;

[0126] v) the amino acid sequence of a thermophilic hydrogenase 1 (SEQ ID NO: 10 and / or 11) or an amino acid sequence having at least 60% homology thereto;

[0127] vi) the amino acid sequence of a hydrogenase of Hydrogenovibrio maritima (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto;

[0128] vii) the amino acid sequence of a Thiocapsa roseopersicina hydrogenase (SEQ ID NOs: 14 and 15) or an amino acid sequence having at least 60% homology thereto;

[0129] viii) the amino acid sequence of the hydrogenase from Alteromonas macleodii (SEQ ID NO: 16 and / or

[0130] or 17) or an amino acid sequence having at least 60% homology thereto;

[0131] ix) the amino acid sequence of Allochromatium vinosum membrane-bound hydrogenase (SEQ ID NO: 18 and / or 19) or an amino acid sequence having at least 60% homology thereto;

[0132] x) the amino acid sequence of Salmonella enterica serovar Typhimurium LT2 nickel-iron hydrogenase 5 (SEQ ID NO: 20 and / or 21) or an amino acid sequence having at least 60% homology thereto;

[0133] xi) the amino acid sequence of Desulfovibrio vulgaris Miyazaki F hydrogenase (SEQ ID NO: 23 and / or 24) or an amino acid sequence having at least 60% homology thereto;

[0134] xii) the amino acid sequence of Clostridium beijerinckii SM10 (CbA5H) [FeFe]-hydrogenase (KX147468) (SEQ ID NO: 25), the amino acid sequence of Clostridium beijerinckii ATCC 51743 [FeFe]-hydrogenase (Cbei_4110) (SEQ ID NO: 26), the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_1773) (SEQ ID NO: 27), or the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_3796) (SEQ ID NO: 28), or an amino acid sequence that is at least 60% homologous thereto;

[0135] xiii) the amino acid sequence of Clostridium pasteurianum [FeFe]-hydrogenase (hydA) (SEQ ID NO: 29) or an amino acid sequence at least 60% homologous thereto;

[0136] xiv) the amino acid sequence of Chlamydomonas reinhardtii [FeFe]-hydrogenase (hyd1) (SEQ ID NO: 30) or an amino acid sequence having at least 60% homology thereto;

[0137] xv) the amino acid sequence of Chlorella variabilis [FeFe]-hydrogenase (SEQ ID NO: 31 and / or 32) or an amino acid sequence having at least 60% homology thereto;

[0138] xvi) an amino acid sequence of a portion of a soluble hydrogenase from Ralstonia eutropha (SEQ ID NO: 33 and / or 34) or an amino acid sequence having at least 60% homology thereto;

[0139] xvii) an amino acid sequence of a portion of a soluble hydrogenase from Rhodococcus opacus (SEQ ID NO: 35 and / or 36) or an amino acid sequence having at least 60% homology thereto;

[0140] xviii) the amino acid sequence of Desulfovibrio fructosovorans membrane-bound hydrogenase (SEQ ID NO: 37 and / or 38) or an amino acid sequence having at least 60% homology thereto;

[0141] xix) the amino acid sequence of Clostridium acetobutylicum iron-ferrohydrogenase (SEQ ID NO: 39) or an amino acid sequence having at least 60% homology thereto;

[0142] xx) Amino acid sequence of nickel-iron selenide hydrogenase from Desulfomicrobium baculatum (SEQ ID NO:

[0143] 40 and / or 41) or an amino acid sequence having at least 60% homology thereto;

[0144] xxi) an amino acid sequence of a portion of a soluble hydrogenase from Hydrogenophilus thermoluteolus (SEQ ID NO: 42 and / or 43) or an amino acid sequence having at least 60% homology thereto;

[0145] xxii) Amino acid sequence of Desulfovibrio gigas periplasmic [NiFe] hydrogenase (SEQ ID NO: 44

[0146] and / or 45) or an amino acid sequence having at least 60% homology thereto; or

[0147] xxiii) Amino acid sequence of the soluble α subunit of Pyrococcus furiosus (SEQ ID NO: 46)

[0148] or an amino acid sequence having at least 60% homology thereto;

[0149] or a functional fragment, derivative or variant thereof.

[0150] More preferably, when the supported biocatalyst comprises a hydrogenase, the hydrogenase is selected from or comprises:

[0151] i) the amino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2) or an amino acid sequence having at least 60% homology thereto;

[0152] ii) the amino acid sequence of Escherichia coli hydrogenase 2 (SEQ ID NO: 3 and / or 4) or an amino acid sequence having at least 60% homology thereto;

[0153] iii) an amino acid sequence of a portion of the membrane-bound hydrogenase of Ralstonia eutropha (SEQ ID NO: 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto;

[0154] iv) an amino acid sequence of a portion of the regulatory hydrogenase of Ralstonia eutropha (SEQ ID NO: 8 and / or 9) or an amino acid sequence having at least 60% homology thereto;

[0155] v) the amino acid sequence of a thermophilic hydrogenase 1 (SEQ ID NO: 10 and / or 11) or an amino acid sequence having at least 60% homology thereto;

[0156] vi) the amino acid sequence of a hydrogenase of Hydrogenovibrio maritima (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto;

[0157] vii) the amino acid sequence of a Sulphuria auberginea hydrogenase (SEQ ID NOs: 14 and 15) or an amino acid sequence having at least 60% homology thereto;

[0158] viii) the amino acid sequence of an Alteromonas mesquite hydrogenase (SEQ ID NO: 16 and / or 17) or an amino acid sequence having at least 60% homology thereto;

[0159] ix) the amino acid sequence of Allochromatium vinosum membrane-bound hydrogenase (SEQ ID NO: 18 and / or 19) or an amino acid sequence having at least 60% homology thereto;

[0160] x) the amino acid sequence of Salmonella typhimurium LT2 nickel-iron hydrogenase 5 (SEQ ID NO: 20 and / or 21) or an amino acid sequence having at least 60% homology thereto;

[0161] xi) the amino acid sequence of Desulfovibrio vulgaris F hydrogenase from Miyazaki (SEQ ID NO: 23 and / or 24) or an amino acid sequence having at least 60% homology thereto;

[0162] xii) the amino acid sequence of Clostridium beijerinckii SM10 (CbA5H) [FeFe]-hydrogenase (KX147468) (SEQ ID NO: 25), the amino acid sequence of Clostridium beijerinckii ATCC 51743 [FeFe]-hydrogenase (Cbei_4110) (SEQ ID NO: 26), the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_1773) (SEQ ID NO: 27), or the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_3796) (SEQ ID NO: 28), or an amino acid sequence that is at least 60% homologous thereto; or

[0163] xiii) the amino acid sequence of Clostridium acetobutylicum iron-ferrohydrogenase (SEQ ID NO: 39) or an amino acid sequence having at least 60% homology thereto.

[0164] Still more preferably, when the supported biocatalyst comprises a hydrogenase, the hydrogenase is selected from or comprises:

[0165] i) the amino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2) or an amino acid sequence having at least 60% homology thereto;

[0166] ii) the amino acid sequence of Escherichia coli hydrogenase 2 (SEQ ID NO: 3 and / or 4) or an amino acid sequence having at least 60% homology thereto;

[0167] iii) an amino acid sequence of a portion of the membrane-bound hydrogenase of Ralstonia eutropha (SEQ ID NO: 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto;

[0168] iv) an amino acid sequence of a thermophilic hydrogenase 1 (SEQ ID NO: 10 and / or 11) or an amino acid sequence having at least 60% homology thereto;

[0169] v) the amino acid sequence of a hydrogenase from Hydrogenovibrio maritima (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto;

[0170] vi) the amino acid sequence of Alteromonas mesquite hydrogenase (SEQ ID NO: 16 and / or 17) or an amino acid sequence having at least 60% homology thereto; or

[0171] vii) the amino acid sequence of Salmonella typhimurium LT2 nickel-iron hydrogenase 5 (SEQ ID NO: 20 and / or 21) or an amino acid sequence having at least 60% homology thereto;

[0172] or a functional fragment, derivative or variant thereof.

[0173] Most preferably, when the supported biocatalyst comprises a hydrogenase, the hydrogenase comprises: the amino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2) or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0174] Also most preferably, in another embodiment, when the supported biocatalyst comprises a hydrogenase, the hydrogenase comprises: the amino acid sequence of Clostridium beijerinckii SM10 (CbA5H) [FeFe]-hydrogenase (KX147468) (SEQ ID NO: 25), the amino acid sequence of Clostridium beijerinckii ATCC 51743 [FeFe]-hydrogenase (Cbei_4110) (SEQ ID NO: 26), the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_1773) (SEQ ID NO: 27) or the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_3796) (SEQ ID NO: 28), or an amino acid sequence having at least 60% homology thereto.

[0175] Preferably, when the supported biocatalyst comprises carbon monoxide dehydrogenase, the carbon monoxide dehydrogenase is selected from or includes:

[0176] i) the amino acid sequence of Hildenborough Desulfovibrio vulgaris carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 47) or an amino acid sequence having at least 60% homology thereto;

[0177] ii) the amino acid sequence of Desulfovibrio miyazaki vulgaris carbon monoxide dehydrogenase (DvMF) (SEQ ID NO: 48) or an amino acid sequence having at least 60% homology thereto;

[0178] iii) the amino acid sequence of Desulfovibrio psychrotolerans carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 49) or an amino acid sequence having at least 60% homology thereto;

[0179] iv) the amino acid sequence of Desulfoluna spongiiphila carbon monoxide dehydrogenase (SAMN05216233) (SEQ ID NO: 50) or an amino acid sequence having at least 60% homology thereto;

[0180] v) the amino acid sequence of Halodesulfovibrio spirochaetisodalis carbon monoxide dehydrogenase (SP90) (SEQ ID NO: 51) or an amino acid sequence having at least 60% homology thereto;

[0181] vi) Amino acid sequence of Desulfovibrio desulfuricans carbon monoxide dehydrogenase (Ddes)

[0182] (SEQ ID NO: 52) or an amino acid sequence having at least 60% homology thereto;

[0183] vii) Amino acid sequence of Desulfurivibrio alkaliphilus carbon monoxide dehydrogenase (DaAHT2)

[0184] (SEQ ID NO: 53) or an amino acid sequence having at least 60% homology thereto;

[0185] viii) the amino acid sequence of Pseudodesulfovibrio aespoeensis carbon monoxide dehydrogenase (Daes) (SEQ ID NO: 54) or an amino acid sequence having at least 60% homology thereto;

[0186] ix) the amino acid sequence of Desulfovibrio alaskensis carbon monoxide dehydrogenase (Dde_3028) (SEQ ID NO: 55) or an amino acid sequence having at least 60% homology thereto;

[0187] x) the amino acid sequence of Desulfovibrio ferrophilus carbon monoxide dehydrogenase (DFE_2686) (SEQ ID NO: 56) or an amino acid sequence having at least 60% homology thereto;

[0188] xi) the amino acid sequence of Carboxydothermus hydrogenoformans carbon monoxide dehydrogenase 2 (cooS2) (SEQ ID NO: 57) or an amino acid sequence having at least 60% homology thereto;

[0189] xii) Amino acid sequence of carbon monoxide dehydrogenase (cooS) from Desulfofundulus salinum (SEQ ID

[0190] NO: 58) or an amino acid sequence having at least 60% homology thereto;

[0191] xiii) the amino acid sequence of Caldanaerobacter subterraneus carbon monoxide dehydrogenase (TTE1708) (SEQ ID NO: 59) or an amino acid sequence having at least 60% homology thereto;

[0192] xiv) Carbon monoxide dehydrogenase from Thermanaeromonas toyohensis

[0193] (SAMN00808754_0706) (SEQ ID NO: 60), or an amino acid sequence having at least 60% homology thereto;

[0194] xv) Amino acid sequence of Desulfobulbus sp. carbon monoxide dehydrogenase (JT06_17280) (SEQ ID

[0195] NO: 61) or an amino acid sequence having at least 60% homology thereto;

[0196] xvi) the amino acid sequence of Desulfotomaculum copahuensis carbon monoxide dehydrogenase (A6M21_00615) (SEQ ID NO: 62) or an amino acid sequence having at least 60% homology thereto;

[0197] xvii) the amino acid sequence of Pelotomaculum propionicicum carbon monoxide dehydrogenase (cooS2) (SEQ ID NO: 63) or an amino acid sequence having at least 60% homology thereto;

[0198] xviii) Amino acid sequence of carbon monoxide dehydrogenase (cooS2) from anaerobic methylomusa anaerophila (SEQ

[0199] ID NO: 64) or an amino acid sequence having at least 60% homology thereto;

[0200] xix) Amino acid sequence of carbon monoxide dehydrogenase (cooS2) from Sporomusa silvacetica (SEQ ID

[0201] NO: 65) or an amino acid sequence having at least 60% homology thereto;

[0202] xx) Amino acid sequence of Heliobacillus mobilis carbon monoxide dehydrogenase (cooS) (SEQ ID NO:

[0203] 66) or an amino acid sequence having at least 60% homology thereto;

[0204] xxi) the amino acid sequence of Desulfocucumis palustris carbon monoxide dehydrogenase (DCCM_2691) (SEQ ID NO: 67) or an amino acid sequence having at least 60% homology thereto;

[0205] or a functional fragment, derivative or variant thereof.

[0206] Preferably, when the supported biocatalyst comprises formate dehydrogenase, the formate dehydrogenase is selected from or includes:

[0207] i) the amino acid sequence of the major subunit (fdnG) of the nitrate-inducible formate dehydrogenase of Escherichia coli (SEQ ID NO: 68) or an amino acid sequence having at least 60% homology thereto;

[0208] ii) the amino acid sequence of Shigella flexneri formate dehydrogenase-N nitrate-inducible alpha subunit (fdnG) (SEQ ID NO: 69) or an amino acid sequence having at least 60% homology thereto;

[0209] iii) the amino acid sequence of formate dehydrogenase-Nα subunit (fdnG) from Enterobacteriaceae (SEQ ID NO: 70) or an amino acid sequence having at least 60% homology thereto;

[0210] iv) the amino acid sequence of Salmonella typhimurium molybdenum purine oxidoreductase (fdnG) (SEQ ID NO: 71) or an amino acid sequence having at least 60% homology thereto;

[0211] v) the amino acid sequence of the major subunit (fdnG) of the nitrate-inducible formate dehydrogenase from Citrobacter rodentium (SEQ ID NO: 72) or an amino acid sequence having at least 60% homology thereto;

[0212] vi) amino acid sequence of Escherichia alba formate dehydrogenase-Nα subunit (fdnG) (SEQ ID

[0213] NO: 73) or an amino acid sequence having at least 60% homology thereto;

[0214] vii) the amino acid sequence of formate dehydrogenase-N, alpha subunit (fdnG) from Enterobacteriaceae (SEQ ID NO: 74) or an amino acid sequence having at least 60% homology thereto;

[0215] viii) the amino acid sequence of formate dehydrogenase-Nα subunit (fdnG) of Enterobacteriaceae 4M9 (SEQ ID NO: 75) or an amino acid sequence having at least 60% homology thereto;

[0216] ix) The amino acid sequence of Erwinia sp. formate dehydrogenase-Nα subunit (fdnG) (SEQ ID NO:

[0217] 76) or an amino acid sequence having at least 60% homology thereto;

[0218] x) the amino acid sequence of Jejubacter calystegiae formate dehydrogenase-Nα subunit (fdnG) (SEQ ID NO: 77) or an amino acid sequence having at least 60% homology thereto;

[0219] xi) the amino acid sequence of the Moellerella wisconsensis formate dehydrogenase Nα subunit (M992_0960) containing selenocysteine ​​(SEQ ID NO: 78) or an amino acid sequence having at least 60% homology thereto;

[0220] or a functional fragment, derivative or variant thereof.

[0221] Preferably, when the supported biocatalyst comprises an oxidoreductase, the oxidoreductase comprises or consists of one or more amino acid sequences having at least 60% homology to the specified sequence, each amino acid sequence independently having at least 70%, such as at least 80%, more preferably at least 90%, for example at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% homology to the specified sequence. More preferably, each amino acid sequence independently has at least 70%, such as at least 80%, more preferably at least 90%, for example at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% identity to the specified sequence. For the avoidance of doubt, if the oxidoreductase has two or more amino acid sequences, the percent homology of each of the two or more sequences relative to their respective specified sequences may be the same or different, preferably the same. As described herein, the percent homology and / or percent identity are each preferably determined over the full length of the specified reference sequence.

[0222] It will be apparent to one skilled in the art that an oxidoreductase can be a single polypeptide or can comprise multiple polypeptides. An oxidoreductase can be part of a multi-domain polypeptide, such as one or more domains. For example, one skilled in the art will appreciate that oxidoreductases such as hydrogenases and carbon monoxide dehydrogenases typically comprise two or more subunits. As used herein, the term "oxidoreductase" refers to one or more subunits of the protein of interest. For example, when the oxidoreductase is Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2), the oxidoreductase can comprise (i) SEQ ID NO: 1 but not SEQ ID NO: 2; (ii) SEQ ID NO: 2 but not SEQ ID NO: 1; or (iii) SEQ ID NO: 1 and SEQ ID NO: 2. For example, when the oxidoreductase is E. coli hydrogenase 2 (SEQ ID NO: 3 and / or 4), the oxidoreductase may comprise (i) SEQ ID NO: 3 but not SEQ ID NO: 4; (ii) SEQ ID NO: 4 but not SEQ ID NO: 3; or (iii) SEQ ID NO: 3 and SEQ ID NO: 4. Typically, when the oxidoreductase is a hydrogenase having two or more subunits, the oxidoreductase comprises the two or more subunits.

[0223] The oxidoreductase may be used in the form of a monomer or a polymer. For example, when the oxidoreductase comprises a hydrogenase that can exist in the form of a monomer or a dimer, the oxidoreductase may be provided in the form of a monomer or a dimer. For example, E. coli hydrogenase 1 may be purified as a dimer or a monomer or a mixture thereof. When the oxidoreductase comprises E. coli hydrogenase 1 (i.e., SEQ ID NO: 1 and / or 2), the first polypeptide may be provided as a monomer (1×SEQ ID NO: 1 and / or 1×SEQ ID NO: 2) or a dimer (2×SEQ ID NO: 1 and / or 2×SEQ ID NO: 2), or a mixture thereof. When the oxidoreductase is provided as a mixture of monomers and dimers, the mixture typically comprises about 1% to about 99% monomers and about 99% to about 1% dimers. Sometimes, the amounts of monomer and dimer can be roughly similar, so the first polypeptide can contain about 30% to about 70% monomer and about 70% to about 30% dimer, such as about 40% to about 60% monomer and about 60% to about 40% dimer. Sometimes, the oxidoreductase contains about 1% to about 10% monomer / about 90% to about 99% dimer, such as about 1% to about 5% monomer / about 95% to about 99% dimer. Sometimes, the oxidoreductase contains about 1% to about 10% dimer / about 90% to about 99% monomer, such as about 1% to about 5% dimer / about 95% to about 99% monomer.

[0224] It will be apparent to those skilled in the art that the supported biocatalyst may comprise related proteins, which may, for example, be co-purified with the oxidoreductase. For example, when the supported biocatalyst comprises a hydrogenase of the amino acid sequence of SEQ ID NO: 1 and / or 2 (or a functional fragment, derivative or variant thereof), the supported biocatalyst may further comprise a natural cytochrome electron transfer partner, such as the cytochrome of SEQ ID NO: 22 (or a functional fragment, derivative or variant thereof). Thus, in embodiments where the oxidoreductase comprises SEQ ID NO: 1 and / or 2 (or a functional fragment, derivative or variant thereof), the supported biocatalyst may also comprise SEQ ID NO: 22 (or a functional fragment, derivative or variant thereof).

[0225] Polypeptides used in the present invention

[0226] The method of expressing protein in cell (such as microorganism) expression system is a conventional method well known to those skilled in the art. For example, conventional purification methods can be used to separate oxidoreductase from its host organism. For example, host cells can be grown in suitable culture medium. The lysis of cells allows access to the internal components of the cell. Detergents such as Triton X (such as Triton X-114, (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol, available from SigmaAldrich) can be used to dissolve membrane proteins. Standard chromatographic techniques (such as size exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography) can be used to separate and purify soluble or dissolved proteins. Alternatively, oxidoreductase can be encoded in one or more nucleotide vectors and then expressed in suitable host cells (such as microbial cells, such as Escherichia coli). Purification tags such as HIS (six histidine) tags can be encoded (usually at the C-terminus or N-terminus of the relevant polypeptide) and can be used to separate labeled proteins using affinity chromatography (such as using nickel-NTA chromatography or cobalt-NTA chromatography). If desired, a protease recognition sequence can be incorporated between the oxidoreductase and the affinity purification tag to allow the tag to be removed after expression. Such techniques are routine to those skilled in the art and are described, for example, in Sambrook et al. ("Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press).

[0227] As described herein, oxidoreductases can be functional fragments, derivatives or variants of enzymes or amino acid sequences. As will be appreciated by those skilled in the art, fragments of amino acid sequences include deletion variants of such sequences, in which one or more, for example at least 1, 2, 5, 10, 20, 50 or 100 amino acids are deleted. Deletion can occur at the C-terminus or N-terminus of the native sequence, or in the native sequence. Typically, the deletion of one or more amino acids will not affect the residues that are close to the active site of the enzyme. Derivatives of amino acid sequences include sequences that are post-translationally modified, including sequences that are modified in vivo or in vitro. Many different protein modifications are known to those skilled in the art, and include modifications that introduce new functions to amino acid residues, modifications that protect reactive amino acid residues, or modifications that couple amino acid residues to chemical moieties (such as reactive functional groups on linkers or substrates (surfaces)) to connect these amino acid residues.

[0228] Derivatives of amino acid sequences include addition variants of these sequences, wherein one or more (e.g., at least 1, 2, 5, 10, 20, 50, or 100 amino acids) are added or introduced into the native sequence. The addition may occur at the C-terminus or N-terminus of the native sequence, or within the native sequence. Typically, the addition of one or more amino acids will not affect the residues immediately surrounding the active site of the enzyme.

[0229] The variant of amino acid sequence includes the sequence in which one or more amino acids (such as at least 1, 2, 5, 10, 20, 50 or 100 amino acid residues) in the native sequence are replaced by one or more non-natural residues. Therefore, such variants can include point mutations, or can be more profound, such as natural chemical connection can be used to splice non-natural amino acid sequences into part of the native sequence to produce variants of natural enzymes. The variant of amino acid sequence includes sequences carrying natural amino acids and / or non-natural amino acids. The variant, derivative and functional fragment of the above-mentioned amino acid sequence retain at least some activity / function of the natural / wild-type sequence. Preferably, when compared with the natural / wild-type sequence, the variant, derivative and functional fragment of the above-mentioned sequence have increased / improved activity / function.

[0230] Variants of enzymes, such as the oxidoreductases described herein, may preferably be modified to have an enhanced catalytic activity towards their respective substrates. Preferably, the catalytic activity is increased by at least 2-fold, such as at least 5-fold, for example at least 10-fold, such as at least 100-fold, preferably at least 1000-fold. The catalytic activity may be determined by any suitable method. For example, the catalytic activity may be correlated with the Michaelis constant K M (Activity increase is usually associated with K M value) or the catalytic rate constant k cat (Activity increase is usually associated with k cat value increase).

[0231] Measuring K M and k cat It is routine for those skilled in the art. For example, the K of a polypeptide to a substrate M The determination can be performed spectrophotometrically, for example, by mixing in 50 mM Tris-HCl buffer (pH 8.0, containing 1 mM substrate, 5 mM benzyl viologen (oxidized; ε = 8.9 mM –1 cm –1 ), 90 μM dithionite and 10 to 30 pmol enzyme) under anaerobic conditions at 30° C. The absorbance at 578 nm is measured. An example of a solution assay for determining the absorbance of oxidized and reduced flavin is described in the Examples.

[0232] Standard methods in the art can be used to determine homology. For example, the UWGCG package provides the BESTFIT program (e.g., used under its default settings) that can be used to calculate homology (Devereux et al (1984) Nucleic Acids Research 12, p387-395). PILEUP and BLAST algorithms can be used to calculate homology or arrange sequences (e.g., identify equivalent residues or corresponding sequences (usually on their default settings)), for example, as described in Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, SF et al (1990) J Mol Biol 215: 403-10. Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0233] Similarity can be measured using paired identities or by applying a scoring matrix (such as BLOSUM62) and converting to equivalent identities. Because they represent functional changes rather than evolutionary changes, when determining homology, the position of intentional mutations will be obscured. By applying a position-specific scoring matrix, similarity can be determined more sensitively, for example, using PSIBLAST on a comprehensive database of protein sequences. Different scoring matrices can be used to reflect the chemical-physical properties of amino acids, rather than the replacement frequency (such as charge) on an evolutionary time scale. Conservative replacement replaces amino acids with other amino acids having similar chemical structures, similar chemical properties or similar side chain volumes. The amino acid introduced can have polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge similar to the amino acid replaced with it. Alternatively, conservative replacement can introduce another aromatic or aliphatic amino acid to replace pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 main amino acids defined in Table A below. In cases where the amino acids have similar polarity, this can also be determined by reference to the hydrophilicity scale of the amino acid side chains in Table B.

[0234] Table A - Chemical properties of amino acids

[0235]

[0236] Table B - Hydrophilicity Scale

[0237]

[0238] Preferably, sequence homology can be evaluated based on sequence identity. Any of a variety of sequence alignment methods can be used to determine the identity percentage, including but not limited to global methods, local methods and hybrid methods, such as segment methods. The scheme for determining the identity percentage is a routine procedure within the scope of those skilled in the art. The global method aligns the sequence from the beginning to the end of the molecule, and determines the optimal alignment by accumulating the scores of single residue pairs and applying gap penalties. Preferred methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22 (22) 4673-4680 (1994)) and iterative optimization (Gotoh, J. Mol. Biol. 264 (4) 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs common to all input sequences. Preferred methods include match-box (Match-box, Depiereux and Feytmans, CABIOS 8 (5) 501-509 (1992)); Gibbs sampling (Lawrence et al., Science 262 (5131) 208-214 (1993)); and Alignment-M (Van Walle et al., Bioinformatics, 20 (9) 1428-1435 (2004)). Thus, the percent sequence identity is determined by conventional methods. See, for example, Altschul et al. (Bull. Math. Bio. 48: 603-16, 1986) and Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992). Briefly, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) as shown below (amino acids are represented by the standard single letter code) to optimize the alignment score.

[0239] Alignment score used to determine sequence identity

[0240]

[0241] The percent identity is then calculated as:

[0242] 100×(T / L)

[0243] in,

[0244] T = total number of identical matches

[0245] L = the length of the longer sequence plus the number of gaps introduced into the longer sequence to align the two sequences

[0246] Carrier material

[0247] As discussed, the disclosed methods involve the use of a supported biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a support material.

[0248] The benefit of a support for the oxidoreductase is that the supported biocatalyst can be easily removed from the reaction mixture. For example, the support can be removed by precipitation, filtration, centrifugation, etc. Many such methods are known to those skilled in the art, for example, filtration can be achieved by removing solid components from a liquid composition using simple filter paper; or a mixed solid / liquid composition can be allowed to settle and then the liquid can be decanted from the settled solid.

[0249] The oxidoreductase (or its functional fragment or derivative) is usually fixed on a carrier material. As used herein, the term "fixation" includes adsorption, entrapment and / or crosslinking between the carrier and the polypeptide. Adsorption includes non-covalent interactions, which include electrostatic interactions, hydrophobic interactions, etc. Charged adsorption enhancers (such as polymyxin B sulfate) can be used to enhance adsorption. Encapsulation includes containing the enzyme on the surface of the carrier, such as in a polymer film or in a hydrogel. Crosslinking includes covalent bonding, directly between the enzymes (e.g., by amide coupling, such as by EDC / NHS and / or other coupling agents conventional to those skilled in the art) or using one or more covalent crosslinking agents, such as thiol-terminated joints or crosslinking reagents. A fixation method comprising or consisting of adsorption is preferred. A combination of some or all of the above fixation methods can be used. For example, the enzyme can be connected (e.g., covalently linked) to a binding group for non-covalent adsorption to a carrier material. For example, the enzyme can be covalently linked to a hydrophobic group (e.g., an aromatic or heteroaryl group) for non-covalent interaction with the support material (e.g., via hydrophobic interactions, π-π stacking, etc.) Suitable binding groups are known in the art and include molecules such as pyrene and its derivatives.

[0250] In the disclosed methods, the support material is typically conductive or semiconductive.

[0251] Preferably, the support or each support independently comprises: a material comprising carbon (including doped carbon materials), a metal or metal alloy, a metal oxide (including mixed metal oxides), a metal hydroxide (including layered double hydroxides), a metal chalcogenide, a semiconductor material (including carbonitrides, silicates, germanium compounds and gallium compounds, such as silicon carbide, doped silicon and / or doped germanium) or a conductive polymer or a mixture thereof. As will be appreciated by those skilled in the art, suitable support materials may include: mixtures of the materials described herein, such as mixtures of metal oxides or mixed metal oxides; mixtures of carbon with metal oxides (e.g., mixtures of carbon with silicon dioxide, aluminum oxide, etc.). As used herein, the term "mixture" includes both atomic mixtures (such as alloys) and heterogeneous mixtures (such as particle mixtures of one or more materials disclosed herein).

[0252] Any suitable support material may be used.

[0253] More preferably, the or each support material independently comprises:

[0254] i) carbon; and / or

[0255] ii) a metal or metal alloy selected from the group consisting of gold, silver, tungsten, iridium, platinum, palladium, copper, titanium, brass and steel; and / or

[0256] iii) A material selected from titanium oxide, indium oxide, tin oxide and indium tin oxide.

[0257] Still more preferably, the support material or each support material comprises a carbon material. Still more preferably, the support material or each support material independently comprises a carbon material comprising graphite, carbon nanotubes, carbon black, activated carbon, carbon nanopowder, glassy carbon, carbon fiber, carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite, doped or surface modified carbon or doped diamond.

[0258] Examples of doped carbon materials include carbon doped with boron, nitrogen, oxygen, phosphorus, silicon, sulfur, first or second row transition metals (e.g., scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver and cadmium; particularly cobalt, nickel and copper), late transition metals (e.g., aluminum, gallium, indium and tin) and / or lanthanides (e.g., cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and lutetium). The dopant material may optionally be present in the material at a concentration of from about 0.001 atomic % (at %) to about 10 atomic %, for example from 0.01 atomic % to about 1 atomic %, such as about 0.1 atomic %. Preferred examples of doped carbon materials include carbon doped with boron, nitrogen, cobalt, nickel and / or copper. The carbon in the doped carbon material may be selected from graphite, carbon nanotubes, carbon black, activated carbon, carbon nanopowder, glassy carbon, carbon fiber, carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite and diamond (optionally surface-modified diamond).

[0259] For example, when the carbon material includes doped graphene, the graphene may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals. When the carbon material includes doped carbon nanotubes, the carbon nanotubes may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals. When the carbon material includes doped diamond, the diamond may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen and silicon. When the carbon material includes doped carbon black, the carbon black may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals. For example, when the carbon material includes doped activated carbon, the activated carbon may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals.

[0260] Examples of surface-modified carbon materials include: carbon materials containing acidic surface groups (e.g., carboxylic acids, carboxylic anhydrides, lactones, hydroxyls); basic surface groups (e.g., amides, imides, lactams, carbonyls, amines, imines, pyrroles, and pyridyls) and carbon materials containing surface groups (e.g., nitro, diazo, nitroso, fluorine, chlorine, bromine, and iodine). Functional groups on carbon materials can be introduced by methods such as oxidation (e.g., using reagents such as nitric acid, sulfuric acid, hydrogen peroxide, potassium permanganate, air, ozone, plasma, etc.), amidation, silanization, silylation, polymer grafting, polymer encapsulation, surfactant adsorption, and encapsulation. Such modifications can generally be applied to carbon materials disclosed herein. In some embodiments, acid oxidation is preferred to produce carboxylic acid groups on the carbon surface. Therefore, in some embodiments, preferred carrier materials include: carbon black having carboxylic acid surface groups, activated carbon having carboxylic acid surface groups, and carbon nanotubes having carboxylic acid surface groups.

[0261] Most preferably, the or each support material independently comprises a carbon material comprising graphite (or highly oriented pyrolytic graphite or pyrolytic graphite), activated carbon or carbon black; most preferably activated carbon or carbon black.

[0262] Preferably, the carrier or each carrier is a conductive particle. Preferred conductive particles include materials described herein. Preferably, when the carrier or each carrier includes particles, the particle size of the particles is from about 1 nm to about 100 μm, such as from about 10 nm to about 10 μm, for example, from about 100 nm to about 1 μm. The method for determining the particle size is a conventional method in the art, including, for example, dynamic light scattering. Conductive particles suitable for the method of the present invention include conductive carbon black particles, such as "Black Pearls 2000" particles available from Cabot Group (Boston, Mass., USA); and activated carbon, such as activated carbon "DARCO" (about 100 mesh).

[0263] Target compound

[0264] As described herein, the disclosed methods include reducing a reducible functional group (eg, a nitrogen-containing functional group) in a target compound.

[0265] Exemplary nitrogen-containing functional groups include: nitro (R-NO 2 ), azido (RN 3 ), hydroxylamine (R-NR'OH), nitroso (R-NO), nitrile (R-CN), diazo (R-CR'=N 2 ), diazonium (RN 2 + ), isonitrile (R-NC); isothiocyanate (R-NCS), isocyanate (RN=C=O), hydrazone (R-CR'=N-NR'), hydrazine (R-NR'-NR' 2 ), amidino (RC(NR')NR' 2 ), azo (RN = N = R'), and guanidine (R-NR'-C(NR')-NR' 2 ) etc.; wherein R represents the remaining part of the target compound, and each R' independently represents a group, such as H or a hydrocarbon group, such as an alkyl group, such as C 1-6 or C 1-4 Alkyl, such as methyl or ethyl; typically H. More typically, the nitrogen-containing functional group is selected from nitro (R-NO 2 ), azido (RN 3 ), hydroxylamine (R-NR'OH), nitroso (R-NO), nitrile (R-CN), diazo (R-CR'=N 2 ), diazonium (RN 2 +), isonitrile (R-NC); isothiocyanate (R-NCS), isocyanate (RN=C=O), hydrazone (R-CR'=N-NR'), and hydrazine (R-NR'-NR' 2 Still more typically, the nitrogen-containing functional group is selected from nitro (R-NO 2 ), azido (RN 3 ), hydroxylamine (R-NR'OH), nitrile (R-CN), diazo (RN 2 + ), isonitrile (R-NC). Still more typically, the nitrogen-containing functional group is selected from nitro (R-NO 2 ) and azido (RN 3 ). Most typically, the nitrogen-containing functional group is a nitro group.

[0266] The target compound can be any target compound, and the selection of an appropriate target compound according to the disclosed method is an operating parameter of the disclosed method and is well within the capabilities of those skilled in the art.

[0267] The method of the present invention is particularly suitable for the production of complex products, such as the synthesis or derivatization of natural products, and drug production. Therefore, the target compound can be a pharmaceutical intermediate, such as an oxidized form of a therapeutic drug, wherein the drug comprises an amine group, and wherein the oxidized form of the drug comprises an oxidized form of the amine group, such as a nitro group. The characterization of the product obtained from the method of the present invention is fully within the capabilities of those skilled in the art. For example, the product can be characterized by chemical analysis techniques, such as IR spectroscopy, NMR, GC (including chiral phase GC), polarimetry, mass spectrometry, HPLC, etc. Exemplary methods are provided in the embodiments.

[0268] The target compound is typically an organic compound and typically has a molecular weight of about 20 g / mol to about 2000 g / mol, such as about 50 g / mol to about 1000 g / mol, for example about 100 g / mol to about 700 g / mol.

[0269] Typically, the nitrogen-containing functional group is attached (e.g., covalently attached as a substituent) to an aryl group contained in the target compound (R), although non-aromatic groups are also included in the methods of the present invention. Aryl groups include hydrocarbyl aryls (e.g., benzene) and heteroaryls (e.g., pyridine), which may be further substituted. Exemplary non-aromatic groups include alkyl, alkenyl, carbocyclyl, and heterocyclyl groups, which may be further substituted. The conversion of aromatic and aliphatic compounds is demonstrated in the examples using a series of oxidoreductases, demonstrating the broad applicability of the disclosed methods to a variety of target compounds.

[0270] The target compound may generally contain a plurality of such groups bonded together and optionally further substituted. The substituents that may be present on the target compound (e.g., the groups in the target compound disclosed herein) are not limited and include, for example, H, halogen (e.g., F, Br, Cl, I), -OR' (e.g., OH or OMe), -NR' 2 , SR' (e.g. SH), SOR', SO 2 R', C(O)R', C(O)OR' and C(O)NR' 2 Other substituents that may be present on the subject compounds (eg, on the groups in the subject compounds disclosed herein) include optionally substituted alkyl groups, such as C 1 To C 6 (For example, C 1 To C 4 ) alkyl, which is unsubstituted or substituted by OR' or halogen (eg F); CN; optionally substituted alkenyl, such as C 1 To C 6 (For example, C 1 To C 4 ) alkyl, which is unsubstituted or substituted by OR' or halogen (eg F, such as CF 3 In addition to the nitrogen-containing functional groups, the groups in the target compounds disclosed herein may generally contain 1, 2, 3 or more substituents.

[0271] As used herein, R may comprise or consist of any suitable aromatic or aliphatic group or combination of groups. Suitable groups are defined herein.

[0272] For the avoidance of doubt, multiple nitrogen-containing functional groups may be contained in the target molecule and may be reduced according to the methods disclosed herein.

[0273] Alkyl (such as C 1 To C 20 Alkyl) is a straight or branched chain alkyl group containing 1 to 20 carbon atoms. Similarly, C 1 To C 10 Alkyl is a straight or branched chain alkyl group containing 1 to 10 carbon atoms. 1 To C 10 Alkyl groups are usually C 1 To C 6 Alkyl or C 1 To C 4 Alkyl. C 1 To C 4 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. As used herein, the term "alkyl" includes "alkylene," which is defined as a bidentate moiety derived from an alkane by removing two hydrogen atoms.

[0274] Alkenyl (such as C 2 To C 20 Alkenyl) is a straight chain or branched alkenyl group containing 2 to 20 carbon atoms. Similarly, C 2 To C 10 Alkenyl is a straight-chain or branched alkenyl group containing 2 to 10 carbon atoms and having one or more, for example one or two, typically one, double bonds. 2 To C 10 Alkenyl is usually C 2 To C 6 Alkenyl or C 2 To C 4 Alkenyl. C 2 -C 4 Examples of alkenyl groups include ethenyl, propenyl, and butenyl. As used herein, the term "alkenyl" includes "alkenylene," which is defined as a bidentate moiety derived from an alkene by removing two hydrogen atoms.

[0275] Carbocyclyl is a cyclic hydrocarbon typically containing 3 to 20 (e.g., 3 to 10) carbon atoms. Carbocyclyl may be saturated or partially unsaturated, but is typically saturated; when unsaturated, the group typically contains 1 or 2, e.g., 1, double bonds. Examples of saturated carbocyclyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0276] Heterocyclyl is a cyclic group generally containing 3 to 20, for example 3 to 10 atoms selected from C, O, N and S in the ring, including at least one heteroatom, generally one or two heteroatoms. Heterocyclyl may be saturated or partially unsaturated, but is generally saturated; when unsaturated, the group generally contains 1 or 2, for example 1, double bonds. Examples of heterocyclic groups include piperazine, piperidine, morpholine, 1,3-oxazinane, pyrrolidine, imidazolidine, oxazolidine; tetrahydropyrazine, tetrahydropyridine, dihydro-1,4-oxazine, tetrahydropyrimidine, dihydro-1,3-oxazine, dihydropyrrole, dihydroimidazole, dihydrooxazole, dihydroindole, 2,3-dihydrobenzofuran, 2,3-dihydrobenzo[b]thiophene, 2,3-dihydro 1H-benzo[d]imidazole, 2,3-dihydrobenzo[d]oxazole, 2,3-dihydrobenzo[d]thiazole, benzo[d][1,3]dioxole, 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine and 4,5,6,7-tetrahydrothiazolo[4 ,5-c]pyridine, 1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinoline, chroman, isochroman, thiochroman, isothiochroman, 1,2,3,4-tetrahydroquinoxaline, 1,2,3,4-tetrahydroquinazoline, 1,4-dihydro-2H-benzo[d][1,3]oxazine, 3,4-dihydro-2H-benzo[b][1,4]oxazine, 3,4-dihydro-2H-benzo[b][1,4]thiazine, 1,4-dihydro-2H-benzo[d][1,3]thiazine, 4H-benzo[d][1,3]dioxin and 2,3-dihydrobenzo[b][1,4]dioxin, including quaternized derivatives thereof.

[0277] The alkylaryl group is usually C 6 To C 20 Aryl, more usually C 6 To C 10 Aryl, which is a substituted or unsubstituted monocyclic or fused polycyclic aromatic group containing 6 to 20 (e.g., 6 to 10) carbon atoms in the ring portion. Examples include monocyclic groups (such as phenyl) and fused bicyclic groups (such as naphthyl and indenyl). Phenyl (benzene) is preferred.

[0278] Heteroaryl is typically a 5-10 membered heteroaryl, which is a substituted or unsubstituted monocyclic or fused polycyclic aromatic radical, containing 5-10 atoms in the ring portion, including at least one heteroatom, such as 1, 2 or 3 heteroatoms, typically selected from O, S and N. Examples of heteroaryl include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, pyrazine, indole, benzothiophene, benzofuran, benzoxazole, benzothiazole, benzimidazole, imidazo[1,2-a]pyridine, [1,2,4]triazolo[1,5-a]pyridine, imidazo[1,2-a]pyrazine, quinoline, isoquinoline, quinazoline and quinoxaline. The heteroaryl radical may be a fused polycyclic system, including, for example, a fused bicyclic system, in which the heteroaryl radical is fused to an aryl radical as defined herein.

[0279] In addition to substitution with the reducible nitrogen-containing functional groups defined herein, further substituents may be present as described above.

[0280] In some embodiments, the selection of oxidoreductase is determined by the reduction potential of the target compound. In some embodiments, the oxidoreductase is capable of oxidizing a molecular reducing agent at a potential more negative than the reduction potential of the target compound. The reduction potential of the target compound can be determined directly, for example, by using the electrochemistry described in the examples. Similarly, the potential at which the oxidoreductase is capable of oxidizing a given molecular reducing agent can be determined directly. For example, protein film voltammetry can be used. In short, in the presence of a molecular reducing agent, the oxidoreductase is fixed on an electrode optionally comprising a support material described herein, and the voltage potential applied to the electrode is changed, and at the potential at which the oxidoreductase is capable of oxidizing the molecular reducing agent, a current corresponding to the oxidation of the reducing agent is observed.

[0281] Typically, the nitrogen-containing functional group is attached to an unsaturated carbon atom. Typically, the target compound comprises an aromatic nitrogen-containing functional group. In other words, the nitrogen-containing functional group is typically attached to an aromatic group (hydrocarbyl aromatic group or heteroaryl group). More typically, the target compound comprises an aromatic nitro group; an aromatic azido group or an aromatic nitrile group. For example, the target compound can be a nitroaromatic compound. In some embodiments, the nitrogen-containing functional group is covalently attached to an aromatic group that is additionally substituted at the meta or para position relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently attached to an aromatic group that is ortho-substituted relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently attached to an aromatic group that is not ortho-substituted relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently attached to an aromatic group that is not substituted at the ortho position relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently attached to an aromatic group that is not substituted at the ortho position relative to the nitrogen-containing functional group. t Bu group) ortho-substituted aryl group.

[0282] In some embodiments, the target compound comprises multiple functional groups, for example, the target compound may comprise a reducible nitrogen-containing functional group and a reducible non-nitrogen functional group, such as a C=C double bond. Generally, the methods provided herein are capable of selectively reducing nitrogen-containing functional groups without reducing non-nitrogen functional groups. Without being bound by theory, the inventors believe that the reducible nitrogen-containing functional group may have an appropriate reduction potential to be reduced by a supported biocatalyst comprising an oxidoreductase described herein.

[0283] It is therefore apparent that the disclosed methods include forming a reaction product comprising a reduced functional group, for example comprising a reduced nitrogen-containing functional group as described herein. Thus, also provided herein is a method of forming a reaction product comprising a reduced nitrogen-containing functional group, the method comprising: contacting a target compound comprising an oxidized nitrogen-containing functional group with a supported biocatalyst in the presence of a molecular reducing agent as described herein, the biocatalyst comprising an oxidoreductase supported on a support material as described herein, under the conditions that:

[0284] - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and

[0285] - the nitrogen-containing functional groups are reduced at the support material;

[0286] - thereby forming a reaction product.

[0287] Typically, the reaction product comprises an aromatic amine, ie, an amine group or a quaternary ammonium group bonded to a hydrocarbyl aryl or heteroaryl group as defined herein.

[0288] Preferred aspects

[0289] In some preferred embodiments, the disclosed method is a method for reducing a reducible nitrogen-containing functional group in a target compound; wherein the nitrogen-containing functional group is selected from a nitro group, an azido group, a hydroxylamine group, a nitrile group, a diazo group, and an isonitrile group; and wherein the reduction converts the nitrogen-containing functional group into an amine group;

[0290] The method comprises: contacting the target compound with a biocatalyst in the presence of a molecular reductant selected from molecular hydrogen and / or CO and / or formate; wherein the biocatalyst comprises an oxidoreductase selected from hydrogenase and / or carbon monoxide dehydrogenase and / or formate dehydrogenase or a functional fragment or derivative thereof supported on a conductive or semiconductive carrier material, under the condition that:

[0291] - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and

[0292] - The nitrogen-containing functional groups are reduced to amine groups at the support material.

[0293] In some further preferred embodiments, the disclosed method is a method for reducing a reducible nitrogen-containing functional group in a target compound; wherein the nitrogen-containing functional group is selected from a nitro group, an azide group, or a nitrile group; and wherein the reduction converts the nitrogen-containing functional group into an amine group;

[0294] The method comprises: contacting a target compound with a biocatalyst; wherein the biocatalyst comprises a hydrogenase or a functional fragment or derivative thereof supported on a conductive or semi-conductive carrier material, wherein the carrier material comprises: carbon, a metal or a metal alloy, a metal oxide or a mixed metal oxide, a metal hydroxide, a metal chalcogenide, a semiconductor material or a conductive polymer, or a mixture thereof;

[0295] In the presence of molecular hydrogen or an isotope thereof, provided that:

[0296] - hydrogen or an isotope thereof is oxidized by a hydrogenase or a functional fragment or derivative thereof; and

[0297] - The nitrogen-containing functional groups are reduced to amine groups at the support material.

[0298] In a further preferred embodiment, the disclosed method is a method for reducing a nitro group in a nitroaromatic target compound; wherein said reduction converts said nitro group into an amine group;

[0299] The method comprises contacting a nitroaromatic target compound with a biocatalyst; wherein the biocatalyst comprises a hydrogenase or a functional fragment or derivative thereof supported on a carbon-based carrier material;

[0300] In the presence of molecular hydrogen or an isotope thereof, provided that:

[0301] - hydrogen or an isotope thereof is oxidized by a hydrogenase or a functional fragment or derivative thereof; and

[0302] - The nitrogen-containing functional groups are reduced to amine groups at the support material.

[0303] Reaction conditions

[0304] In the disclosed methods, the target compound is preferably initially added or present in the reaction medium at a concentration of 1 μM to 1 M, such as 5 μM to 800 mM, for example 10 μM to 600 mM, such as 25 μM to 400 mM, for example 50 μM to 200 mM, such as 100 μM to about 100 mM, for example about 250 μM to about 10 mM, such as about 500 μM to about 1 mM.

[0305] As mentioned above, when the molecular reducing agent is a gas (such as hydrogen or CO), the disclosed method is generally carried out under a gas atmosphere; that is, in the presence of a gas (e.g., in the head space of the reactor). Preferably, the gas atmosphere includes hydrogen or an isotope thereof and / or CO or an isotope thereof, and optionally an inert gas. O may be present. 2 or its isotopes. Preferred inert gases include nitrogen, argon, helium, neon, krypton, xenon, radon and sulfur hexafluoride (SF 6 ) and mixtures thereof, more preferably nitrogen and / or argon, most preferably nitrogen.

[0306] When the atmosphere includes hydrogen and inert gases and / or O 2 When the mixture is prepared, hydrogen is preferably present in a concentration of 1-100%, and the remaining gas comprises an inert gas as defined herein and / or O 2 The preferred gas atmosphere includes 80-100% H 2 , the remaining gas includes one or more inert gases; and 0-20% H 2 The remaining gas includes one or more inert gases and / or O 2 (For example, 1-4% H 2 ). When the gas atmosphere includes CO and inert gas and / or O 2 When the mixture is prepared, CO is preferably present in a concentration of 1-100%, and the remaining gas comprises an inert gas as defined herein and / or O 2 The preferred gas atmosphere comprises 80-100% CO, with the remainder comprising one or more inert gases; and 0-20% CO, with the remainder comprising one or more inert gases and / or O 2 (such as 1-4% CO in the air).

[0307] The gas atmosphere can also optionally include non-inert gases, such as ammonia, carbon dioxide and hydrogen sulfide. However, preferably, the gas atmosphere does not contain ammonia, carbon dioxide and hydrogen sulfide. The method of the present invention can be carried out at any suitable pressure: selecting a suitable pressure is an operating parameter of the method of the present invention, which can be controlled by the operator. Sometimes, the method of the present invention is carried out at ambient pressure (e.g., about 1 bar). Sometimes, the method of the present invention is carried out at reduced pressure (e.g., less than 1 bar) or high pressure (e.g., greater than 1 bar). For example, increasing the operating pressure can increase the solubility of hydrogen in the reaction medium. Preferably, the method of the present invention is carried out at a pressure of about 0.1 bar to about 20 bar, such as about 1 bar to about 10 bar, such as about 2 bar to about 8 bar, such as about 4 bar to about 6 bar, such as about 5 bar.

[0308] The disclosed method can be carried out under aerobic or anaerobic conditions. As used herein, "aerobic conditions" refers to a gas atmosphere that is not strictly anaerobic, such as containing at least trace amounts of O 2Suitable O 2 Levels are typically greater than 100 ppm, such as greater than 1000 ppm (0.1%), such as greater than 1% O 2 , for example greater than 2% O 2 . Usually, O 2 Levels do not exceed those in atmospheric O 2 level, i.e. 21% of O 2 , however, does not exclude higher O 2 level.

[0309] The provided methods are typically performed in aqueous compositions that may optionally contain, for example, a buffer salt. For some applications, no buffer is required, and the methods of the present invention may be performed without any buffer. Preferred buffer salts that may be used in the methods of the present invention include: Tris; Phosphate; Citric Acid / Na 2 HPO 4 ; Citric acid / sodium citrate; Sodium acetate / acetic acid; Na 2 HPO 4 / NaH 2 PO 4 ; imidazole (glyoxaline) / HCl; sodium carbonate / sodium bicarbonate; ammonium carbonate / ammonium bicarbonate; MES; Bis-Tris; ADA; aces; PIPES; MOPSO; Bis-Tris propane; BES; MOPS; TES; HEPES; DIPSO; MOBS; TAPSO; Trizma; HEPPSO; POPSO; TEA; EPPS; zwitterionic buffer (Tric ine); Gly-Gly; Bicine; HEPBS; TAPS; AMPD; TABS; AMPSO; CHES; CAPSO; AMP; CAPS and CABS. It is routine for those skilled in the art to select an appropriate buffer for the desired pH value, and guidance can be obtained, for example, at http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html. Buffer salts are preferably used in solution at a concentration of 1 mM to 1 M, preferably 10 mM to 100 mM, such as about 50 mM. The most preferred buffer for use in the methods of the invention comprises 50 mM phosphate (pH 8.0).

[0310] The provided methods are generally carried out in aqueous compositions. However, in the compositions used in the disclosed methods, non-aqueous components may optionally be used in place of or in addition to water. For example, one or more organic solvents (e.g., alcohols, DMSO, acetonitrile, etc.) or one or more ionic liquids may be used or included in the compositions.

[0311] The disclosed methods can be implemented in mixed solvent systems comprising multiple (e.g., 2, 3, 4, 5 or more) aqueous or non-aqueous solvents (e.g., solvents described herein). In some embodiments, the disclosed methods are implemented in a single-phase solvent system. In some embodiments, the disclosed methods are implemented in an immiscible solvent mixture (e.g., a two-phase or three-phase solvent system).

[0312] The disclosed methods are typically carried out at a temperature of about 10°C to about 80°C, such as about 20°C to about 60°C, for example about 30°C to about 50°C.

[0313] The disclosed methods are typically performed at a pH of about pH 4 to about pH 10, such as about pH 5 to about pH 9, for example about pH 6 to about pH 8, for example about pH 7.

[0314] The disclosed method can be implemented in the device provided herein. The device generally includes a reaction vessel. The reaction vessel generally includes: one or more inlets for molecular hydrogen or hydrogen-containing liquid (e.g., hydrogen-saturated liquid, such as a buffer solution described herein); and / or one or more inlets for reagents; and / or one or more outlets for products. Other devices such as pressure controllers, temperature controllers, mixing devices, flow controllers, etc. may be included. The device can be used as part of a device for converting an initial reagent into a final product, and is therefore configured to implement an intermediate reaction step. The device can be controlled by equipment such as a computer controller. The device may include a device for detecting cofactor conversion, reagent utilization and / or product generation, such as a spectrophotometric device. The device can be configured to operate in a flow mode (i.e., continuous mode) or in a batch mode.

[0315] Thus, the methods provided herein can be performed in a flow setting, such as in a flow reaction cell. Alternatively, the methods provided herein can be performed in a batch setting, such as in a batch reaction cell.

[0316] system

[0317] Also provided herein is a system for implementing the methods disclosed herein.

[0318] Preferably, the system comprises:

[0319] i) a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material;

[0320] ii) a molecular reducing agent; and

[0321] iii) a target compound comprising a reducible nitrogen-containing functional group;

[0322] Wherein, the system is configured such that, in use, (a) the molecular reductant is oxidized by an oxidoreductase or a functional fragment or derivative thereof; and (b) the nitrogen-containing functional group is reduced at the support material.

[0323] Typically, the system is designed or configured such that the oxidoreductase or functional fragment or derivative transfers electrons to the support material, and reduction of the nitrogen-containing functional group involves direct electron transfer from the support material to the target compound, such as to a reducible functional group of the target compound.

[0324] In the systems provided herein, the oxidoreductase is generally as defined herein. The support material is generally as defined herein. The molecular reducing agent is generally as defined herein. The target compound and functional groups contained therein are generally as defined herein. The system can be configured to operate as described in the methods provided herein. The system can be configured to operate under the reaction conditions defined herein.

[0325] Typically, the system can further include a device for controlling the concentration of the molecular reducing agent present in the reaction medium (e.g., by controlling the gas atmosphere in the system, e.g., by using a gas flow system). The system is typically configured as a flow cell containing the reagents described herein. The system (e.g., a flow cell) can include features of the provided apparatus described herein, such as one or more inlets for the molecular reducing agent (e.g., hydrogen or CO gas or formate) and / or one or more inlets for the reagents; and / or one or more outlets for the product; and / or one or more pressure controllers, temperature controllers, mixing devices, flow controllers, etc.

[0326] The following examples illustrate the present invention. However, they do not limit the present invention in any way. In this respect, it is important to understand that the specific assay used in the examples section is only designed to provide an indication of the effect of the inventive method. There are many assays that can be used to determine the reaction efficiency, so the negative result of any specific assay is not deterministic.

[0327] Example

[0328] Example 1

[0329] This example demonstrates the complete reduction of reducible nitrogen-containing functional groups according to the disclosed methods.

[0330] As described herein, the hydrogen-oxidizing oxidoreductase Escherichia coli hydrogenase 1 (Hyd1) was expressed, purified and immobilized on a carrier material. The carrier material used was carbon black nanopowder (BP2000, Cabot Corporation). Hyd1 is a good H 2 Oxidants (H 2 →2H + +2e - ), and resistant to O 2 . 1 Control experiments used an equal amount of Hyd1 in solution but without support material. Reduction of an exemplary nitrogen-containing functional group (the nitro group of 2-methyl-5-nitropyridine, Compound A) was quantified by 1H NMR spectroscopy ( Fig.10 ). Reaction conditions: at 1 bar H 2 Under pressure, 0.25 mL of the reaction contained 5 mM Tris-HCl solution of Compound A (50 mM, pH 8.0) containing 2 vol% DMSO.

[0331] The results are shown in Table 1. Hyd1 (6 μL) immobilized by adsorption on 0.5 mg of carbon support material catalyzed the complete 6-electron reduction of the nitro group of compound A to amine analog B (2-methyl-5-aminopyridine), with a conversion rate of 100% after 6 hours. In contrast, in the absence of any support material, the reduction of the nitro group was ineffective, and after 2 hours and 40 minutes, even at an elevated temperature of 30°C, 71% of the target compound A remained in the oxidized form. No complete reduction was observed, and 21% of the target compound was reduced to form hydroxylamine compound C (2-methyl-5-hydroxyaminopyridine).

[0332]

[0333] Table 1.

[0334]

[0335] Thus, this example demonstrates that the presence of the support material described herein results in improved nitrogen-based reduction in terms of more complete reduction and higher reduction efficiency. Without being bound by theory, the inventors believe that when modified with an oxidoreductase (e.g., a hydrogenase, such as Hyd1), the support material acts as an "active site" for promoting complete reduction of nitrogen-containing functional groups. Without being bound by theory, the inventors propose that the mechanism of nitro group reduction occurs through a series of electron and / or hydride transfers from the carbon material to the nitro group.

[0336] Example 2

[0337] Example 1 was repeated with other target compounds containing nitrogen-containing functional groups: 4-nitrophenol (i) and 1-methyl-2-nitrobenzene (ii); see Scheme 1. Again, the supported biocatalyst was able to efficiently and completely reduce the nitro group, demonstrating the chemical versatility of the disclosed method.

[0338] Solution 1

[0339]

[0340] pass 1 H NMR determination of reaction efficiency ( Fig.11 ). (i) The best reduction efficiency of the reaction was observed at pH 7, where the reaction proceeded to completion after 21 hours.

[0341] Example 3

[0342] Escherichia coli Hyd-1 modified carbon particles are loaded into a packed bed flow column as follows: 50 mg of activated carbon is loaded into a stainless steel flow column (30 × 4 mm) using a cartridge packer (ThalesNano). Before sealing, 0.2 mL (1.9 mg) of a solution of Hyd1 is injected into the column. The column is sealed with a column packer and stirred on a vortex mixer and then stored at 4 ° C for 1 hour. The column is then stored at 4 ° C for 1 hour, then installed in a reactor (H-Cube) and rinsed with sodium phosphate buffer (100 mM, pH 6.0) to remove any unadsorbed enzyme.

[0343] Reaction (i) of Example 2 was repeated under the following flow conditions: a liquid feed (Knauer H-Cube piston pump) containing 10 mM 4-nitrophenol in sodium phosphate buffer (100 mM, pH 6.0) was pumped through the H-Cube, where H 2 Produced by built-in water reservoir and electrolyzer (gas feed pressure set at 2 bar H 2 ). The liquid feed flow rate was set at 0.1 ml per minute to give a 1.6 second residence time (tRes) through the biocatalyst column. The reaction was carried out at 28°C. Aliquots were periodically removed for analysis by UV-Vis spectroscopy and HPLC.

[0344] Under industrially relevant conditions, successful and complete reduction of the nitro group to the corresponding amine group was observed, as shown in Scheme 1. The reaction was carried out for about 92 hours with high conversion (about 80%). This is consistent with a Hyd1 TTN of greater than 1.4 million.

[0345] Example 4

[0346] Example 1 was repeated with a different model target compound having an aliphatic azide functional group instead of a heteroaromatic nitro group. The target compound was 2-azido-1-phenylethanone (1 below). The reaction conditions were: 5 mM target compound (1) in buffer and cosolvent at room temperature, in H 2 After the reaction mixture was subjected to Boc derivatization conditions, the conversion was determined using HPLC (see Fig.12 As shown in Table 2, nearly 100% conversion was observed after 21 hours.

[0347] Additional control experiments were performed to confirm the role of the oxidoreductase and support material in the supported biocatalyst. No reduction of the azide group was observed in control experiments in which: (i) the carbon support material was omitted and an equal amount of E. coli Hyd1 solution was used; (ii) the oxidoreductase was omitted and an equal amount of carbon support material was used; or (iii) both the carbon support material and the hydrogenase were omitted.

[0348] Solution 2

[0349]

[0350] Table 2

[0351]

[0352] Example 5

[0353] The system of Example 1 was used to reduce nitrogen-containing functional groups of other target compounds. This example demonstrates that: (i) the disclosed method can be used to reduce aromatic azides with high efficiency and selectivity; (ii) further substitution on the aromatic group is well tolerated; and (iii) undesirable side reactions, such as dehalogenation of the para-chloro substituent, are avoided. Such undesirable side reactions are a common problem when noble metal catalysts, such as palladium on carbon (Pd / C), are used to catalyze the reduction of aromatic nitrogen-containing functional groups, such as azides. 2，3 .

[0354] The results are shown in Table 3. Reaction conditions: 0.25 mL reaction volume, 5 mM azide (target compound), 19 ug Hyd1 enzyme immobilized on 25 ug carbon black nanoparticle support material, mixed under 1 bar atmospheric pressure.

[0355] Table 3

[0356]

[0357] No dehalogenation products were detected

[0358] The conversion was determined using GC-FID. The conversion was calculated by comparing the peak areas of the azide and the corresponding aniline.

[0359] GC-FID method:

[0360] Column: CP-Chirasil-Dex CB (Agilent), 25 m long, 0.25 mm diameter, 0.25 μm (film thickness), with a 10 m non-deactivated fused silica shield of the same diameter

[0361] Carrier: Helium (CP grade), 170kPa (constant pressure)

[0362] Inlet temperature: 200℃

[0363] Injection conditions: splitless, split 60 mL / min, splitless time 0.8 min, purge 5 mL / min. Injection volume = 0.5 μL.

[0364] Detection: FID(H 2 =35mL / min, air =350mL / min, supplemented with N 2 =40mL / min, temperature = 200°C) oven heating curve:

[0365] Time (minutes) Temperature

[0366] 0→5 Maintain at 70℃

[0367] 5→30 Increase the temperature to 120℃ at a rate of 2℃ / min

[0368] 30→36 Increase the temperature to 180℃ at a rate of 10℃ / min

[0369] 36→45 Keep at 180℃ for 5 minutes

[0370] Compound retention time:

[0371]

[0372]

[0373] Example 6

[0374] The selectivity of the disclosed method in reducing nitrogen-containing functional groups compared to other reducible functional groups (such as unsaturated C=C double bonds) was demonstrated by repeating Example 1 using cinnamoyl azide (3). 1 H NMR spectroscopy to determine the product distribution (see Fig.13 ).

[0375] Solution 3

[0376]

[0377] A control experiment was carried out using a conventional noble metal catalyst (Pd / C) under the same reaction conditions. Both the alkenyl and azide groups of compound 3 were reduced to form compound 5.

[0378] The spectrum of the reaction product using Pd / C as a catalyst showed no peaks in the olefin region (i.e., 5-7 ppm), confirming that Pd / C reduced the olefin. In contrast, the spectrum of the Hyd1 / C-catalyzed reaction contained an olefin peak that was down-shifted relative to the starting azide, indicating the formation of the corresponding cinnamamine.

[0379] Thus, this example demonstrates that the disclosed systems and methods have improved chemoselectivity in the selective reduction of nitrogen-containing functional groups compared to conventional catalysts.

[0380] Example 7

[0381] Experiments were performed to explore the mechanism and product distribution of the reactions described in Examples 1-6.

[0382] Figure 1 The electrochemistry of 1 mM nitrobenzene in pH 8 buffer on a pyrolytic graphite edge (PGE) electrode is shown. The reductive wave at more negative potentials indicates irreversible reduction of the nitro group to the amino group. Figure 1 The onset potential of the reduction of nitrobenzene to aniline is shown to be significantly more positive than that of the hydrogen pair (assuming pH 8 and 1 bar H 2 ). This confirms that the electrons donated by the hydrogenase that oxidizes dihydrogen can be used by PGE to reduce nitrobenzene to aniline.

[0383] Figure 2 It was demonstrated that, similar to PGE, carbon black nanopowder (BP2000) was able to reduce nitrobenzene under the described conditions at a potential more positive than that of hydrogen.

[0384] Figure 3 The IR difference spectrum of 2 mM nitrobenzene in 1 vol% DMSO in 50 mM tris-HCl is shown, showing two NO stretch absorption bands of the nitro group (dark grey dashed lines) and three bands originating from DMSO (light grey dotted lines). Figure 4 It is shown that when a reduction potential equal to the hydrogen pair potential is applied to the BP working electrode, the NO stretching band (black dashed line) is disappearing. This indicates that the reductive wave starting at around -0.4 V (vs SHE) corresponds to the reduction of the nitro group of nitrobenzene.

[0385] The electrochemical behavior of nitrophenol in aqueous electrolyte (100 mM sodium phosphate, pH 6) on the BP electrode was also evaluated. Figure 5A). The irreversible reductive wave is clearly observed to start at around -0.3 V (vs SHE). When the potential is scanned at a lower rate (i.e. closer to steady-state conditions, Figure 5 B), it is clear that the reductive wave starts at a significantly more positive potential than the hydrogen pair under these conditions (pH 6, 1 bar hydrogen, equal to E 0 (H + / H 2 )=-0.355V vs SHE). This shows again that with H 2 It is possible that electrons provided by oxidative hydrogenases (such as Hyd1) reduce nitrophenols on the BP surface.

[0386] The electrochemistry of 5 mM 2-azido-1-phenylethanone on the BP electrode was further studied in a spectroelectrochemical cell. In the cyclic voltammogram, a reductive wave ( Figure 6 ). Similar to the case of nitrobenzene and nitrophenol, this is more than the hydrogen pair under these conditions (pH 8, 1 bar hydrogen, equal to E 0 (H + / H 2 )=-0.473V vsSHE) is significantly corrected, which again shows that the use of H 2 It is possible that the electrons provided by oxidative hydrogenases (such as Hyd1) reduce 2-azido-1-phenylethanone on the BP surface.

[0387] Figure 7 The IR difference spectrum of 5 mM 2-azido-1-phenylethanone in 1 vol% DMSO in 50 mM tris-HCl is shown, showing the N=N=N stretching absorption band of the azide group of 2-azido-1-phenylethanone (red line) and the C=O stretching of the carbonyl group (blue line). Figure 8 Shown are the infrared difference spectra of 5 mM 2-azido-1-phenylethanone in 50 mM tris-HCl (pH 8) containing 1 vol% DMSO at different potentials (vs SHE) applied to the BP-modified activated carbon paper working electrode. Figure 8 It is shown that when a reduction potential approximately equal to the hydrogen pair potential is applied to the BP working electrode, the N=N=N stretching band (about 2100 cm -1 ) is disappearing. This indicates that the reductive wave starting at around -0.4 V (vs SHE) is indeed the reduction of the azide group of 2-azido-1-phenylethanone and confirms that BP can utilize H 2 The electrons provided by hydrogenase are used to reduce the azide group.

[0388] Fig. 9 The scan rate is 10 mV s at 0 rpm. -1Zoomed region of the cyclic voltammogram of the PGE electrode modified with BP200 in 100 mM sodium phosphate (pH 6) containing 1 vol% DMSO and 1 mM tetracyanobenzene obtained at 2 0.5 % . This indicates that tetracyanobenzene can be reduced on the carbon surface, with the reductive wave starting at a potential slightly more positive than the hydrogen pair at pH 6 (assuming H 2 The pressure was 4 bar). This indicates that the supported biocatalysts described herein (eg, carbon-supported hydrogenases) can reduce nitrile substrates.

[0389] Example 8

[0390] Further experiments were performed to demonstrate the broad applicability of the disclosed method. As shown in Table 4, by controlling the reaction conditions, a variety of target compounds can be successfully processed. Selecting appropriate reaction conditions is well within the capabilities of those skilled in the art, and selecting reaction conditions to produce the desired results is an operating parameter of the method, which can be controlled by the technician.

[0391] Reaction conditions: 10 mM substrate and cosolvent in 100 mM sodium phosphate buffer, pH 6 (unless otherwise stated), at room temperature in a pressure vessel at 2 bar H 2 The mixture was mixed on a shaking plate under constant pressure for 24 h.

[0392] Table 4

[0393]

[0394]

[0395] a Entries 9 and 10 were carried out in 50 mM tris-HCl (pH 8). In addition to the observed conversion to 3-vinylaniline b Apart from 1, 2-dihydro-1-nitropropene and 1,2-dihydro-1-nitropropene, no other by-products were identified. The conversion was determined using HPLC.

[0396] HPLC Method:

[0397] Column: Shim-pack GIS 5μm HILIC reverse phase column (Shimadzu), 150mm long, 4.6mm in diameter; equipped with 5μm Shim-pack GIS (G) guard column, 4mm long, 10mm in diameter

[0398] Mobile phase: 80% 20 mM ammonium acetate buffer (pH 7.7), 20% acetonitrile, flow rate 1 ml / min

[0399] Oven temperature: 40℃

[0400] Injection conditions: injection volume = 10 μL.

[0401] Detection: UV at 260nm and 310nm

[0402]

[0403] Example 9

[0404] This example describes various nitro reductions using the catalyst "CaHydA1 / C" formed by immobilizing Clostridium acetobutylicum [FeFe]-hydrogenase A1 (CaHydA1) onto carbon black nanopowder (BP2000). CaHydA1 is a good H 2 The oxidant, operates without overpotential and can therefore provide a large driving force.

[0405] Table 5 shows the successful conversion of various substituents using CaHydA1 / C. No side products were identified.

[0406] Reaction conditions: 10 mM substrate and the specified cosolvent in 100 mM sodium phosphate buffer (pH 6) were mixed in a pressure vessel at 2 bar H 2 The mixture was mixed on a shaking plate under constant pressure for 24 h.

[0407] Table 5

[0408]

[0409] Example 10

[0410] Some compounds with reducible nitrogen-containing functional groups have difficult-to-obtain reduction potentials. Such compounds can be readily reduced according to the methods disclosed herein by using oxidoreductases operating at low overpotentials.

[0411] Examples of such compounds include 1-nitrohexane and 1-nitropentane. Fig.14 As shown in , the reduction potentials of these compounds are only slightly more positive than that of the hydrogen pair (pH 6, 1 bar hydrogen, equal to E 0 (H + / H 2 )=-0.355V vs SHE).

[0412] By using a hydrogenase with a small overpotential, such as [FeFe] hydrogenase CaHydA1, compounds 1-nitrohexane and 1-nitropentane can be easily reduced according to the published methods (see Example 9). Using an oxidoreductase with a significant overpotential (e.g., E. coli Hyd1 to H 2 Control experiments in which oxidation had an overpotential of about 80 mV did not result in effective reduction. The overpotential at which the hydrogenase was effective was determined directly. 1

[0413] Embodiment 11

[0414] The reduction of other substrates was demonstrated using carbon particles modified with E. coli Hyd-1. The reactions were carried out in sodium phosphate buffer at pH 6 under a flow of hydrogen at atmospheric pressure at room temperature. The reaction products were analyzed using NMR. Substrates 8-13, 15-24 and 26-29 were run using 10% v / v MeCN as a co-solvent.

[0415] The reactions are shown in the table below. All reactions were fully converted to the corresponding amines within the times shown (ie, 100% conversion).

[0416] This example therefore demonstrates the broad applicability of the methods disclosed herein. A wide variety of substituents on the aryl group carrying a reducible nitrogen-containing group (nitro) are tolerated, and complete reduction of the nitro group is readily achieved under the experimental conditions.

[0417] Table 6

[0418]

[0419] Table 6 (continued)

[0420]

[0421] *The amount of catalyst was doubled for substrates 8, 12, 14, 17-20, 23, 24, 26, and 29. For substrate 27, four times more catalyst was used.

[0422] Example 12

[0423] The reduction of other substrates was demonstrated using carbon particles modified with E. coli Hyd-2. The reactions were carried out at room temperature in a pressure vessel under 2 bar of hydrogen in sodium phosphate buffer at pH 6. The reaction products were analyzed using NMR.

[0424] The substrates are shown in the table below. Both substrates were efficiently reduced and only the amine was detected as the reaction product, implying complete conversion.

[0425] This example further demonstrates the broad applicability of the methods disclosed herein to aliphatic compounds as well as aromatic compounds.

[0426] Table 7

[0427]

[0428] References

[0429] 1M.J.Lukey,A.Parkin,MMRoessler,BJMurphy,J.Harmer,T.Palmer,F.Sargent and FAArmstrong,J.Biol.Chem.,2010,285,3928–38.

[0430] 2X.Zhao,SECleary,C.Zor,N.Grobert,HAReeve and KAVincent,Chem.Sci.,,

[0431] DOI:10.1039 / D1SC00295C.

[0432] 3C. A. Marques, M. Selva and P. Tundo, J. Org. Chem., 1993, 58, 5256–5260.

[0433] 4US2020 / 0262784 A1, 2020.

[0434] 5M. Rueping, C. Vila and U. Uria, Org. Lett., 2012, 14, 768–771.

[0435] Sequence Listing

[0436] SEQ ID NO: 1 - Escherichia coli hydrogenase 1 large subunit (hyaB)

[0437] MSTQYETQGYTINNAGRRLVVDPITRIEGHMRCEVNINDQNVITNAVSCGTMFRGLEIILQGRDPRDAWAFVERICGVCTGVHALASVYAIEDAIGIKVPDNANIIRNIMLATLWCHDHLVHFYQLAGMDWIDVLDALKADPRKTSELAQSLSSWPKSSPGYFFDVQNRLKKFVEGGQLGIFRNGYWGHPQYKLPPEANLMGFAHYLEALDFQREIVKIHAVFGGKNPHPNWIVGGMPCAINIDESGAVGAVNMERLNLVQSIITRTADFINNVMIPDALAIGQFNKPWSEIGTGLSDKCVLSYGAFPDIANDFGEKSLLMPGGAVINGDFNNVLPVDLVDPQQVQEFVDHAWYRYPNDQVGRHPFDGITDPWYNPGDVKGSDTNIQQLNEQERYSWIKAPRWRGNAMEVGPLARTLIAYHKGDAATVESVDRMMSALNLPLSGIQSTLGRILCRAHEAQWAAGKLQYFFDKLMTNLKNGNLATASTEKWEPATWPTECRGVGFTEAPRGALGHWAAIRDGKIDLYQCVVPTTWNASPRDPKGQIGAYEAALMNTKMAIPEQPLEILRTLHSFDPCLACSTHVLGDDGSELISVQVR

[0438] SEQ ID NO: 2 - Escherichia coli hydrogenase 1 small subunit (hyaA)

[0439] MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGAGMAPKIAWALENKPRIPVVWIHGLECTCCTESFIRSAHPLAKDVILSLISLDYDDTLMAAAGTQAEEVFEDIITQYNGKYILAVEGNPPLGEQGMFCISSGRPFIEKLKRAAAGASAIIAWGTCASWGCVQAARPNPTQATPIDKVITDKPIIKVPGCPPIPDVMSAIITYMVTFDRLPDVDRMGRPLMFYGQRIHDKCYRRAHFDAGEFVQSWDDDAARKGYCLYKMGCKGPTTYNACSSTRWNDGVSFPIQSGHGCLGCAENGFWDRGSFYSRVVDIPQMGTHSTADTVGLTALGVVAAAVGVHAVASAVDQRRRHNQQPTETEHQPGNEDKQASEQ ID NO: 3 - Escherichia coli hydrogenase 2 large subunit (hybC)

[0440] MSQRITIDPVTRIEGHLRIDCEIENGVVSKAWASGTMWRGMEEIVKNRDPRDAWMIVQRICGVCTTTHALSSVRAAESALNIDVPVNAQYIRNIILAAHTTHDHIVHFYQLSALDWVDITSALQADPTKASEMLKGVSTWHLNSPEEFTKVQNKIKDLVASGQLGIFANGYWGHPAMKLPPEVNLIAVAHYLQALECQRDANRVVALLGGKTPHIQNLAVGGVANPINLDGLGVLNLERLMYIKSFIDKLSDFVEQVYKVDTAVIAAFYPEWLTRGKGAVNYLSVPEFPTDSKNGSFLFPGGYIENADLSSYRPITSHSDEYLIKGIQESAKHSWYKDEAPQAPWEGTTIPAYDGWSDDGKYSWVKSPTFYGKTVEVGPLANMLVKLAAGRESTQNKLNEIVAIYQKLTGNTLEVAQLHSTLGRIIGRTVHCCELQDILQNQYSALITNIGKGDHTTFVKPNIPATGEFKGVGFLEAPRGMLSHWMVIKDGIISNYQAVVPSTWNSGPRNFNDDVGPYEQSLVGTPVADPNKPLEVVRTIHSFDPCMACAVHVVDADGNEVVSVKVL

[0441] SEQ ID NO: 4 - Escherichia coli hydrogenase 2 small subunit (hybO)

[0442] MTGDNTLIHSHGINRRDFMKLCAALAATMGLSSKAAAEMAESVTNPQRPPVIWIGAQECTGCTESLLRATHPTVENLVLETISLEYHEVLSAAFGHQVEENKHNALEKYKGQYVLVVDGSIPLKDNGIYCMVAGEPIVDHIRKAAEGAAAIIAIGSCSAWGGVAAAGVNPTGAVSLQEVLPG

[0443] KTVINIPGCPPNPHNFLATVAHIITYGKPPKLDDKNRPTFAYGRLIHEHCERRPHFDAGRFAK

[0444] EFGDEGHREGWCLYHLGCKGPETYGNCSTLQFCDVGGVWPVAIGHPCYGCNEEGIGFHKG

[0445] IHQLANVENQTPRSQKPDVNAKEGGNVSAGAIGLLGGVVGLVAGVSVMAVRELGRQQKK

[0446] DNADSRGE

[0447] SEQ ID NO: 5 - Ralstonia eutropha membrane-bound hydrogenase part (HoxG)

[0448] MSAYATQGFNLDDRGRRIVVDPVTRIEGHMRCEVNVDANNVIRNAVSTGTMWRGLEVILK

[0449] GRDPRDAWAFVERICGVCTGCHALASVRAVENALDIRIPKNAHLIREIMAKTLQVHDHAVH

[0450] FYHLHALDWVDVMSALKADPKRTSELQQLVSPAHPLSSAGYFRDIQNRLKRFVESGQLGPF

[0451] MNGYWGSKAYVLPPEANLMAVTHYLEALDLQKEWVKIHTIFGGKNPHPNYLVGGVPCAI

[0452] NLDGIGAASAPVNMERLSFVKARIDEIIEFNKNVYVPDVLAIGTLYKQAGWLYGGGLAATN

[0453] VLDYGEYPNVAYNKSTDQLPGGAILNGNWDEVFPVDPRDSQQVQEFVSHSWYKYADESV

[0454] GLHPWDGVTEPNYVLGANTKGTRTRIEQIDESAKYSWIKSPRWRGHAMEVGPLSRYILAYA

[0455] HARSGNKYAERPKEQLEYSAQMINSAIPKALGLPETQYTLKQLLPSTIGRTLARALESQYCG

[0456] EMMHSDWHDLVANIRAGDTATANVDKWDPATWPLQAKGVGTVAAPRGALGHWIRIKDG

[0457] RIENYQCVVPTTWNGSPRDYKGQIGAFEASLMNTPMVNPEQPVEILRTLHSFDPCLACSTHV

[0458] MSAEGQELTTVKVR

[0459] SEQ ID NO: 6 - Membrane - bound hydrogenase part of Ralstonia eutropha (HoxK)

[0460] MVETFYEVMRRQGISRRSFLKYCSLTATSLGLGPSFLPQIAHAMETKPRTPVLWLHGLECTC

[0461] CSESFIRSAHPLAKDVVLSMISLDYDDTLMAAAGHQAEAILEEIMTKYKGNYILAVEGNPPL

[0462] NQDGMSCIIGGRPFIEQLKYVAKDAKAIISWGSCASWGCVQAAKPNPTQATPVHKVITDKPI

[0463] IKVPGCPPIAEVMTGVITYMLTFDRIPELDRQGRPKMFYSQRIHDKCYRRPHFDAGQFVEEW

[0464] DDESARKGFCLYKMGCKGPTTYNACSTTRWNEGTSFPIQSGHGCIGCSEDGFWDKGSFYD

[0465] RLTGISQFGVEANADKIGGTASVVVGAAVTAHAAASAIKRASKKNETSGSEH

[0466] SEQ ID NO: 7 - Alcaligenes eutropha membrane-bound hydrogenase portion (HoxZ)

[0467] MSTKMQADRIADATGTDEGAVASGKSIKATYVYEAPVRLWHWVNALAIVVLAVTGFFIGS

[0468] PPATRPGEASANFLMGYIRFAHFVAAYIFAIGMLGRIYWATAGNHHSRELFSVPVFTRAYW

[0469] QEVISMLRWYAFLSARPSRYVGHNPLARFAMFFIFFFLSSVFMILTGFAMYGEGAQMGSWQE

[0470] RMFGWVIPLLGQSQDVHTWHHLGMWFIVVFVIVHVYAAIREDIMGRQSVVSTMVSGYRTF

[0471] KD

[0472] SEQ ID NO: 8 - Alcaligenes eutropha regulatory hydrogenase portion (HoxB)

[0473] MNAPVCTGLASAKPGVLNVLWIQSGGCGGCSMSLLCADTTDFTGMLKSAGIHMLWHPSLS

[0474] LESGVEQLQILEDCLQGRVALHALCVEGAMLRGPHGTGRFHLLAGTGVPMIEWVSRLAAV

[0475] ADYTLAVGTCAAYGGITAGGGNPTDACGLQYEGDQPGGLLGLNYRSRAGLPVINVAGCPT

[0476] HPGWVTDALALLSARLLTASDLDTLGRPRFYADQLVHHGCTRNEYYEFKASAEKPSDLGC

[0477] MMENMGCKGTQAHADCNTRLWNGEGSCTRGGYACISCTEPGFEEPGHPFHQTPKVAGIPI

[0478] GLPTDMPKAWFVALASLSKSATPKRVKLNATADHPLIAPAIRKTRLK

[0479] SEQ ID NO: 9 - Ralstonia eutropha regulatory hydrogenase part (HoxC)

[0480] MERLVVGPFNRVEGDLEVNLEVASGRVCSARVNATMYRGLEQILLHRHPLDALVYAPRVC

[0481] GICSVSQSVAASRALADLAGVTVPANGMLAMNLMLATENLADHLTHFYLFFMPDFTREIY

[0482] AGRPWHTDATARFSPTHGKHHRLAIAARQRWFTLMGTLGGKWPHTESVQPGGSSRAIDAA

[0483] ERVRLLGRVREFRCFLEQTLYAAPLEDVVALDSEVALWRWHAQAPQAGDLRCFLTIAQDA

[0484] ALDQMGPGPGTYLSYGAYPQPEGGFCFAQGVWRSAQGRLDALDLAAISEDATSAWLVDQ

[0485] GGARHPANGLTAPAPDKVGAYTWNKAPRLAGAVLETGAIARQLAGAQPLVRDAVARCGA

[0486] TVYTRVLARLVELARVVPLMEDWLQSLEIGAPYWASAHLPDQGAGVGLTEAARGSLGHW

[0487] VSVRDGRIDNYQIVAPTSWNFSPRDIAGQPGAVEKALEGAPVLQGETTPVAVQHIVRSFDPC

[0488] MVCTVH

[0489] SEQ ID NO: 10 - Hyperthermophilic hydrogenase large subunit (mbhL3)

[0490] MKIEKLVLTRVEGEASLNLVWERGVIKDAKISFYSTRGIEKVLRERPFMDALVINPRICGICG

[0491] HAHLIATVRAIENAIGIKEIPEKAKITRLVTQITEMVQNHVKWFYLFVMPDFLKFKESLSQFE

[0492] PFKGERWKRAVQFSSQIVKIIALFGGQWPHSSYAVPGGITSNFSEREVLKALNLVKEGKTFF

[0493] EKNVSEDLELFLNLCEEFNLLSIGKAYNRFLSGGGLAYCFNPSYKKGKGKVCKFNVRYVQE

[0494] LEAADYSKANPVRYKGLPYETGPLARELISKNPLVLKLYKNYGDSYAVRVAARLAEIKDLL

[0495] EILEKLLKELMNHLEEPSCLWEDRSDESGEGFGVVEAARGTLIHRVVIEKGKIKDYKVITPS

[0496] QWNLGPRCKKYLGVAEKAIVGLDSELKAQMVLRSFDLCSVCTTK

[0497] SEQ ID NO: 11 - Small subunit of hyperthermophilic hydrogenase (mbhS1)

[0498] METFWEVFKRHGVSRRDFLKFATTITGLMGLAPSMVPEVVRAMETKPRVPVLWIHGLECT

[0499] CCSESFIRSATPLASDVVLSMISLEYDDTLSAAAGEAVEKHRERIIKEYWGNYILAVEGNPPL

[0500] GEDGMYCIIGGRPFVEILKESAEGAKAVIAWGSCASWGCVQAAKPNPTTAVPIDKVIKDKPI

[0501] IKVPGCPPIAEVMTGVIMYMVLFDRIPPLDSQGRPKMFYGNRIHDTCYRRSFFNAGQFVEQF

[0502] DDEGAKKGWCLYKVGCRGPTTYNSCGNMRWYNGLSYPIQSGHGCIGCAENNFWDNGPFY

[0503] ERIGGIPVPGIESKADKVGAIAAAAAAGGAIIHGIASKIRKSGEKEE

[0504] SEQ ID NO: 12 - Hydrogenovibrio marinus hydrogenase part (hoxK)

[0505] MSSQVETFYEVMRRQGITRRSFLKYCSLTAAALGLSPAYANKIAHAMETKPRTPVIWLHGL

[0506] ECTCCSESFIRSAHPLAKDVVLSMISLDYDDTLMAASGHAAEAILDEIKEKYKGNYILAVEG

[0507] NPPLNQDGMSCIIGGRPFSEQLKRMADDAKAIISWGSCASWGCVQAAKPNPTQATPVHKFL

[0508] GGGYDKPIIKVPGCPPIAEVMTGVITYMLTFDRIPELDRQGRPKMFYSQRIHDKCYRRPHFD

[0509] AGQFVEEWDDEGARKGYCLYKVGCKGPTTYNACSTVRWNGGTSFPIQSGHGCIGCSEDGF

[0510] WDKGSFYSRDTEMNAFGIEATADDIGKTAIGVVGAAVVAHAAISAVKAAQKKGDK

[0511] SEQ ID NO: 13 - Hydrogenovibrio marinus hydrogenase part (hoxG)

[0512] MSVLNTPNHYKMDNSGRRVVIDPVTRIEGHMRCEVNVDENNVIQNAVSTGTMWRGLEVIL

[0513] RGRDPRDAWAFVERICGVCTGCHALASVRAVEDALDIKIPHNATLIREIMAKTLQIHDHIVH

[0514] FYHLHALDWVNPVNALKADPQATSELQKLVSPHHPMSSPGYFKDIQIRIQKFVDSGQLGIFK

[0515] NGYWSNPAYKLSPEADLMAVTHYLEALDFQKEIVKIHAIFGGKNPHPNYMVGGVPCAINID

[0516] GDMAAGAPINMERLNFVKSLIEQGRTFNTNVYVPDVIAIAAFYRDWLYGGGLSATNVMDY

[0517] GAYPKTPYDKSTDQLPGGAIINGDWGKIHPVDPRDPEQVQEFVTHSWYKYPDETKGLHPW

[0518] DGITEPNYELGSKTKGSRTNIIEIDESAKYSWIKSPRWRGHAVEVGPLARYILAYAQGVEYV

[0519] KTQVHTSLNRFNAVCRLLDPNHKDITDLKAFLGSTIGRTLARALESEYCGDMMLDDFNQLI

[0520] SNIKNGDSSTANTDKWDPSSWPEHAKGVGTVAAPRGALAHWIVIEKGKIKNYQCVVPTTW

[0521] NGSPRDPKGNIGAFEASLMGTPMERPDEPVEVLRTLHSFDPCLACSTHVMSEEGEEMATVK

[0522] VR

[0523] SEQ ID NO: 14 -大亚基红假单胞菌氢化酶(hupL)

[0524] MSVTTANGFELDTAGRRLVVDPVTRIEGHLRCEVNLDENNVIRNAVSTGTMWRGLEVILR

[0525] GRDPRDAWAFTERICGVCTGTHALTSVRAVEDALGIPIPENANSIRNIMHVTLQAHDHLVHF

[0526] YHLHALDWVDVVSALGADPKATSALAQSISDWPKSSPGYFRDVQNRLKRFVESGQLGPFM

[0527] NGYWGSPAYKLPPEANLMAVTHYLEALDFQKEIVKIHTVYGGKNPHPNWLVGGMPCAIN

[0528] VDGTGAVGAINMERLNLVSSIIDQTIAFIDKVYIPDLIAIASFYKDWTYGGGLSSQAVMSYG

[0529] DIPDHANDMSSKNLLLPRGAIINGNLNEIHEIDLRNPEEIQEFVDHSWFSYKDETRGLHPWD

[0530] GVTEPNFVLGPNAVGSRTRIEALDEQAKYSWIKAPRWRGHAMEVGPLARYVIGYAKGIPEF

[0531] KEPVDKVLTDLGQPLEAIFSTLGRTAARGLEASWAAHKMRYFQDKLVANIRAGDTATANV

[0532] DNWDPKTWPKEARGVGTTEAPRGALGHWIVIKDGKIDNYQAVVPTTWNGSPRDPAGNIG

[0533] AFEASLLNTPLAKADEPLEILRTLHSFDPCLACATHIMGPDGEELTRIKVR

[0534] SEQ ID NO: 15 - Hydrogenase small subunit (hupS) of Thiocapsa roseopersicina

[0535] MPTTETYYEVMRRQGITRRSFLKFCSLTATALGLSPTFAGKIAHAMETKPRIPVVWLHGLEC

[0536] TCCSESFIRSAHPLVSDVILSMISLDYTILIMAAAGHQAEAILEEVRHKHAGNYILAVEGNPPL

[0537] NQDGMSCIIGGRPFLEQLLEMADSCKAVISWGSCASWGCVQAARPNPTRATPVHEVIRDKP

[0538] VIKVPGCPPIAEVMTGVLTYILTFDRLPELDRQGRPLMFYGQRIHDKCYRRPHFDAGQFVES

[0539] WDDEGARRGYCLYKVGCKGPTTYNACSTIRWNGGVSFPIQSGHGCIGCSEDGFWDKGSFY

[0540] QHVTDTHAFGIEANADRTGIAVATRRGAAHRAHAAVSVVKRVQQKKEEDQS

[0541] SEQ ID NO: 16 -大亚基氢化酶(HyaB)交替单胞菌

[0542] MENTASNNRLVVDPITRIEGHLRIEAEMDGNTIKQAFSSGTSVRGIELILQGRDPRDAWAFA

[0543] QRICGVCTLVHGMASVRAVEDAIRKAWRSNAKLGVAIGKPSMTSMPKGPMQHGKKGHRQ

[0544] SRTSIGVLSEAEMAIPQNAQLIRNIMIATQYVHDHVMHFYHLHALDWVDVVSALDADPTRT

[0545] AALAGQLSDYPRSSPGYFKDMKQKVKTLVESGQLGIFSNAYWGHPGYKLPPEVNLMALAH

[0546] YLDALTWQREVVKVHTIFGGKNPHPNFVVGGVPSPINLNASTGINTSRLVQLQDAITQMKS

[0547] FVDQVYYPDIVAIAGYYKEWGTRGEGLGNFLTYGDLPMTSMDDPDSFLFPRGAILGRDLSK

[0548] VHDLDLDDPSEIQEFVSSSWYRYSGGNASGLHPFNGQTTLEYTGPKPPYKHLNVGAEYSWL

[0549] KSPRWKGHAMEVGPLARVLMMYAKKDAAAQDIVNRSLSILDLETSALFSTLGRTLARAVE

[0550] TKIVVNQLQSWYDQLLDNIAKGDTDTFNPLYFDPTNWPIKGQGVGVMEAPRGALGHWLV

[0551] MQNGKIENYQCVVPTTWNAGPRDPNSQAGAYEAALQDKHTLHDPDQPLEILRTLHSFDPC

[0552] LACAVHVMDETGEERLRLKVR

[0553] SEQ ID NO: 17 - Hydrogenase small subunit (hoxK) of Alteromonas macleodii

[0554] MALPTLNKQLQASGISRRTFLKFCATTASLLALPQSAVADLATALGNARRPSVIWLPFQECT

[0555] GCTEAILRSHAPTLESLIFDHISLDYQHTIMAAAGEQAEDARRAAMNAHKGQYLLLVDGSV

[0556] PVGNPGYSTISGMSNVDMLRESAKDAAGIIAIGTCASFGGIPKANPNPTGAVAVSDIITDKPI

[0557] VNISGCPPLPIAITAVLVHYLTFKRFPDLDELQRPLAFFGESIHDRCYRRPFFEQRKFAKSFDD

[0558] EGAKNGWCLFELGCKGPETFNACATVKWNQGTSFPIESGHPCLGCSEPDFWDKSSFYQALG

[0559] PWEWYKSKPGKGAQKHAGKNS

[0560] SEQ ID NO: 18 - Membrane-bound hydrogenase large subunit (hydL) of Allochromatium vinosum

[0561] MSERIVVDPITRIEGHLRIEAQMDGATIAQAYSSGTMVRGIETILKGRDPRDAWAFVQRICG

[0562] VCTLVHGIASVRAVEDALRIELPLNAQLIRNLMIGAQYIHDHVMHFYHLHALDWVDVVSA

[0563] LSADPRATSELAQSISAWPKSSPGYFADTQKRIKTFVESGQLGIFANGYWGHPAYRLPPEAN

[0564] LMAVAHYLEALAWQRDTAKFHAIFGGKNPHPNFVVGGVPSPIDLDSDSALNAKRLAEVRN

[0565] LIQSMRTFVDQVYVPDTLAIAGFYKDWGERGEGLGNFLCYGDLPTGASLDPATFLFPRGAIL

[0566] DRDLSTIHEVDLEATGEIQEFVNHSWYEYSVGNDRGLHPYEGQTNLEYDRRGGVAPPYKQ

[0567] LDVSDGYSWLKAPRWKGRSVEVGPLARVLMLYATGHDQARELVDSTLSRLDLPVDALYS

[0568] TLGRTAARALESKILVDAMQGWYDGLIANVKSGDTKTFNETLWEPSSWPSRAQGVGIMEA

[0569] PRGALGHWIVIEDGRIANYQAVVPSTWNAGPRDGRGQAGAYEAALQDNHQLVDVKQPIEI

[0570] LRTIHSFDPCIACAVHLADPESGESFQVRVV

[0571] SEQ ID NO: 19 - Small subunit of membrane - bound hydrogenase (hydS) from Allochromatium vinosum

[0572] PSVVWLSFQECTGCTESLTRAHAPTLEDLILDFISLDYHHTLQAASGEAAEAARLQAMDEN

[0573] RGQYLVIVDGSIPGPDANPGFSTVAGHSNYSILMETVEHAAAVIAVGTCAAFGGLPQARPNP

[0574] TGAMSVMDLVRDKPVINVPGCPPIPMVITGVIAHYLVFGRLPEVDGYGRPLAFYGQSIHDRC

[0575] YRRPFYDKGLFAESFDDEGAKQGWCLYRLGCKGPTTYNACATMKWNDGTSWPVEAGHP

[0576] CLGCSEPQFWDAGGFYEPVSVPLTLGPATLLGAGAAGAVVGGGLAALSRKKGRDAAATR

[0577] QPVTVDELEQKL

[0578] SEQ ID NO: 20 - Salmonella typhimurium NiFe hydrogenase (hybO)

[0579] MTGDNTLITSHGINRRDFMKLCAALAATMGLSSKAAAEMAESVSNPQRPPVIWIGAQECTG

[0580] CTESLLRATHPTVENLVLETISLEYHEVLSAAFGHQVEENKHNALEKYKGQYVLVVDGSIPL

[0581] KDNGIYCMVAGEPIVDHIRKAADGAAAIIAIGSCSAWGGVAAAGVNPTGAVSLQEVLPGKT

[0582] VINIPGCPPNPHNFLATVAHIITYGTPPKLDAKNRPTFAYGRLIHEHCERRPHFDAGRFAKEF

[0583] GDEGHRQGWCLYHLGCKGPETWGNCSTLQFCDVGGVWPVAIGHPCYGCNEEGIGFHKGI

[0584] HQLAHVENQTPRSEKPDVNMKEGGNISAGAVGLLGGVVGLVAGVSVMAVRELGRQQKK

[0585] DNADSRGE

[0586] SEQ ID NO: 21 - Salmonella typhimurium hydrogenase (hyaB)

[0587] MSNQYQTQGYTVNDAGRRLIVDPITRIEGHMRCEVNIDEQNVITNAVSCGTMFRGLEIILQG

[0588] RDPRDAWAFVERICGVCTGVHALASVYAIEDAIGIQVPDNANIIRNIMLATLWCHDHLVHF

[0589] YQLAGMDWIDVLNALKADPRATSQLAQSLSAWPMSSPGYFFDVQNRLKKFVDGGQLGIFR

[0590] NGYWGHPQYKLSPEANLMGFAHYLEALDFQREIVKIHTIFGGKNPHPNWIVGGMPCAINLD

[0591] QSGAVGAINMERLNLVQSIITRTADFINNVMVPDALAIGQFNKAWSQIGTGLSDKCVLSYG

[0592] AFPDIANDFSQQSLLMPGGAVINGDFKNVMPVDLADPQQIQEFVDHAWYRYPDDRLGRHP

[0593] FDGITDPWYNPGDVKGSDTHIQQLNEQERYSWIKAPRWRGHAMEVGPLARTLIAYHKGDA

[0594] ATIESVDRMMSALKLPLSGIQSTLGRILCRAHEAQWAVGKLQYFFDRLMTNLKNGDLATA

[0595] NTEKWEPASWPQHCRGIGFTEAPRGALGHWASIRDQKIELYQCVVPTTWNASPRDPKKQIG

[0596] AYEAALMGTQMAIPDQPLEILRTLHSFDPCLACSTHVLGDDGSELIAVQVR

[0597] SEQ ID NO: 22 - Escherichia coli Ni / Fe hydrogenase 1 cytochrome subunit, type B (strain K12) (hyaC)

[0598] MQQKSDNVVSHYVFEAPVRIWHWLTVLCMAVLMVTGYFIGKPLPSVSGEATYLFYMGYIR

[0599] LIHFSAGMVFTVVLLMRIYWAFVGNRYSRELFIVPVWRKSWWQGVWYEIRWYLFLAKRPS

[0600] ADIGHNPIAQAAMFGYFLMSVFMIITGFALYSEHSQYAIFAPFRYVVEFFYWTGGNSMDIHS

[0601] WHRLGMWLIGAFVIGHVYMALREDIMSDDTVISTMVNGYRSHKFGKISNKERS

[0602] SEQ ID NO: 23 - Desulfovibrio miyazaki vulgaris F periplasmic [NiFe] hydrogenase small subunit (hydA)

[0603] MKISIGLGKEGVEERLAERGVSRRDFLKFCTAIAVTMGMGPAFAPEVARALMGPRRPSVVY

[0604] LHNAECTGCSESVLRAFEPYIDTLILDTSLSLDYHETIMAAAGDAAEAALEQAVNSPHGFIAV

[0605] VEGGIPTAANGIYGKVANHTMLDICSRILPKAQAVIAYGTCATFGGVQAAKPNPTGAKGVN

[0606] DALKHLGVKAINIAGCPPNPYNLVGTIVYYLKNKAAPELDSLNRPTMFFGQTVHEQCPRLP

[0607] HFDAGEFAPSFESEEARKGWCLYELGCKGPVTMNNCPKIKFNQTNWPVDAGHPCIGCSEPD

[0608] FWDAMTPYQ

[0609] SEQ ID NO: 24 - Desulfovibrio miyazaki vulgaris F periplasmic [NiFe] hydrogenase large subunit (hydB)

[0610] MSGCRAQNAPGGIPVTPKSSYSGPIVVDPVTRIEGHLRIEVEVENGKVKNAYSSSTLFRGLEII

[0611] LKGRDPRDAQHFTQRTCGVCTYTHALASTRCVDNAVGVHIPKNATYIRNLVLGAQYLHDH

[0612] IVHFYHLHALDFVDVTAALKADPAKAAKVASSISPRKTTAADLKAVQDKLKTFVESGQLGP

[0613] FTNAYFLGGHPAYYLDPETNLIATAHYLEALRLQVKAARAMAVFGAKNPHTQFTVVGGVT

[0614] CYDALTPQRIAEFEALWKETKAFVDEVYIPDLLVVAAAYKDWTQYGGTDNFITFGEFPKDE

[0615] YDLNSRFFKPGVVFKRDFKNIKPFDKMQIEEHVRHSWYEGAEARHPWKGQTQPKYTDLHG

[0616] DDRYSWMKAPRYMGEPMETGPLAQVLIAYSQGHPKVKAVTDAVLAKLGVGPEALFSTLG

[0617] RTAARGIETAVIAEYVGVMLQEYKDNIAKGDNVICAPWEMPKQAEGVGFVNAPRGGLSH

[0618] WIRIEDGKIGNFQLVVPSTWTLGPRCDKNKLSPVEASLIGTPVADAKRPVEILRTVHSFDPCI

[0619] ACGVHVIDGHTNEVHKFRIL

[0620] SEQ ID NO: 25 - Clostridium beijerinckii SM10 (CbA5H) [FeFe]-hydrogenase (KX147468)

[0621] MGDNKKSFIQSALGSVFSVFSEEELKELSNGRKIAICGKVNNPGIIEVPEGATLNEIIQLCGGL

[0622] INKSNFKAAQIGLPFGGFLTEDSLDKEFDFGIFYENIARTIIVLSQEDCIIQFEKFYIEYLLAKIK

[0623] DGSYKNYEVVKEDITEMFNILNRISKGVSNMREIYLLRNLAVTVKSKMNQKHNIMEEIIDKF

[0624] YEEIEEHIEEKKCYTSQCNHLVKLTITKKCIGCGACKRACPVDCINGELKKKHEIDYNRCTH

[0625] CGACVSACPVDAISAGDNTMLFLRDLATPNKVVITQMAPAVRVAIGEAFGFEPGENVEKKI

[0626] AAGLRKLGVDYVFDTSWGADLTIMEEAAELQERLERHLAGDESVKLPILTSCCPSWIKFIEQ

[0627] NYGDMLDVPSSAKSPMEMFAIVAKEIWAKEKGLSRDEVTSVAIMPCIAKKYEASRAEFSVD

[0628] MNYDVDYVITTRELIKIFENSGINLKEIEDEEIDTVMGEYTGAGIIFGRTGGVIEAATRTALEK

[0629] MTGERFDNIEFEGLRGWDGFRVCELEAGDIKLRIGVAHGLREAAKMLDKIRSGEEFFHAIEI

[0630] MACVGGCIGGGGQPKTKGNKQAALQKRAEGLNNIDRSKTLRRSNENPEVLAIYEKYLDHP

[0631] LSNKAHELLHTVYFPRVKKD

[0632] SEQ ID NO: 26 - Clostridium beijerinckii ATCC 51743 [FeFe]-hydrogenase (Cbei_4110)

[0633] MSKYMVIDGNRIEFDKEKNILDLVRKAGIDLPTLCYYTDLSVYGACRMCVVEDERGSILTS

[0634] CSTPPKDMMSIRTNTPKLQKYRKVILELLLATHCRDCTICEKNGKCKLQKLASRFGLTNIRF

[0635] KSIEGKKDLDTSSKSIIRDPNKCILCGDCVRMCNEIQSVGAIDFANRGSNMVVSPAFGKSLAE

[0636] TDCVNCGQCATVCPTGAIVVKSDIKNVWKAIYNHKQRVIAQVAPAVRVALGEEFGIKPGEN

[0637] VMGKIVAAMRKLGFENIYDTSLSADLTVIEESKEFLKKLESDDNKFPLFTSCCPAWVRYVE

[0638] NKYTELLPYVSSCKSPMEMFGSVVKAYFKEKDSLENRETISVAVMPCTAKKAEAAREEFIR

[0639] DNIPDVDYVITTAELCAMIKEIGIQFDEIEAEASDIPLSLYSGAGVIFGVTGGVTEAVIREVVK

[0640] DKSSRVLKDIEFIGVRGMKGVKTCELQVKDESIRIGIVSGLRNAEDLIEKIKSGEEHFDFIEVM

[0641] ACPGGCIAGAGQPFGLMEEKNERAKGLYKIDKVTQIKRSEENLVVKSLYEGLLKNRTKELL

[0642] HVHYDKSEH

[0643] SEQ ID NO: 27 - Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_1773)

[0644] MGDNKKSFIQSALGSVFSVFSEEELKELSNGRKIAICGKVNNPGIIEVPEGATLNEIIQLCGGL

[0645] INKSNFKAAQIGLPFGGFLTEDSLDKEFDFGLFYENIARTIIVLSQEDCIIQFEKFYIEYLLAKIK

[0646] DGSYKNYEVVKEDITEMFNILNRISKGVSNMREIYLLRNLAVTVKSKMNQKHNIMEEIIDKF

[0647] YEEIEEHIEEKKCYTSQCNHLVKLTITKKCIGCGACKRACPVDCINGELKKKHEIDYNRCTH

[0648] CGACVSACPVDAISAGDNTMLFLRDLATPNKVVITQMAPAVRVAIGEAFGFEPGENVEKKI

[0649] AAGLRKLGVDYVFDTSWGADLTIMEEAAELQERLERHLAGDESVKLPILTSCCPSWIKFIEQ

[0650] NYGDMLDVPSSAKSPMEMFAIVAKEIWAKEKGLSRDEVTSVAIMPCIAKKYEASRAEFSVD

[0651] MNYDVDYVITTRELIKIFENSGINLKEIEDEEIDTVMGEYTGAGIIFGRTGGVIEAATRTALEK

[0652] MTGERFDNIEFEGLRGWDGFRVCELEAGDIKLRIGVAHGLREAAKMLDKIRSGEEFFHAIEI

[0653] MACVGGCIGGGGQPKTKGNKQAALQKRAEGLNNIDRSKTLRRSNENPEVLAIYEKYLDHP

[0654] LSNKAHELLHTVYFPRVKKD

[0655] SEQ ID NO: 28 - Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_3796)

[0656] MDIGFVNIDKELCTGCQQCVEVCPVNAIQGKKGQPQNIDYDVCVSCGQCIQVCNSYGFENR

[0657] ENSHLIEEKRRDRGVLESVKEPVFAAFNKGNAAKVKEALHDEELFTIVQCAPAVRVSLGEE

[0658] FGLKAGSLTAGKMAAALRRLGFNRVYDTNFGADLTIMEEGSELIKRVTEGGELPMFTSCCP

[0659] AWVKFMEQSYPELLNHLSSCKSPQQMAGTIFKTYGAKIDKVNPKKIYNVAIMPCTCKQFEC

[0660] DREEMQDSGFKDVDIVITTREFAQLIRDNEIDFKNLKDEEFDLPLGSYTGAGNIFGVTGGVM

[0661] EAALRSGYEMLTKKSIPNLELNFVRGSEGIRVAEVKLPKITLKVAVVSGLKNVVQILEDIKE

[0662] GKCDFDFIEVMTCPEGCVSGGGQPKFILDIDRRNALVSRKKGIYKHDSELEIRKSHENPFIKK

[0663] LYEEFLIEPLGEKSHHLLHTKFVSRKKEEI

[0664] SEQ ID NO: 29 - Clostridium pasteurianum [FeFe]-hydrogenase (hydA)

[0665] MKSEYNDLFKSLIDAYYKDDFDEFIKKALSDSTVNKEELSNIISSFCGVELKYTDKDTYIKDL

[0666] KNAIKNYNSDHKIVTKIRDCSVDCADENGKTSCQKSCPFDAILIDEANKTSYIDKDLCTDCG

[0667] FCVEGCPNGSILDKVEFIPLANLLKEKQPVIAAVAPAITGQFGDDVTIDQLRTAFKKVGFAD

[0668] MIEVAFFADMLTLKEACEFNAHVKSKDDLMITSCCCPMWVGMLKRVYKDMVKYVSPSVS

[0669] PMIAAGRVIKTLNSNCKVVFVGPCIAKKAESKNKDIEGDIDFVLTFEEVKNIFESLNINPSELP

[0670] EDPSTDYASREGRLYARTGGVSISVSEAVAKLFPEKKDLFKSVQANGVIECKKILEKAQNGE

[0671] VAANFIEGMGCVGGCVGGPKALIPKEKGREKVNEFAENSNVKISLESDQMKKILNMLNITS

[0672] AKDFMDEEKIKIFEREF

[0673] SEQ ID NO: 30 - Chlamydomonas reinhardtii [FeFe]-hydrogenase (hyd1)

[0674] MSALVLKPCAAVSIRGSSCRARQVAPRAPLAASTVRVALATLEAPARRLGNVACAAAAPA

[0675] AEAPLSHVQQALAELAKPKDDPTRKHVCVQVAPAVRVAIAETLGLAPGATTPKQLAEGLR

[0676] RLGFDEVFDTLFGADLTIMEEGSELLHRLTEHLEAHPHSDEPLPMFTSCCPGWIAMLEKSYP

[0677] DLIPYVSSCKSPQMMLAAMVKSYLAEKKGIAPKDMVMVSIMPCTRKQSEADRDWFCVDA

[0678] DPTLRQLDHVITTVELGNIFKERGINLAELPEGEWDNPMGVGSGAGVLFGTTGGVMEAALR

[0679] TAYELFTGTPLPRLSLSEVRGMDGIKETNITMVPAPGSKFEELLKHRAAARAEAAAHGTPGP

[0680] LAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKLITKMQAGEAKYDFVEIMACPAGCVG

[0681] GGGQPRSTDKAITQKRQAALYNLDEKSTLRRSHENPSIRELYDTYLGEPLGHKAHELLHTH

[0682] YVAGGVEEKDEKK

[0683] SEQ ID NO: 31 - Chlorella [FeFe]-hydrogenase (HYDA1)

[0684] MEALVRRGLQSPDQALRLLGVARAICVGLSRSLPALAASAEQFAEQYPKLKLKVNGREVT

[0685] VPEGTSVLNACREAGAYVPTLCTHPRLPTTPGTCRICMVETGGGQLKPACATPAWEGMEV

[0686] QTATDKVQESIRGVLSLMKANHPSDCMNCDASGRCEFQDLISRYNVKDVLPKLKTYSHEW

[0687] DAEVQADFEHFHDSSSTALTLDLEKCIKCGRCVTMCGQVQQMNVLGMINRSRMAHPGVLI

[0688] EEALDHSKCIECGQCSSVCPVGAIVEHSEWRQVLDALENKQKVMVVQTAPSVRVSIGEELG

[0689] LAPGTVETGQMVAAQRALGFDYVFDSDFSADLTIMEEGTELLQRLGAAWRAETAAQDAA

[0690] AGSWAAAKQGHGEGEAHGHAPGPLPMFTSCCPAWINLVEKSYPELIPHLSSCKSPQMMMG

[0691] AVVKHYWAKKKGLKPEDVCLVGIMPCTAKKHETERKEFRNENGAYDCDYVITTREFGHM

[0692] LRHKKIPMPSLKPEEFDNPLGEATGAAALFGATGGVMEAAIRTAYEIAAGEPLPKLEVEAVR

[0693] GVKGVKEATLTLPANDTTLKAGVAGKEIRVAVASGIGNARHLLQRIQAGEAHYDFVEVMA

[0694] CPGGCIGGGGQPKTHDPDAVLKRMGAIYQVDKSLALRKSHENPSIHKIYAEFLGQPGGELS

[0695] HKLLHTHYTDHSVDTLPSVRELGGSGEVAKRAALTAAGEMRYKRIAMVGDPSAKR

[0696] SEQ ID NO: 32 - Chlamydomonas [Fe - Fe] hydrogenase (HYD2)

[0697] MATIHINGHTVSVPEGTSILTAATQLGIHIPTLCTHPRLPTTPGTCRLCLVEVAGGALKPACA

[0698] TPVCHGLEVTTDSPQVKDSIRGVLALLKANHPADCMTCDVSGRCEFQARPGFWGGAWAG

[0699] QRMACPRQRPMLPMLGCRCLKCGRCVTACGLVQEMDVLGMKGRSRERHPAVLTEAMDL

[0700] SKCISCGQCAVMCPVGAITERAEWREVEDQLDAKRKAGRGAGRAGLMVCVTAPAVRVAI

[0701] GEELGLAPGTITTGQMVAAQRQLGFDYVFDVNFGADLTIMEEGTELLQRLRHAWGLDLPA

[0702] EGSGGAGAGPLPMFTSCCPGWVTACEKSFPELLPHLSTCKSPQQMMGAVVKSHFAAKLGK

[0703] RAQDICLVSVMPCTAKKYEAERGEMVREGEGPDVDYVITTREFGRLLRERHIPLASLPESAF

[0704] DNPLGEGSGAGVIFGNTGGVMEAALRTAYELAAGQPLPKLEEEAIRGLRGIKQATVTLPPT

[0705] APAGMASRQLRVAVASGIGQARHLLERMHTGHSPHFDFVEVMACPGGCVGGGGQPKSAD

[0706] PLVLLKRMGAVYSIDERSAIRKSHENPSIQKLYKACAEFLGEPGGSLSHQLLHTTYINRSTAS

[0707] QPTYTAFQRMDEPCNPKLQQAAAAGRGSSSSALPGTSA

[0708] SEQ ID NO: 33 - Soluble hydrogenase part of Ralstonia eutropha (HoxH)

[0709] MSRKLVIDPVTRIEGHGKVVVHLDDDNKVVDAKLHVVEFRGFEKFVQGHPFWEAPMFLQR

[0710] ICGICFVSHHLCGAKALDDMVGVGLKSGIHVTPTAEKMRRLGHYAQMLQSHTTAYFYLIVP

[0711] EMLFGMDAPPAQRNVLGLIEANPDLVKRVVMLRKWGQEVIKAVFGKKMHGINSVPGGVN

[0712] NNLSIAERDRFLNGEEGLLSVDQVIDYAQDGLRLFYDFHQKHRAQVDSFADVPALSMCLV

[0713] GDDDNVDYYHGRLRIIDDDKHIVREFDYHDYLDHFSEAVEEWSYMKFPYLKELGREQGSV

[0714] RVGPLGRMNVTKSLPTPLAQEALERFHAYTKGRTNNMTLHTNWARAIEILHAAEVVKELL

[0715] HDPDLQKDQLVLTPPPNAWTGEGVGVVEAPRGTLLHHYRADERGNITFANLVVATTQNNQ

[0716] VMNRTVRSVAEDYLGGHGEITEGMMNAIEVGIRAYDPCLSCATHALGQMPLVVSVFDAAG

[0717] RLIDERAR

[0718] SEQ ID NO: 34 - Soluble hydrogenase part of Ralstonia eutropha (HoxY)

[0719] MRAPHKDEIASHELPATPMDPALAANREGKIKVATIGLCGCWGCTLSFLDMDERLLPLLEKVTLLRSSLTDIKRIPERCAIGFVEGGVSSEENIETLEHFRENCDILISVGACAVWGGVPAMRNVFELKDCLAEAYVNSATAVPGAKAVVPFHPDIPRITTKVYPCHEVVKMDYFIPGCPPDGDAIFKVLDDLVNGRPFDLPSSINRYD

[0720] SEQ ID NO:35—Partial Rhodococcus opacus SH hydrogenase (HoxH)

[0721] MSTKLVIDPVTRIEGHGKVTVHLDDNNNVVDAHLHVVEFRGFEKLVQGHPFWEAPMLMQRICGICFVSHHLCGAKALDDMVGVGLKSGIDVTPTAEKIRRLGHYAQMLQSHATAYFYLIVPE MLFGMDAAPEQRNVLGLIEANPELVKRVVMLRKWGQEVIKAVFGRRMHGISSVPGGVNKNLSVAECQRFLKGEEGLPSVDEVIEYAQEGVQLFYDFHEQNRVQVDSFANVSALSMSLVDADG NVDYYHGKLRIIDDDKNVVQEFDYHDYLDHFSEAVEEWSYMKFPFLKALGRERGSVRVGPLGRLNVTNSLTPLAQEALERFHAYTNGKANNMTLHTNWARAIEILHAAELIKELLNDPDLQ KEQLLLTPADNAWTGEGVGVVEAPRGTLLHHYRADQEGDITFANLVVATTQNNQVMNRTVRSVAEDYLGGQGEVTEGMMNAIEVGIRAYDPCLSCATHALGQMPLIVSVHDTEGHVINERVR

[0722] SEQ ID NO:36 - Partial Rhodococcus opacus SH hydrogenase (HoxY)

[0723] MKHSEKNEIASHELPTTPLDPVLAAGRESKIKVAMIGLCGCWGCTLSFLDMDERLLVLLDKVTLHRSSLSDIKRITERCAIGFIEGGVANEENIETLEHYRENC DVLISVGACAVWGGVPAMRNVFELKDCLSEVYIDSATSVPGAKPVVPFHPDIPRITDKVYPCHEVVKMDYFIPGCPPDADAIFKVLDDLVNGRPFDLPSSINQYD

[0724] SEQ ID NO:37—Desulfovibrio fructivorans nickel-iron hydrogenase large subunit.

[0725] MAESKPTPQSTFTGPIVVDPITRIEGHLRIMVEVENGKVKDAWSSSQLFRGLEIILKGRDPRDAQHFTQRACGVCTYVHALASSRCVDDAVKVSIPANARMMRNLVMASQYLHDHLVHFYHLHALDWVDVTAALKADPNKAAKLAASIDTARTGNSEKALKAVQDKLKAFVESGQLGIFTNAYFLGGHKAYYLPPEVNLIATAHYLEALHMQVKAASAMAILGGKNPHTQFTVVGGCSNYQGLTKDPLANYLALSKEVCQFVNECYIPDLLAVAGFYKDWGGIGGTSNYLAFGEFATDDSSPEKHLATSQFPSGVITGRDLGKVDNVDLGAIYEDVKYSWYAPGGDGKHPYDGVTDPKYTKLDDKDHYSWMKAPRYKGKAMEVGPLARTFIAYAKGQPDFKKVVDMVLGKLSVPATALHSTLGRTAARGIETAIVCANMEKWIKEMADSGAKDNTLCAKWEMPEESKGVGLADAPRGSLSHWIRIKGKKIDNFQLVVPSTWNLGPRGPQGDKSPVEEALIGTPIADPKRPVEILRTVHAFDPCIACGVHVIEPETNEILKFKVC

[0726] SEQ ID NO: 38 - Desulfovibrio fructosovorans nickel-iron hydrogenase small subunit.

[0727] LTAKHRPSVVWLHNAECTGCTEAAIRTIKPYIDALILDTISLDYQETIMAAAGETSEAALHEALEGKDGYYLVVEGGLPTIDGGQWGMVAGHPMIETCKKAAAKAKGIICIGTCSPYGGVQKAKPNPSQAKGVSEALGVKTINIPGCPPNPINFVGAVVHVLTKGIPDLDENGRPKLFYGELVHDNCPRLPHFEASEFAPSFDSEEAKKGFCLYELGCKGPVTYNNCPKVLFNQVNWPVQAGHPCLGCSEPDFWDTMTPFYEQG

[0728] SEQ ID NO: 39 - Clostridium acetobutylicum iron-iron hydrogenase.

[0729] MKTIILNGNEVHTDKDITILELARENNVDIPTLCFLKDCGNFGKCGVCMVEVEGKGFRAACVAKVEDGMVINTESDEVKERIKKRVSMLLDKHEFKCGQCSRRENCEFLKLVIKTKAKASKPFLPEDKDALVDNRSKAIVIDRSKCVLCGRCVAACKQHTSTCSIQFIKKDGQRAVGTVDDVCLDDSTCLLCGQCVIACPVAALKEKSHIEKVQEALNDPKKHVIVAMAPSVRTAMGELFKMGYGKDVTGKLYTALRMLGFDKVFDINFGADMTIMEEATELLGRVKNNGPFPMFTSCCPAWVRLAQNYHPELLDNLSSAKSPQQIFGTASKTYYPSISGIAPEDVYTVTIMPCNDKKYEADIPFMETNSLRDIDASLTTRELAKMIKDAKIKFADLEDGEVDPAMGTYSGAGAIFGATGGVMEAAIRSAKDFAENKELENVDYTEVRGFKGIKEAEVEIAGNKLNVAVINGASNFFEFMKSGKMNEKQYHFIEVMACPGGCINGGGQPHVNALDRENVDYRKLRASVLYNQDKNVLSKRKSHDNPAIIKMYDSYFGKPGEGLAHKLLHVKYTKDKNVSKHE

[0730] SEQ ID NO: 40 -大亚基的嗜热栖热菌镍 - 铁 - 硒氢化酶。

[0731] It should be noted that there may be some inaccuracies in the content of the original text, especially in the description of "SEQ ID NO: 40 -大亚基的嗜热栖热菌镍 - 铁 - 硒氢化酶" which seems to have some errors in the name of the enzyme. It is recommended to check and correct it according to the actual situation.MSQAATPAADGKVKISIDPLTRVEGHLKIEVEVKDGKVVDAKCSGGMFRGFEQILRGRDPRDSSQIVQRICGVCPTAHCTASVMAQDDAFGVKVTTNGRITRNLIFGANYLQSHILHFYHLAALDYVKGPDVSPFVPRYANADLLTDRIKDGAKADATNTYGLNQYLKALEIRRICHEMVAMFGGRMPHVQGMVVGGATEIPTADKVAEYAARFKEVQKFVIEEYLPLIYTLGSVYTDLFETGIGWKNVIAFGVFPEDDDYKTFLLKPGVYIDGKDEEFDSKLVKEYVGHSFFDHSAPGGLHYSVGETNPNPDKPGAYSFVKAPRYKDKPCEVGPLARMWVQNPELSPVGQKLLKELYGIEAKNFRDLGDKAFSIMGRHVARAEETWLTAVAVEKWLKQVQPGAETYVKSEIPDAAEGTGFTEAPRGALLHYLKIKDKKIENYQIVSATLWNANPRDDMGQRGPIEEALIGVPVPDIKNPVNVGRLVRSYDPALGCAVH

[0732] SEQ ID NO: 41 - Small subunit of Ni - Fe - Se hydrogenase from Desulfomicrobium baculatum.

[0733] MTEGAKKAPVIWVQGQGCTGCSVSLLNAVHPRIKEILLDVISLEFHPTVMASEGEMALAHMYEIAEKFNGNFFLLVEGAIPTAKEGRYCIVGETLDAKGHHHEVTMMELIRDLAPKSLATVAVGTCSAYGGIPAAEGNVTGSKSVRDFFADEKIEKLLVNVPGCPPHPDWMVGTLVAAWSHVLNPTEHPLPELDDDGRPLLFFGDNIHENCPYLDKYDNSEFAETFTKPGCKAELGCKGPSTYADCAKRRWNNGINWCVENAVCIGCVEPDFPDGKSPFYVAE

[0734] SEQ ID NO: 42 - HoxH of the SH part from Hydrogenophilus thermoluteolus.

[0735] MTQHAPQAVSPRPSLPANATRRVAIDPLSRVEGHGKVTIWLDDDGQVVEARLHIVEFRGFEAFIVGRPYWEAPVVVQRLCGICPVSHHLAAAKALDRLVGVTQLPPTAEKMRRLMHYGQVLQSHALHFFYLAAPDLLLGFSADPAQRNVFGLAAQKRELARQGILVRQFGQECIEATAGKRIHGTSAVPGGIHKNLSRRERMALLSRAPEIRSWCEAAVALIERLFTEHAPFFAQFGSFQTKTFSLVAADGSLDLYDGTFRVKEANGAILIDHYDPNDYDQLLVEAVRPWSYMKFPYLKAYGEPDGFYRVGPSARLINCDRLTTARAEAARQRFLTFDQGTVAHSTLGYHWARLIEMLHCAELIEALLTDADLEGGELRARGQRQHRGVGVIEAPRGTLIHHYEVGDDDLITYCNLIVSTTHNNAVMNQAVTTAAKAFLSGVTLTEALLNHIEVAVRAFDPCLSCATH

[0736] SEQ ID NO: 43 - HoxY of the SH part of Hydrogenophilus thermoluteolus

[0737] MTSAAPSAMPPRKIRIATASLAGCFGCHMSFADIDTRLLALAEWVTFDRSPLTDWKTVGECDIALIEGGVCNAENVEVLRAYRRAARILVAVGACAINGGLPAQRNQHRVERLLTQVFEADRHLAPGSRVPNDPELPLLLEHVHPIHEIVRVDYYLPGCPPTAEVIWTFLTDLLVGREPHFPYPTLRYD

[0738] SEQ ID NO: 44 - Periplasmic [NiFe] hydrogenase small subunit of Desulfovibrio gigas

[0739] LTAKKRPSVVYLHNAECTGCSESVLRTVDPYVDELILDVISMDYHETLMAGAGHAVEEALHEAIKGDFVCVIEGGIPMGDGGYWGKVGGRNMYDICAEVAPKAKAVIAIGTCATYGGVQAAKPNPTGTVGVNEALGKLGVKAINIAGCPPNPMNFVGTVVHLLTKGMPELDKQGRPVMFFGETVHDNCPRLKHFEAGEFATSFGSPEAKKGYCLYELGCKGPDTYNNCPKQLFNQVNWPVQAGHPCIACSEPNFWDLYSPFYSA

[0740] SEQ ID NO: 45 - Periplasmic [NiFe] hydrogenase large subunit of Desulfovibrio gigas.

[0741] MSEMQGNKIVVDPITRIEGHLRIEVEVEGGKIKNAWSMSTLFRGLEMILKGRDPRDAQHFTQRACGVCTYVHALASVRAVDNCVGVKIPENATLMRNLTMGAQYMHDHLVHFYHLHALDWVNVANALNADPAKAARLANDLSPRKTTTESLKAVQAKVKALVESGQLGIFTNAYFLGGHPAYVLPAEVDLIATAHYLEALRVQVKAARAMAIFGAKNPHTQFTVVGGCTNYDSLRPERIAEFRKLYKEVREFIEQVYITDLLAVAGFYKNWAGIGKTSNFLTCGEFPTDEYDLNSRYTPQGVIWGNDLSKVDDFNPDLIEEHVKYSWYEGADAHHPYKGVTKPKWTEFHGEDRYSWMKAPRYKGEAFEVGPLASVLVAYAKKHEPTVKAVDLVLKTLGVGPEALFSTLGRTAARGIQCLTAAQEVEVWLDKLEANVKAGKDDLYTDWQYPTESQGVGFVNAPRGMLSHWIVQRGGKIENFQLVVPSTWNLGPRCAEGKLSAVEQALIGTPIADPKRPVEILRTVHSYDPCIACGVH

[0742] SEQ ID NO: 46 - Soluble hydrogenase alpha subunit of Pyrococcus furiosus.

[0743] MIIELDEFTRVEGNGKAEIVIENGEVKDARVKIVEGPRFFEILTLGRDYWDVPDLEARICAICYIAHSVASVRAIEKALGIDVPESVEKLRELALWGEIIESHALHLYLLALPDVFGYPDAISMIPRHGELVKEGLTIKAFGNAIRELIGGREIHGINIKPGGFGRYPSEEELEKIAEHSKSLIKFARRIVGIFASQEAGGAVGEVLMATSDYLWGDELIINGERVQYYEVDEVPVGYSFAKHSYYKGNPVFVGALPRLLLKGESIEGEAARMLEEYRDKLESKYVIYNNLAQAIELLYALERVPQLVEEILSEGIERGNGEISQESGEGVGYVEAPRGVLVHHYRIENGKVVWSNTITPTAFNQRLMELSLLEEA

[0744] KRLYGSESEENMKKRLEVIVRAFDPCISCSVHFVKL

[0745] SEQ ID NO: 47 - Desulfovibrio vulgaris Hildenborough carbon monoxide dehydrogenase (cooS)

[0746] MSSSKTIRSRSIWDDAHAMLEKAKAEGISTVWDRAAEQTPACKFCELGTTCRNCIMGPCRI

[0747] ANRKDGKMRLGVCGADADVIVARNFGRFIAGGAAGHSDHGRDLIETLEAVAEGKAPGYTI

[0748] RDVAKLRRIAAELGVADAATRPAHDVAADLVTICYNDFGSRRNALAFLARAPQVRRDLWQ

[0749] RLGMTPRGVDREIAEMMHRTHMGCDNDHTSLLVHAARTALADGWGGSMIGTELSDILFGT

[0750] PRPRQSTVNLGVLRKDAVNILVHGHNPVVSEMILAATREPAVRQAAQDAGAADINVAGLC

[0751] CTGNELLMRQGIPMAGNHLMTELAIVTGAADAIVADYQCIMPSLVQIAACYHTRFVTTSPK

[0752] GRFTGATHVEVHPHNAQERCREIVMLAIDAYTRRDPARVDIPSQPVSIMSGFSNEAILEALG

[0753] GTPKPLIDAVVAGQIRGFVGIVGCNNPKIRQDSANVTLTRELIRRDIMVLATGCVTTAAGKA

[0754] GLLVPEAASKAGEGLAAVCRSLGVPPVLHMGSCVDNSRILQLCALLATTLGVDISDLPVGA

[0755] SSPEWYSEKAAAIAMYAVASGIPTHLGLPPNILGSENVTAMALHGLQDVVGAAFMVEPDPV

[0756] KAADMLEAHIVARRARLGLTS

[0757] SEQ ID NO: 48 - Desulfovibrio vulgaris Miyazaki carbon monoxide dehydrogenase (DvMF)

[0758] MNSGTPDTERTLWEDARQMLRKAEAENIPTAWDRMREQTPHCKFCELGTTCRNCVMGPC

[0759] RIANRKDGKMRYGVCGADADVIVARNFGRFIAGGAAGHSDHGRDLIETLEAVVEGHAPGY

[0760] TIRDEAKLRRIAGELGVADADTRPTLDVARDLVDVCYADFGSRRTELAFLKRAPKARRELW

[0761] ARLGMTPRGIDREIAEMMHRTHMGCDNDPASLLTHAARTALADGWGGSMIGTELSDILFG

[0762] TPMPRQSTVNLGVLREDAVNILVHGHNPVVSEMILAAAREPAMQQAARDAGAAALNVAG

[0763] LCCTGNELLMRQGIPMAGNHLMTELAIVTGAADAIVADYQCIMPSLVRIAACYHTRFVTTS

[0764] SKGRFTGATHIEVHPHNAQEKCREIVQLAIEAFTRRDPARVSIPVQPVPITTGFSNEAILAALG

[0765] GTPAPLLDAVKAGQIRGFVGIVGCNNPKIMQDSGNVGLAKELIRRDIMVLATGCVTTAAGK

[0766] AGLLMPEAADMAGPGLSAVCKALGVPPVLHMGSCVDNSRILQLCGLLADELGVDISDLPV

[0767] GASSPEWYSEKAAAIGLYAVASGIPTHLGLPPNILGSDVVTGMAVDGLNGLVGAAFMVEA

[0768] DPVKAADLLEAHIVARRQKLGLSA

[0769] SEQ ID NO: 49 - Desulfovibrio frigidus carbon monoxide dehydrogenase (cooS)

[0770] MTREQRSVEELSIWEDARSMLRKARAEGVETAWDRLEQQTPHCTFCELGTTCRNCVMGPC

[0771] RISPKATKSGKLQRGVCGADADVIVARNFGRFIAGGSAGHSDHGRDLIEVLEAIVEGETTDY

[0772] AIRDEAKLRRIAAELGIDAAERPLMDVARDLVDECYSDFGSRRKSVGFLARAPEKRRALWE

[0773] KLGMTPRGVDREIAEMMHRTHMGCDNDAPNTLIHAARCALADGWSGSMIGTELSDIIFGV

[0774] PTPSMSTANLGILKKDTVNILVHGHNPVVSEMILAAAREPQLIAAAKAHGASGITVGGLCCT

[0775] GNELLMRQGIPMAGNHLMTELAIVTGAVEAVVVDYQCIMPSLVQIAGCYHTRFITTAGKAR

[0776] FTGAIHFDIHPHNAMEEARKIVQMAVDAFAERDPKRVEIPVEPVRIMTGFSNEAILNALGGS

[0777] LTPLLDAVKAGSIRGFVAIVGCNNPKIQQDSANVGLAKALIERDIMVLATGCVTTAAGKAG

[0778] LLVPEAASMAGPGLKAVCTALGIPPVLHMGSCVDNSRIMHLCGLIANELGVDISDLPVGASS

[0779] PEWYSEKAAAIGMYAVASGVYTHLGLPPNILGSQTVTDLALNGLEGLVGASFAVEPDPLKA

[0780] ADLLDARIRAKREALGLKP

[0781] SEQ ID NO: 50 - Carbon monoxide dehydrogenase of Desulfosarcina spongiae (SAMN05216233)

[0782] MAREKRKIEELSMWEDARAMIRKAQAEGIETVWDRLEEQTPHCRFCELGTTCRNCTMGPC

[0783] RISPKKGGKMQRGVCGADADVVVARNFGRFIAGGAAGHSDHGRDLIEVLAGVAEGECPDY

[0784] GIRDKAKLVRIAEELGIKSEGRSEMDIAGDLAEVLFGDFGSRKAEVAFLKRAPESRRETWKQ

[0785] LGMTPRGVDREIAEMMHRTHMGCDNDAPSTLIHAARTALADGWAGSMIGTELSDIIFGTPT

[0786] PSESTANLGVLKKDQVNILVHGHNPVVSEMILAAARMPELVARAKDVGASGINVGGLCCT

[0787] GNELLMRQGIPMAGNHLMTELSIVTGAVEAVVVDYQCIMPSMVQISGCYHTRFITTSGKAR

[0788] FTGATHFDIHPENAMEKAREIVALAVDAFAERDASRVEIPHEPVPIMTGFSNEAILAAVGGT

[0789] PDPLIDAMKRGAIRGVVGIVGCNNPKFTQDSMNVGLAKALLKKDILVLVTGCVTTAAGKA

[0790] GLLMPEGAEMAGPGLKEVCGALGIPPVLHMGSCVDNARILQLCALLAETCGVDISDLPVAA

[0791] SSPEWYSEKAAAIGLYAVASGIETHLGHPPNILGSDTVTHLATEGLEDLVGARFIIEKDPEEA

[0792] AERLDKRITAKRVGLGWNP

[0793] SEQ ID NO: 51 - Desulfovibrio halophilus carbon monoxide dehydrogenase (SP90)

[0794] MAKEPKKPEELSIWQDAHAMIRKAKAESIETAWDRLERQTPHCKFCDLGTTCRNCIMGPCR

[0795] ISTKPGAKMNLGVCGADADVVVARNFGRFIAGGSAGHSDHGRDLIEVLEAIVDGDTKHYTI

[0796] TEPEKLIRLAAEVGIATEGRELNDVANDLVDECYKDFGSRRSSLAFLSRAPEQRRALWDRV

[0797] GITPRGVDREIAEMMHRTHMGCDNDAPNTLLHSARCALSDGWGGSMIGTELSDVIFGTPTP

[0798] SRSTSNLGVIKEDKVNILVHGHNPVVSEMILAAARRPELVEKAKAAGAAGINVAGLCCTGN

[0799] ELLMRQGIPMAGNHLMTELAIVTGAVEAVVVDYQCIMPSLVTAAHCYHTRFITTAEKAKFT

[0800] GAMHFEVHPHNALEQATAIVEEAIRAYTERDKGRVEIPAESVEIMTGFSNEAILSALGGTLTP

[0801] LLDAIKAGKIRGIVGIVGCNNPKIKQDSANVGLAKELLKRDILVLVTGCVTTAAGKAGLLVP

[0802] EGIEMAGEGIKEVCGALGIPPVLHLGSCVDNSRIMHLCGLVAKELGVDISDLPVGASSPEWY

[0803] SEKAAAIGMYAVASGIMTHLGLPPNILGSETVTNIALEGLEDIVGAHFVVEDDYVKAAELLD

[0804] ARIRMKRVGLGLSE

[0805] SEQ ID NO: 52 - Desulfovibrio desulfuricans carbon monoxide dehydrogenase (Ddes)

[0806] MDDRQTDINNMSIWEDAQKMLHKARAEGIETAWDRLAQQTPHCRFCELGTTCRNCVMGP

[0807] CRISAKAAPGGKLSRGVCGADAHVIVARNFGRFIAGGSAGHSDHGRDVIETLEAVVEGKAE

[0808] GYEIRDPAKLLRIAGELGIAVDGLAVNEVAALVVDACYSDFGSRRGAVNFISRVPAKRRQV

[0809] WEKLGITPRGVDREIAEMMHRTHMGCDNDAPNTMLHAARCALSDGWAGSMIATELCDILF

[0810] GTPSPRMSTVNLGVIKKETVNILVHGHNPVVSEMILAVSREPEVLEQARQAGAAGITVAGL

[0811] CCTGNELLMRQGIPMAGNHLMTELAIVTGAVEAVVVDYQCIMPSLVQVAACYHTRFIDTA

[0812] PKARFTGAVHMNFTPENARQEASAILHMAIEAFAARDPGRVDIPVSPVNIMTGFSNEAILEA

[0813] LGGSLDPLLDAVKAGTVRGFAAVVGCNNPKVRQDSANVGIIKELIKKDIMVLVTGCITTAA

[0814] GKAGLLLPEGAAMAGPGLQKLCASLGIPPVLHMGSCVDNARIMHLCGLIANSLGVDISDLP

[0815] VVASAPEWYSEKAAAIGLYAVASGVYTHLGLPPNILGSDVVTDIALNGLEGLVGASFVVEA

[0816] DPVKAADLLDRRIRTKRSALGLDA

[0817] SEQ ID NO: 53 - Desulfuromonas acetoxidans carbon monoxide dehydrogenase (DaAHT2)

[0818] MAEKNKKSIEEKSLWEDARAMLRKAEQDGVETAWDRLEQQTPHCKFCELGTSCRNCVMG

[0819] PCRITAKMPRGVCGADADVVVARNFGRFIAGGSAGHSDHGRDLIEVLEAIVEGETKDYQIK

[0820] DEAKLRRIAGELGVADAGGGALAAVARAVMEILYADFGSRKKEVAFLGRVPEKRRAIWQS

[0821] LGMTPRGVDREIAEMMHRTHMGCDNDAASTLIHAARTALADGWAGSMIGTELSDVIFGTP

[0822] SPKKSTVNLGVINQEQVNILVHGHNPVVSEMIVAAANDPEMLARAEAAGAGGINVAGLCC

[0823] TGNEILARRGIPMAGNHLMTELAVVTGAVEAVVVDYQCIMPSLLQAAGCYHTQFITTANK

[0824] ARFSGAVHFDIKPATALAQAKEIVQLAIDGFSKRDPQRVEIPSQPVEIMTGFSNEAILNALGG

[0825] SLTPLLEAIKAGKIRGVVGIVGCNNPKFQQDSGNIGLTKALIQRDILVLVTGCVTTAAGKAG

[0826] LLLPQAIDQAGPGLQEICGALGIPPVVHFGSCVDNSRIIHLCALIAEELGVDISDLPVAASSPE

[0827] WYSEKAAAIGLYAVASGIYTHLGLPPNILGSPAVTDLALNGLESLVGASFVVEPDPDKAAD

[0828] LLEARIVSKRKALGLPK

[0829] SEQ ID NO: 54 - Desulfovibrio vulgaris subsp. desulfuricans carbon monoxide dehydrogenase (Daes)

[0830] MAKEPRPVEELSIWEDARAMIRKARKEGIETVHERLDQQTPHCKFCELGTTCRNCTMGPCR

[0831] ISAKAPRGVCGADADVVVARNFGRFVTGGAAGHSDHGRDLIEVLEAIVEGETKDYRITDEP

[0832] KLRAIAAEIGVATEGRTVMEVARDVMDAFYADFGSRRKSISFLCRVPEKRKAIWAKLGMTP

[0833] RGVDREIAEMMHRTHMGCDNDAPNTMIHAARTALADGWGGSMIGTELSDVIFGTPTPKMS

[0834] TANLAVIKKDQVNILVHGHNPVVSEMILAAAREPELIEQAKKLGATGINVAGLCCTGNELL

[0835] MRQGIPMAGNHLMTELAIITGAVEAVVVDYQCIMPSLVQISGCYHTKFIDTAAKARFTGAIH

[0836] FDIHPHNAMEQARKIVGLAVQGFVERDPGRVDIPGEPVDIMTGFSNEAVIKALGGSLDPLIQ

[0837] AIASGDIRGAVGIVGCNNPKFKQDSMNVGLAKELIKKDILVLVTGCVTTAAGKAGLLLPSAI

[0838] DQAGPGLRKICGSLGIPPVLHYGSCVDNARILQLCAALANGLGVDISDLPVGASSPEWYSEK

[0839] AAAIGLYAVASGIYTHLGHPPNILGSETVTNLAVSGLEDLVGACFVIEPDPVKAAELFDIRIR

[0840] AKRKGLGLSE

[0841] SEQ ID NO: 55 - Desulfovibrio alaskensis carbon monoxide dehydrogenase (Dde_3028)

[0842] MAKEPRPVEALSIWEDARAMLTKARAEGIETAHERLQQQTPHCKFCELGVSCRNCTMGPC

[0843] KITPKAPRGVCGADADVIVARNFGRFVAGGSAGHSDHGRDLVEVLESIVEGSTSDYRITDEA

[0844] KLRRIAAELGVTVEDRSTMDIAAELIDIFYADFGSRKKEVAFLCRVPAKRRELWDRLGMTP

[0845] RGVDREIAEMMHRTHMGCDNDAPNTLIHAARTALADGWAGSMIGTELSDVIFGTPQPSMS

[0846] HANLAAIKKDKVNILVHGHNPVVSEMILAAAREPALIAEAEAMGAQGINVAGLCCTGNELL

[0847] MRQGIPMAGNHLMTELAIVTGAVEAVVVDYQCIMPSLVQIAGCYHTLFIDTAQKARFTGA

[0848] VHFDIHPHNALEQAREIVRMAVQAYARRDASRVQIPGEPVNIMTGFSNEAVIAALGGSLDPL

[0849] VQAIAAGDIRGAVGIVGCNNPKFQQDSMNVGLAKELIKKDILVLVTGCVTTAAGKAGLLLP

[0850] EAAEQAGPGLRKICGALGIPPVLHYGSCVDNSRILQLCAALANALGVDISDLPVGASSPEWY

[0851] SEKAAAIGLYAVASGIYTHLGHPPHILGSQTVTDLAVSGLEDLVGASFFIEPDPVKAALAFDL

[0852] RIRAKRKALGLGE

[0853] SEQ ID NO:56 - Desulfovibrio ferrooxidans carbon monoxide dehydrogenase (DFE_2686)

[0854] MAKELKPPQELSIWEDAQKMIEKARRDGVETVWDRYEQQSPHCTFCELGLTCKNCNMGPC

[0855] RISPKEGGKMQRGVCGADAHVIVARNFGRFVAGGAAGHSDHGRDLIEVLEAIVEGKAPGY

[0856] QISDEAKLRRVSAELGINVDGRDVMDVARDLMDICYADFGSRKKEVGFTCRVPEKRKEIW

[0857] RKLGIMPRGVDREIAEMMHRTHMGCDNDAPNTLLHAARTSLADGWAGSMIGTELSDIIFGT

[0858] PAPSASRANLGVLKADHVNLLVHGHNPVVSEMVLAAAREPEMIAKAKAAGAAGINIGGLC

[0859] CTGNELLMRQGIPMAGNHLMTELAIVTGAVDAMVVDYQCIMPSLVQTAACYHTKFITTAD

[0860] KAKFTGATHVSFHPDNAVKQARVVVEMAIEAYERRDQGRVEIPCEPVDITTGFSIEALLSAL

[0861] GGTLDPVLDAVKAGKIRGFTAIVGCNNPRIKHDSANVGLAKALIKKDILVLATGCVTTAAG

[0862] KAGLLMPEGASMAGPGLQEVCGALGIPPVLHMGSCVDNSRVIQLCAAIANALGVDISDLPV

[0863] WGASPEWYSEKAVAIGLYCVASGIPVQIGTPPHITGSDVVTNLALSGLEDLVGAAFLVEADP

[0864] EKAADIMDERIKAKRKALGLSE

[0865] SEQ ID NO:57 - Carbon monoxide dehydrogenase 2 from thermophilic carbon monoxide - oxidizing bacterium (cooS2)

[0866] MAKQNLKSTDRAVQQMLDKAKREGIQTVWDRYEAMKPQCGFGETGLCCRHCLQGPCRIN

[0867] PFGDEPKVGICGATAEVIVARGLDRSIAAGAAGHSGHAKHLAHTLKKAVQGKAASYMIKD

[0868] RTKLHSIAKRLGIPTEGQKDEDIALEVAKAALADFHEKDTPVLWVTTVLPPSRVKVLSAHG

[0869] LIPAGIDHEIAEIMHRTSMGCDADAQNLLLGGLRCSLADLAGCYMGTDLADILFGTPAPVVT

[0870] ESNLGVLKADAVNVAVHGHNPVLSDIIVSVSKEMENEARAAGATGINVVGICCTGNEVLM

[0871] RHGIPACTHSVSQEMAMITGALDAMILDYQCIQPSVATIAECTGTTVITTMEMSKITGATHV

[0872] NFAEEAAVENAKQILRLAIDTFKRRKGKPVEIPNIKTKVVAGFSTEAIINALSKLNANDPLKP

[0873] LIDNVVNGNIRGVCLFAGCNNVKVPQDQNFTTIARKLLKQNVLVVATGCGAGALMRHGF

[0874] MDPANVDELCGDGLKAVLTAIGEANGLGGPLPPVLHMGSCVDNSRAVALVAALANRLGV

[0875] DLDRLPVVASAAEAMHEKAVAIGTWAVTIGLPTHIGVLPPITGSLPVTQILTSSVKDITGGYF

[0876] IVELDPETAADKLLAAINERRAGLGLPW

[0877] SEQ ID NO: 58 - Desulfovibrio halophilus carbon monoxide dehydrogenase (cooS)

[0878] PMSDLTGNTKTNSLKGGSTETDHSKKSTDPAVQQMLEKAAREGIQTVWDRYQAMRPLCRF

[0879] GETGLCCRHCLQGPCRINPLGREPRVGICGATADVIVIRGLDRAIAAGAAGHSGHAKHLAH

[0880] TLKKAAEGRAPDYLVKDKEKLHSVARRLGIVTDGRPENEIALDVARAALADFHEKDTPVT

[0881] WVTSVLPPDRLEVFSRHGLVPAGIDHEIAEVMHRTSMGCDADASNLLLGGLRCALGDLAG

[0882] CYMATDLSDILFGTPSPVVTECNLGVLKAGAVNVAVHGHNPVLSDIIVLVAKEMEAEARA

[0883] AGAQGINVVGICCTGNEVLMRHGISPCTHSVSQEMAIITGALDALVVDYQCIQPAVATVAE

[0884] CLGTTVITTMDMAKITGTTHVDFSEETAVENARQILRLAIESFTRRRGKPVEIPAMKKKVFA

[0885] GFSVEAILSALGRLDAGDPLKPLIDHLTAGNIRGVCLFAGCNNVKVPQDYNFITTARKLLEQ

[0886] DVLVLASGCSAGALMRHGFMDPASVEELCGGGLKAVLTAIGEANGLGGPLPPVLHVGSCV

[0887] DNSRAAALAVALAGRLGVDLDRLPVVASAAEAMHEKAVSIGAWAVALGLPTHVGVMLPV

[0888] SGGPLVHRILTEEVKGLTGGYFILEPDPESATEKLLEAINERRAGLGLHY

[0889] SEQ ID NO: 59 - Carbon monoxide dehydrogenase of Geothermobacter subterraneus subsp. tengcongensis (TTE1708)

[0890] MTKQCKVSLDPAVCEMVEKARRVKVETVWDRYQAMLPQCGFGETGLCCRHCLQGPCRID

[0891] PFGGEPKLGICGATADVIVARGLDRAIAAGAASHSGHARHLAHTLKLAAKGKARDYTIKDK

[0892] AKLRSVAARLGIPTEGRSIAEIALDVAEAALADFHEKDTPVMWAATTVTKKRARLFEEKGL

[0893] LPKGIDYEVSDIMHRTSYGVDADPVNLLLGGLRCGLADLAGCYMGTDMADILFGTPQPVV

[0894] TEANMGVLKADAVNVAVHGHNPVLSEVIVAVAKEMEAEAKAAGASGINVVGICCTGNEV

[0895] LMRHGIPACTHSVSQEMALVTGALDAMVVDYQCIMPSLATVAECMGTKLITTMEIAKIPGA

[0896] IHIEFSEEKAGEKAREILRLAIETFTRRRGKPVDIPPYKTKVVAGFSVEAIVKALSKLNAEDPL

[0897] KPLIDQIAAGNIRGVCLFAGCNNVKVPQDQNFTAIARRLLKENVLVLATGCGAGALMRHGF

[0898] MDPANVGELCGEGLKAVLTAIGEANGLGGPLPPVLHVGSCVDNSRAVALAVAVADRLGV

[0899] DTDQLPVVASAAEAVAEKAVSIGTYAVALGLPTHVGVMLPVLGGPLVTKVLTDKVKELTG

[0900] GYFIVDLDPESAAEKLLAAIDERRAALGLSVPGGGRR

[0901] SEQ ID NO: 60 - Thermomonas hydrothermalis carbon monoxide dehydrogenase (SAMN00808754_0706)

[0902] MANPCKVSLDPAVCEMVEKARRVKVETVWDRYQAMLPQCGFGETGLCCRHCLQGPCRID

[0903] PFGGEPKTGICGATADVMVARGLDRAIAAGSAAHSGHARHLAHTLKLAAKGKARDYTVK

[0904] DKAKLRSVAARLGIPTEGRSIAEIALDVAEAALADFHEKDTPVMWVATTVTKKRAKLFAEK

[0905] GLLPKGIDYEVADIMHRTLYGTDADPVNLLLGGLRCGLADLAGCYMGTDLSDILFGTPQPV

[0906] VTEANMGVLKADAVNVAVHGHNPVLSDVIVAVAKEMESEAKAAGASGVNVVGICCTGNE

[0907] VMMRHGIPACTHSVSQEMALVTGALDAVVVDYQCIMPSLATVAECMGTKLITTMEITKIPG

[0908] AIHIDFSEETAGENARQIIRLAIESFSRRRGKPVDIPQIKTKVVAGFSVEAIINALAKLNAEDPL

[0909] KPLIDQIVAGNIRGVCLFAGCNNVKVPQDKNFTAIARRLLKENVLVLATGCGAGALMRHGF

[0910] MDPANVGELCGEGLKAVLTAIGEANGLGGPLPAVLHVGSCVDNSRAVAVAVAVADRLGV

[0911] DTDQLPVVASAAEAVSEKAISIGTYAVAIGLPTHVGVMLPVVGGPLVTKVLTEKVKDLTGG

[0912] YFIVDPDPESAAKKLLAAIDERRAALGLSVPGGGRR

[0913] SEQ ID NO: 61 - Desulfobacterium carboxydodehydrogenase (gene: JT06_17280)

[0914] MDKSRDPAVQQMIARAEAQNVTTVWDRYAAMTPQCGFGDTGLCCRHCLQGPCRIDPFGE

[0915] GPREGICGASADVMVARGLDRAIAAGTAAHSGHARHLAHTLKKMAMGKAEAYAVKEPG

[0916] KLEAVAARLGIPTEGRQEREIALDVADAALADFHEKDTPVLWAATVVNPERAKVLADLNL

[0917] VPKGIDHEVSEIMHRTLYGVDADPVNLLLAGLRCGVADLAGCWMGTDLADILFGIPRPTVS

[0918] SANLGTLRADAVNIALHGHNPVLSEILVGAVGAKEEAARAAGATGINLVGICCTGNEVMM

[0919] RHGIPACTHSVSQEMAIMTGAVDAMVVDYQCIMPSVVNVAECTGTRIITTMDIAKITGATH

[0920] VDFSEEAAAAKAHEIIDQAIDQFRRRRNTPVEIPAVKTPVVAGFSVEAIVAALSGIDAEQPLK

[0921] PLVDSIKSGAIRGVCLFAGCNNVKVPQDRNFVRMARRLLRENVLVVATGCGAGALMRHGF

[0922] MDPANVEALCGDGLKAVLTAVGLANDLGGPLPPVLHMGSCVDNSRAVALTVAVAQYLG

[0923] VDTHQLPIVASAAEAVSEKAVSIGVYAVAAGLPTHVGVMLPVLGSRLVTRVLTDKVKDLT

[0924] GGYFIVDLDPDSAADKLLAAIDERRRGLGLG

[0925] SEQ ID NO: 62 - Desulfotomaculum carboxydodehydrogenase (A6M21_00615)

[0926] MQSADPAVREMLDEARRRGVETVWDRYAAQLPQCGFGELGVCCRNCMQGPCRVSPFDD

[0927] GPARGVCGATADTMVARNLVRAIAAGTAAHSGHAKHLAHTLKKSVQGKAPDYPVRDEAK

[0928] LRAVAARQGLEADGVPAAELAARMAEAALANFSEGEEPLAWTTATVTAGRVQAWEKLG

[0929] VLPVGIDSIISEIMHRTHLGVDADAVNLLLGGVACAVADYTGCHLGTDLADILFGTPQPVVS

[0930] EANMGVLKADQVNIALHGHNPVLSEVIVQVARQMEDEAKAAGAAGINLVGVCCTGNEVL

[0931] MRHGIPPATHSISQELPMLTGALDAMVVDYQCVYPSLVGVAECTGTKIITTMGMARITGAT

[0932] HVDFEEENAAAGARQIIRLAIEAFGARRGRPVQIPQYKSQVVAGFSVEAIVAALSKLDAADP

[0933] LKPVIDNIVAGNIQGVCLFAGCNNVKVPQDENYLVMARELAARDVLLLATGCGAGAYARH

[0934] GYLTPEAVGEYAGPGLKAVLTAVGEANGLGGPLPLVLHMGSCVDNSRAVDLAVAVANRL

[0935] GVDTAQLPVVASAPEFKTEKAVAIGTWAVACGLPTHLGLVPPVLGSATVTGLLTGGIKDLL

[0936] GGYFIVETDPVKAAEKLFAAIQERRRGLNLATRSW

[0937] SEQ ID NO: 63 - Propionibacterium anaeum sausage - shaped bacterium carbon monoxide dehydrogenase (cooS2)

[0938] MRSRDSAVSLMLEAAQKEAIETTWDRLDAQLPQCGFGELGVCCRHCMQGPCRISPFEDGP

[0939] QRGICGATADTMVARGLVRAIAAGAAAHSGHAKHLAHTLHKWVKGQAPDYNVKNEAKL

[0940] RAVAARQGISADGLSVRELAAKVVASVFSQFAEGEEPLAWAGATVTTGRVEALAKLGALP

[0941] VGIDSSIAEMMHRTHLGVDADPVNLLLGGISCAIADYTGCHIGTDLADILFGTPVPVVSEAN

[0942] MGVLKENSVNIAVHGHNPVLSEVIVRIAGEMQDEAKAAGADGIVLVGICCTGNEVLMRHGI

[0943] PPVTHGVSQELPILTGALDAIVVDYQCVYPSIVEVARCYGTKVITTMSIAKIQGSTHLEFVEE

[0944] DAAGKAKEIIGVAIEAFKARKGRPVNIPQVKSKVVAGFSVEAIVAALAKLDAADPLKPVIDN

[0945] IVNGNIQGVVLFAGCNNVKVPQDHNYLVMAEELAKRDVLLLATGCGAGAYARQGLMTPE

[0946] ATEKYAGPGLKIVLTAIGEANGLGGPLPLVLHMGSCVDNTRAVDLTVAIANKLGVDIDKLP

[0947] VAASAPEFKTEKAVAIGTWAVASGLPVHLGLVPPVLGGPQVTSILTAGIKDLLGGYFIVETD

[0948] PQKAAEKLFAAIQERRAGLGLGTRQW

[0949] SEQ ID NO: 64 - Anaerobic methyl bacterium carbon monoxide dehydrogenase (cooS2)

[0950] MEAMKAKKAVKATDCAVGEMLEVARKQGIETVWDRYARQIPQCGFGDSGLCCRNCTQGP

[0951] CRIDPFGDGPAAGVCGITADSMVARNLIRAIAAGAASHSGHAKHLAHVLKKVAAGKLTDY

[0952] SVKDENKLRAVATRLGIPTEGKSVQELAAAVASAALEEFSEREEPLAWAATTLTSSQVEKL

[0953] GGLGVLPAGIDSAISEILQRTTLGVDADPVNLLLGGVKGAVADFAGCHLGTDLADILFGTPT

[0954] PVFSEANMGVLKENAVNIALHGHNPVVSELIVVAARQEDILAQAKSAGAEGINLVGICCTG

[0955] NEVLMRQGVAPLTHSLSQEMPILTGALDAIVVDYQCVYPSLANIADCYGTKLITTMDIAKIP

[0956] GAVHMAIEEERGLEQAKEIIKLAVERFKARKGAPTCIPQVKQKVIAGFSTEAIIGALSKLNAA

[0957] DPLKPLIDNIVNGNIQGVVLFAGCNNVKVPQDANFIAIVKELAKRDVLMLATGCGAGALAR

[0958] HGLLTSEATMEYAGPGLKAVFTAIGEANGLGGPLPLVLHVGSCVDNSRPVDIAVALANKLG

[0959] VDVNQLPVAASAPEFKAEKAVAIGTWAVTLGLPTHLGLVPPVLGSKLVTRVLTQDIKDITG

[0960] GYFLVETDPLLAAEKIFDALQERRQKLNIGTRRW

[0961] SEQ ID NO: 65 - Carbon monoxide dehydrogenase (cooS2) from Mycobacterium silvaticum

[0962] MKSQEPLDSSVKEMLDVAKTSNVSTVWDRYQSQLPQCGFGESGICCRNCLQGPCRISPFGD

[0963] GPDVGVCGASADTIVARNLIRGIAGGAASHSGHGKHLAHVLKKVAKGEALAYGVKEAAK

[0964] LNAVAARLGIPTENRSTKEIAGDVAQAALDEFSERETPNAWAATTLTAGRVAKLNELKVM

[0965] PYGIDSIIAEIMHRTTLGVDADPVNLLLGGIKGAIADFAGCHLGTDLADILFGIPAPSVSEAN

[0966] MGVIKEKAVNIALHGHNPLLSDMIVATSREPEILAEAKAAGAEQGIQLMGICCTGNEVLMR

[0967] HGVPTVTTAVSQELPILTGAIDAMIVDYQCVYPSLPTVAACFGTPLITTMDIAKIPGATHIEFD

[0968] EDHASEKAREIIRIAITAFKRRCNKPVHIPPKTSKVVAGFSTETIVGALAKLNADDPLQPLVD

[0969] NIVNGNIQGVVLFAGCSNIKVGHDQNFVAIAKELIARNILVLATGCGAGAFARNGLLDPQAT

[0970] LDYAGDGLKAVLTAIGEANGLNGPLPPILHMGSCVDNSRPVDIAVALANKLKVDIPQLPVV

[0971] ASAPEYKTEKALAIGTWAATLGLPTHLGLVPPVVGSKLVTRILTQDIKELTGGYFIVEPDPIL

[0972] AAEKIFNAIQERRQNLNISTRRW

[0973] SEQ ID NO: 66 - Spirochaeta mobilis carbon monoxide dehydrogenase (cooS)

[0974] MDTRSVDLAVQEMLEVALQNKIETAWDRLNFQQPQCGFGELGLCCRNCTQGPCRIDPFGD

[0975] GPRKGVCGADADTLVARNLARSIVAGTASHSGHSKHLAHVLKKWADGKAPDYPVKDERK

[0976] LLAVAQRHGIEVEGKEIKEIARSVAEVALEDFSERNTPIGFANRTVTKNRAELWNKFGVNPV

[0977] GIDSIIAEMMHRTTLGVDADANNLLLGSIAAGIADYAGCRMGTDLSDILFGTPAPVISEMNM

[0978] AVLDPKQVNVALHGHNPVLSDLLVQVSREMREEAKMAGATGINLVGICCTGSEVLMRHG

[0979] VPSCTHSVSQELPILTGVLDAMVVDYQCVYPSIATLAGCYSTKVITTMSMAKIPGAIHIEIHE

[0980] EGAWAKGKEILRLAIEAFKERAGRLYQIPRYKEKVVAGFSKEAIVAALAKLNQEEPLQPLID

[0981] NIVNGNIQGICLFAGCNNVKVRQDENYIIMAKELAKRNVLLLATGCGAGSLGRLGFMNGEA

[0982] TKAYAGDGMKDVLSAIGEANGLEGPLPPVLHLGSCVDNSRAVDIAVAIAEKLGVDTDMLP

[0983] VVASAPEYKTEKAFSIGTYAVALGLPTHLGMVPPVAGSKQVVKTLTKDIREITGGYFIVETN

[0984] PSEAAKKLFLAIQERRAGLGLLTRLW

[0985] SEQ ID NO: 67 - Desulfovibrio vulgaris carbon monoxide dehydrogenase (DCCM_2691)

[0986] MKSRDKAVNQMIKAAGEREVETVWDRYQTQQPQCGFGELGVCCRNCTQGPCRINPYGDG

[0987] PDHGVCGANADTIVARNLVRAIAAGAAAHSGHARHLAHALKKGLAGKAPAYGLKDEAKL

[0988] RALAARVGLDPELPAEELVNKFMDMALEQFAEGEGPLKWAQSFLTGGCGETFERLGTMPTGIDNTIVEMLHRTHLGVDADPVNLLLAGVSCAIADYAGSHLATDLADIMFGTPGPVFSQANMGVMKEKSVNIALHGHNPVLSDIIVQEAREMEEEARGAGAEGINLMGVCCTGNEILMRHGVPALTHSISQEMPMLTGALDAMVVDYQCVYPSLAGVAECTGTKLITTMDMAKISGAVHLGMHEENAAEKAREIISVAIEQFRARQGRPVNIPSVSSKVVAGFSVEAIVQALSKLDAADPLKPVIDSLAAGSLQGVVLLAGCNNVKVPQDDSFLVIARELAARDVLLLATGCGAGAYARHGYLTPEATLEYAGPGLKAVLTAIGEANGLGGPLPLVLHMGSCVDNSRAVDLAVAVADKLGVDLNKLPVVASAPEFKTEKAVSIGTYAMAIGLPVHLGVVPPVLGSRLVTSVLTEGVKDLFGGYFIVETDPYKAAGAIFAAIQERRSGLGLPTREWE

[0989] SEQ ID NO: 68 - Major subunit of nitrate - inducible formate dehydrogenase from Escherichia coli (fdnG)

[0990]

[0991] SEQ ID NO: 69 - α subunit of formate dehydrogenase - N nitrate - inducible from Shigella flexneri

[0992] (fdnG)MDVSRRQFFKICAGGMAGTTVAALGFAPKQALAQARNYKLLRAKEIRNTCTYCSVGCGLLMYSLGDGAKNAREAIYHIEGDPDHPVSRGALCPKGAGLLDYVNSENRLRYPKYRAPGSDKWQRISWEEAFSRIAKLMKADRDANFIEKNEQGVTVNRWLSTGMLCASGASNETGMLTQKFARSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVMVMGGNAAEAHPVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGILRYLIENNKINAEYVKHYTNASLLVRDDFAFEDGLFSGYDAEKRQYDKSSWNYQFDENGYAKRDETLTHPRCVWNLLKAHVSRYTPDVVENICGTPKADFLKVCEVLASTSAPDRTTTFLYALGWTQHTVGAQNIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSEKQ

[0993] VDLQSYLEANTPKATLADQVNYWSNYPKFFVSLMKSFYGDAAQKENNWGYDWLPKWDQ

[0994] TYDVIKYFNMMDEGKVTGYFCQGFNPVASFPDKNKVVSCLSKLKYMVVIDPLVTETSTFW

[0995] QNHGESNDVDPASIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGEIL

[0996] AGIYHHLRELYQAEGGKGVEPLMKMSWNYKQPHEPQSDEVAKENNGYALEDLYDANGV

[0997] LIAKKGQLLSSFAHLRDDGTTASSCWIYTGSWTEQGNQMANRDNSDPSGLGNTLGWAWA

[0998] WPLNRRVLYNRASADINGKPWDPKRMLIQWNGSKWTGNDIPDFGNAAPGTPTGPFIMQPE

[0999] GMGRLFAINKMAEGPFPEHYEPIETPLGTNPLHPNVVSNPVVRLYEQDALRMGKKEQFPYV

[1000] GTTYRLTEHFHTWTKHALLNAIAQPEQFVEISETQAAAKGINNGDRVTVSSKRGFIRAVAV

[1001] VTRRLKPLNVNGQQVETVGIPIHWGFEGVARKGYIANTLTPNVGDANSQTPEYKAFLVNIE

[1002] KA

[1003] SEQ ID NO: 70 - Formate dehydrogenase - N alpha subunit (fdnG) of Enterobacteriaceae bacteria

[1004] MDVSRRQFFKICAGGMAGTTVAALGFAPKQALAQARNYKLLRAKEIRNTCTYCSVGCGLL

[1005] MYSLGDGAKNAREAIYHIEGDPDHPVSRGALCPKGAGLLDYVNSENRLRYPEYRAPGSDK

[1006] WQRISWEEAFSRIAKLMKADRDANFIEKNEQGVTVNRWLSTGMLCASGASNETGMLTQKF

[1007] ARSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVMVMGGNAAEAH

[1008] PVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLRYLIENDKINA

[1009] EYVKHYTNASLLVRDDFAFEDGLFSGYDAEKRQYDKSSWNYQFDENGYAKRDETLTHPR

[1010] CVWNLLKAHVSRYTPDVVENICGTPKADFLKVCEVLASTSAPDRTTTFLYALGWTQHTVG

[1011] AQNIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSEKQVDL

[1012] QSYLEANTPKATLAGQVNYWSNYPKFFVSLMKSFYGDAAQKENNWGYDWLPKWDQTYD

[1013] VIKYFNMMDEGKVTGYFCQGFNPVASFPDKNKVVSCLSKLKYMVVIDPLVTETSTFWQNH

[1014] GESNDVDPASIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGEILAGIY

[1015] HHLRELYQAEGGKGVEPLMKMSWNYKQPHEPQSDEVAKENNGYALEDLYDANGVLIAK

[1016] KGQLLSSFAHLRDDGTTASSCWIYTGSWTEQGNQMANRDNSDPSGLGNTLGWAWAWPLN

[1017] RRVLYNRASADINGKPWDPKRMLIQWNGSKWTGNDIPDFGNAAPGTPTGPFIMQPEGMGR

[1018] LFAINKMAEGPFPEHYEPIETPLGTNPLHPNVVSNPVVRLYEQDALRMGKKEQFPYVGTTY

[1019] RLTEHFHTWTKHALLNAIAQPEQFVEISETLAAAKGINNGDRVTVSSKRGFIRAVAVVTRRL

[1020] KPLNVNGQQVETVGIPIHWGFEGVARKGYIANTLTPNVGDANSQTPEYKAFLVNIEKA

[1021] SEQ ID NO: 71 - Salmonella typhimurium Molybdopterin Oxidoreductase (fdnG)

[1022] MDVSRRQFFKICAGGMAGTTVAALGFTPKMALAQARNYKLLRAKEIRNSCTYCSVGCGLL

[1023] MYSLGDGAKNAKEAIYHIEGDPDHPVSRGALCPKGAGLLDYVHSEDRLRYPEYRAPGSDK

[1024] WQRISWDDAFTRIAKLMKADRDANFIEKNEQGVTVNRWLSTGMLCASAASNETGMLTQK

[1025] FARSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVMVMGGNAAEA

[1026] HPVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTTIDFLSGVLLYLIENNKIN

[1027] AEYVKHYTNASLLVRDDDFAFDDGLFSGYDAQKRQYDKSSWNYQFDENGYAKRDETLTHP

[1028] RCVWNLLKQHVSRYTPDVVENICGTPKADFLKVCEVLASTSVPDRTTTFLYALGWTQHTV

[1029] GAQNIRTMAMIQLLLLGNMGMGAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSEKQAD

[1030] LQTYLAANTPKATLADQVNYWGNYPKFFVSLMKSFYGDAAQQENDWGFAWLPKWDQSY

[1031] DVIKYFNMMDSGKVTGYFCQGFNPVASFPDKNKVVQSLSKLKYLVVIDPLVTETSTFWQN

[1032] HGESNDVDPTTIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGEILAGI

[1033] YHRLREMYRAEGGKGAEPLLKMSWNYKQPDEPHSEEVAKENNGYALEDLYDANGTLLAR

[1034] KGQLLSSFALLRDDGTTSSSCWIYTGSWTEQGNQMSRRDNADPSGLGNTLGWAWAWPLN

[1035] RRVLYNRASADPQGKPWDPKRMLIQWNGAKWTGNDIPDFNNAAPGSGTNPFIMQPEGLGR

[1036] LFAIDKMAEGPFPEHYEPMETPLGTNPLHPNVVSNPAARLYEEDALRMGKKEQFPYVGTTY

[1037] RLTEHFHTWTKHALLNAIAQPEQFVEISETLAAAKGIANGDYVKVSSKRGFIRAVAVVTRR

[1038] LRTLHVNGQQVETVGIPIHWGFEGVARKGYIANTLTPNVGDANSQTPEYKAFLVNIEKA

[1039] SEQ ID NO: 72 - Main subunit of formate dehydrogenase nitrate - inducible of Citrobacter rodentium (fdnG)

[1040] MDVSRRKFFKICAGGMAGTTVAALGFTPKMALAQARNYKLLRAKEIRNTCTYCSVGCGLL

[1041] MYSLGDGAKNTKEAIYHIEGDPDHPVSRGALCPKGAGLLDYVHSENRLRYPEYRAPGSDK

[1042] WQRISWDEAFSRIAKLMKADRDANFIEKNEQGVTVNRWLSTGMLCASAASNETGMLTQKF

[1043] VRSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVMVMGGNAAEAH

[1044] PVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLLYLIENNKINAE

[1045] YVKHYTNASLLVRDDFAFEDGLFSGYDADKRQYDKSSWNYQFDENGFARRDETLSHPRCV

[1046] WNLLKQHVSRYTPEVVENICGTPKADFLKVCEVLASTSAADRTTTFLYALGWTQHTVGAQ

[1047] NIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSEKHSDWQS

[1048] WLNANTPKATQADQVNYWSNYPKFAVSLMKAFYGDDAQKENDWGFDWLPKWDQAYD

[1049] VIKYFNMMDSGKVTGYICQGFNPVASFPDKNKVVRSLSKLKFMVVIDPLVTETSTFWQNH

[1050] GESNDVDPASIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGEILAGIY

[1051] HRLREMYRTEGGKGAEPLLKMRWSYKQPDHPESEEVAKENNGYALADLYDANGVLIAKK

[1052] GQLLSSFAQLRDDGTTSSSCWIYTGSWTEQGNQMANRDNADPSGLGNTLGWAWAWPLNR

[1053] RVLYNRASADINGKPWDPKRMLIQWNGAKWTGNDIPDFNTAPPGSKTGPFIMQPEGVGRL

[1054] FALDKLAEGPFPEHYEPMETPLGTNPLHPNVVSSPVVRIYEDDVLRLGKKDKFPYVGTTYR

[1055] LTEHFHTWTKHALLNAIAQPEQFVEISETLAAAKGITNGDRVTVSSKRGFIRAVAVVTRRLR

[1056] TLNVNGQQVETVGIPLHWGFEGVARKGYIANTLTPNVGDSNSQTPEYKAFLVNIEKA

[1057] SEQ ID NO: 73 - Formate dehydrogenase - N alpha subunit (fdnG) of Escherichia coli white

[1058] MDVSRRKFFKICAGGMAGTTVAALGFSPKMALAQSRNYKLLRAKEIRNSCTYCSVGCGLL

[1059] MYSLGDGAMNAKEAIYHIEGDPDHPVSRGALCPKGAGLLDYVHSENRLRYPEYRAPGSNK

[1060] WQRISWNEAFDRIAKLMKADRDANFIEKNEQGVTVNRWLSTGMLCASAASNETGMLTQK

[1061] FARSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVMVMGGNAAEA

[1062] HPVGFRWAMEAKNHNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLRYLIENNRIN

[1063] AEYVRHYTNAGLLVRDDFTFEDGLFSGYDAEKRQYDKSSWNYQFDENGHALRDDTLTHP

[1064] RCVWNLLKQHVSRYTPEVVENICGTPKADFLKVCEVLASTSAADRTTTFLYALGWTQHTV

[1065] GAQNIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSEKQTSL

[1066] QSWLADNTPKATLPDQVNYWSNYPKFAVSLMKAFYGDDARKENDWGFDWLPKWDQTY

[1067] DVIKYFNMMDSGKVTGYICQGFNPVASFPDKNKVVRSLSKLKYMVVIDPLVTETSTFWQN

[1068] HGESNDVDPASIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGQILAG

[1069] LFHRLREMYRTEGGKGAEPLLKMRWDYKQPNHPESEEVAKENNGYALEDLYDANGKLIA

[1070] KKGQLLSSFAQLRDDGTTASSCWIYTGSWTEQGNQMANRDNADPSGLGNTLGWAWAWP

[1071] LNRRVLYNRASADINGKPWDPKRMLIQWDGKKWTGNDIPDFNTAAPGSKTGPFIMQPEGL

[1072] GRLFALNKLAEGPFPEHYEPMETPLGTNPLHPNVISSPVVRLYEEDAVRMGKKEQFPYVGT

[1073] TYRLTEHFHTWTKHAKLNAIAQPEQFVEISETLAAAKNIANGDRVTVSSKRGFIRAVAVVT

[1074] RRLRTLHVNGQQVETIGIPLHWGYEGVARKGYIANTLTPNVGDSNSQTPEYKAFLVNIEKA

[1075] SEQ ID NO: 74 - Formate dehydrogenase - N alpha subunit (fdnG) of Enterobacteriaceae bacteria

[1076] MDVSRRKFFKICAGGMAGTTAAALGFAPKMALAQARNFKLLRAKEIRNTCTYCSVGCGLL

[1077] MYSLGDGAKNAKEAIYHIEGDPDHPVSRGALCPKGAGLLDYVHSENRLRYPQYRAPGSDK

[1078] WQRISWDEAFNRIARLMKADRDANFIEKNEQGVTVNRWLSTGMLCASAASNETGMLTQK

[1079] FVRSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVVVMGGNAAEAH

[1080] PVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLLYLIENNKINAE

[1081] YVKHYTNASLLVRDDFAFEEGLFSGYDAEKRQYDKSSWNYQFDENGYAKRDKTLSHPRC

[1082] VWNLLRQHVSRYTPEVVENICGTPKADFLKVCEVLASTSAADRTTTFLYALGWTQHTVGA

[1083] QNIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPSDKQSDLQS

[1084] YLSANTPKATLPDQVNYWSNYPKFFVSLMKSFYGEAAQKENDWGFNWLPKWDQAYDVIK

[1085] YFNMMDNGNVTGYICQGFNPVASFPDKNKVVRSLSKLKYLVVIDPLVTETSTFWQNHGES

[1086] NDVDPAAIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGQDAPGEARNDGEILAGIYHRL

[1087] RELYRREGGKGAEPLLKMSWSYKQPDHPESAEVAKENNGYALADLYDQNGALLAKKGQL

[1088] LNSFALLRDDGSTASSCWIYTGSWTEQGNQMANRDNADPSGLGNTLGWAWAWPLNRRVL

[1089] YNRASADINGKPWDAKRMLIQWNGSKWVGNDIPDFNTAPPGSNTGPFIMQQEGLGRLFAL

[1090] DKLAEGPFPEHYEPMETPLGTNPLHPKVVSSPVVRLYEEDAIRLGKKDKFPYVGTTYRLTEH

[1091] FHTWTKHALLNSIAQPEQFVEISEGLAKSKGIANGDWVKVSSKRGFIRAVAVVTRRLRTLN

[1092] VNGQQVETVGIPLHWGFEGVARKGYIANTLTPNVGDSNSQTPEYKAFLVNIEKA

[1093] SEQ ID NO: 75 - Formate dehydrogenase - Nα subunit (fdnG) of Enterobacteriaceae bacterium 4M9

[1094] MDVSRRGFFKICAGGMAGTTVAVLGFAPKTALAQARNYKLLRAKEIRNTCTYCSVGCGLL

[1095] MYSMGDGSKNAKSSIFHIEGDPDHPVSRGALCPKGAGLLDYIHSDNRLRYPQYRAPGSDK

[1096] WQRISWDDAFKRIARLMKDDRDANFVETNDQGVKVNRWLSTGMLCASAASNETGMLTQ

[1097] KFARSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVVVMGGNAAEA

[1098] HPVGFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLLYLINNNKIH

[1099] ADYVKHYTNASLLIRDDYSFDDGLFSGYDADKRQYDKTSWNYQFDENGHALRDMTLTHP

[1100] RCVWNLLKEHVSRYTPDVVENICGTPKADFLKVCEVLASTSAANRAATFLYALGWTQHTV

[1101] GAQNIRTMAMIQLLLGNMGVAGGGVNALRGHSNIQGLTDIGLLSTSLPGYLTLPQESHHDV

[1102] ETYLAANTPKALLPGQVNYWSNYPKFYVSLMKTFYGDAATRENGWGYDWLPKWDQAYD

[1103] VLKYFDMMDRGEVTGYFCQGFNPVASFPDKNKIVASLSKLKYMVVVDPLVTETSNFWQN

[1104] HGEMNDVDPASIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKGADAPGEAINDGQILAG

[1105] LYDELRRLYRAEGGKGVEPLMKMSWNYKQPHEPESEEVAKENNGYALADLFDANGNLLV

[1106] KKGELLDSFAQLRDDGTTASGCWIYAGSWTRKGNQMANRDNADPSGLGNTLGWTWAWP

[1107] LNRRVLYNRASCDPQGKAWDPHRLLIEWNGEKWVGNDIPDYSAAAPELNAGPFIMQQEGL

[1108] GRLFALNKLAEGPFPEHYEPIETPIGTNPLHPNVVSSPVARMFKADAARIGDRKEFPYIGTTY

[1109] RLTEHFHTWTKHARLNAIAQPEQFVEISEALAKSKGISNGDSVKVSSKRGFIRAKAVVTRRL

[1110] QTFNVNGQQVETVGIPIHWGFEGKTQKGFVANTLTPSVGDANSQTPEYKAFLVNIEKA

[1111] SEQ ID NO: 76 - Formate dehydrogenase - N alpha subunit (fdnG) from Erwinia

[1112] MDVSRRSFFKICAGGMAGTTVAALGFAPTTALAEARSYKLLRAKETRNTCTYCSVGCGLL

[1113] MYSLGDGAKNAKASIFHIEGDPDHPVNRGALCPKGAGLLDYIHSESRLRYPEYRAPGSDKW

[1114] QRISWEDAFERIAKLMKADRDANFISQNKQGTTVNRWLTTGMLCASAASNETGMLTQKFV

[1115] RSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVVVMGGNAAEAHPV

[1116] GFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLHYLITHNKINAEY

[1117] VKHYTNAPLLVRDDFRFEDGLFSGYDAEKRQYDKSSWNYQLDENGFARRDDTLSHPRCV

[1118] WNLLKQHVSRYTPDVVENICGTPQADFLKVCEVLASTSAVDRTTTFLYALGWTQHSVGSQ

[1119] NIRTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLPGYLTLPKDDQHDLES

[1120] YLAANTPKATLPGQVNYWGNYPKFFVSLMKTFWGDTATAQNQWGYDWLPKWDQAYDV

[1121] LNYFDMMDRGEVNGYICQGFNPVASFPDKNKVVASLSKLKYMVVIDPLVTETSNFWQNH

[1122] GESNDVDPSQIQTEVFRLPSTCFAEEDGSIVNSGRWLQWHWKGADAPGEARNDGEILAGIY

[1123] HRLRTLYREQGGVASEPVLNMTWDYKLPDGPESEEVAKENNGYALADIHDASGKLLVKK

[1124] GELLDSFAQLRDDGTTAAGCWIWTGSWTRDGNQMARRDNADPSGLGNTLAWAWAWPLN

[1125] RRVLYNRASADPAGKAWDPKRVLIEWNGKKWVGNDVPDYNTAAPSENVGPFIMQQEGLG

[1126] RLFALNKLAEGPFPEHYEPFETPLGTNPLHPNVISNPVARLYQADAARLGERKAFPYVGTTY

[1127] RLTEHFHTWTKHARLNAIAQPEQFVEISEGLAKAKGIANGDTVKVSSKRGFIRAKAVVTRR

[1128] LRTLEVNGQQVETVGIPLHWGFEGATRKGYLANTLTPSVGDANSQTPEYKAFLVNIEKA

[1129] SEQ ID NO: 77 - Jejubacter calystegiae formate dehydrogenase - Nα subunit (fdnG)

[1130] MDVSRRSFFKICAGGMAGTTVAALGFAPTTALAEARSYKLLRAKETRNTCTYCSVGCGLL

[1131] MYSLGDGAKNAKSSIFHIEGDPDHPVNRGALCPKGAGLLDYIHSESRLRYPEYRAPGSDKW

[1132] QRISWEDAFERIAKLMKADRDANFISQNKQGTTVNRWLTTGMLCASAASNETGMLTQKFV

[1133] RSLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVVVVMGGNAAEAHPV

[1134] GFRWAMEAKNNNDATLIVVDPRFTRTASVADIYAPIRSGTDITFLSGVLHYLIAHNKINAEY

[1135] VKHYTNAPLLVRDDFSFEDGLFSGYDENKRQYDKSSWNYQLDESGFAKRDDTLSHPRCVW

[1136] NLLKQHVSRYTPEVVENICGTPQADFQKVCEVLASTSAVDRTTTFLYALGWTQHSVGSQNI

[1137] RTMAMIQLLLGNMGMAGGGVNALRGHSNIQGLTDLGLLSTSLSGYLTLPKEDQHDLESYL

[1138] TANTPKATLPGQVNYWGNYPKFFVSLMKTFWGDAATAQNQWGYDWLPKWDQAYDVLN

[1139] YFDRMDRGEVNGYICQGFNPVASFPDKNKVVASLSKLKYMIVIDPLVTETSNFWQNHGESN

[1140] DVDSSQIQTEVFRLPSTCFAEEDGSIVNSGRWLQWHWKGADAPGEARNDGEILAGIYHRLR

[1141] TLYREQGGVASEPVLNMTWDYKLPDGPESEEVAKENNGYALADLYDADGKLLVKKGELL

[1142] DSFAQLRDDGTTASGCWIWTGSWTRNGNQMAKRDNADPSGLGNTLDWAWAWPLNRRVL

[1143] YNRASADPAGKAWDPKRVLIEWNGSKWVGNDVPDYNTAAPSQNVGPFIMQQEGLGRLFA

[1144] LNKLAEGPFPEHYEPFETPLGTNPLHPAVISNPVARLYKADAARLGERKAFPYVGTTYRLTE

[1145] HFHTWTKHARLNAIAQPEQFVEISEGLAKAKGIANGDTVKVSSKRGFIRAKAVVTRRLRTL

[1146] DVNGQQVETVGIPLHWGFEGATRKGYLANTLTPSVGDANSQTPEYKAFLVNIEKA

[1147] SEQ ID NO: 78 - Formate dehydrogenase Nα subunit (M992_0960) of Moorella wisconsensis containing selenocysteine

[1148] MNLSRRQFFKICAGGMASTTIAALGFMPGLAMANVREYKLLRSKETRNTCTYCSVGCGLLI

[1149] YSMGDGAMNAKSSIFHIEGDADHPVNRGALCPKGAGLIDYIHSEGRLRYPEYRAPGSNKW

[1150] QRISWDDAFSRIARLMKDDRDTNFIEKNSQGTSVNRWLSTGMLCASAASNETGILTQKFTR

[1151] SLGMLAVDNQARVUHGPTVASLAPTFGRGAMTNHWVDIKNANVIMVMGGNAAEAHPVG

[1152] FRWAMEAKNNNDATLIVVDPRFTRTASVADHYVPIRSGTDITFLSGVLRYLIENNKINVEYV

[1153] KHYTNASLLIRDDFSFDDGLFSGYDAEKRQYDKTSWNYQFDENGFAKRDDSWQHPRCVW

[1154] NLLKQHISRYTPEIVENICGTSQADFLKVCEILATTSSADRTTTFLYALGWTQHTVGSQNIRT

[1155] MAMIQLLLGNMGMAGGGINALRGHSNIQGLTDLGLLSTSLPGYLSLPSEKQTNIESYLQAN

[1156] TPVATLPNQVNYWSNYPKFFVSLMKSFYGDAAQKDNQWGFDWLPKWDQSYDVIKYFDM

[1157] MSKGQVTGYICQGFNPVASFPDKNKVVSCLSKLKYLVIIDPLVTETSTFWQHHGEMNDVDP

[1158] ATIQTEVFRLPSTCFAEEDGSIANSGRWLQWHWKAADAPGEARNDGQILAGLLDKIRQLYA

[1159] SEGGKGTEPLMRMRWDYKQPFHPESEEMAKENNGIALVDLYDADGNLQAKKGQLLNSFA

[1160] LLRDDGSTASSCWIYSGCWTEQGNQMAKRDNSDPSGLGNTLGWAWAWPLNRRIIYNRAS

[1161] ADPQGKPWDKNRVVIEWKGNKWVGNDVPDFNTSAPDVGTSPFIMQPEGLGRLFAIDKMA

[1162] EGPFPEHYEPVETPLGTNPLHPNVVSNPAARIFLEDQQRLGNHQDYPYVGTTYRLTEHFHT

[1163] WTKHARLNAIAQPEQFVEISQTLAQQKGINNGDKVTVSSKRGFIRAIAVVTARLQALHVDG

[1164] KSIETIGIPIHWGFEGQAQKGFIANTLTPSVGDANSQTPEYKAFLVNIEKA。

Claims

1. A method for reducing nitrogen-containing functional groups in a target compound, comprising: The target compound is contacted with a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a support material in the presence of a molecular reducing agent for oxidation by the oxidoreductase or a functional fragment or derivative thereof, under the conditions that: - said molecular reductant is oxidized by said oxidoreductase or a functional fragment or derivative thereof; and - The nitrogen-containing functional groups are reduced at the support material.

2. The method according to claim 1, comprising: The target compound is contacted with the support material.

3. The method according to claim 1 or 2, wherein: The reduction of the nitrogen-containing functional groups involves direct electron transfer from the support material to the target compound.

4. A method according to any one of the preceding claims, comprising: The nitrogen-containing functional groups are reduced to form amine groups or quaternary ammonium groups.

5. A method according to any one of the preceding claims, wherein: The oxidoreductase or a functional fragment or derivative thereof is in electrical contact with the support material.

6. A method according to any one of the preceding claims, wherein: The oxidoreductase or functional fragment or derivative transfers electrons to the carrier material via an intramolecular electron conduction pathway; Preferably, the intramolecular electron conduction pathway includes a series of [FeS] clusters.

7. A method according to any one of the preceding claims, wherein: The molecular reducing agent is selected from hydrogen, carbon monoxide, formate, isotopes thereof and mixtures thereof; Preferably, the molecular reducing agent includes hydrogen or its isotope or consists of hydrogen or its isotope.

8. A method according to any one of the preceding claims, wherein: The supported biocatalyst comprises hydrogenase or a functional fragment or derivative thereof.

9. The method according to claim 8, wherein: The hydrogenase is selected from or includes: i) the amino acid sequence of Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2) or an amino acid sequence having at least 60% homology thereto; ii) the amino acid sequence of Escherichia coli hydrogenase 2 (SEQ ID NO: 3 and / or 4) or an amino acid sequence having at least 60% homology thereto; iii) an amino acid sequence of a portion of the membrane-bound hydrogenase of Ralstonia eutropha (SEQ ID NO: 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto; iv) an amino acid sequence of a portion of the regulatory hydrogenase of Ralstonia eutropha (SEQ ID NO: 8 and / or 9) or an amino acid sequence having at least 60% homology thereto; v) the amino acid sequence of a thermophilic hydrogenase 1 (SEQ ID NO: 10 and / or 11) or an amino acid sequence having at least 60% homology thereto; vi) the amino acid sequence of a hydrogenase of Hydrogenovibrio maritima (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto; vii) the amino acid sequence of a Sulphuria auberginea hydrogenase (SEQ ID NOs: 14 and 15) or an amino acid sequence having at least 60% homology thereto; viii) the amino acid sequence of an Alteromonas mesquite hydrogenase (SEQ ID NO: 16 and / or 17) or an amino acid sequence having at least 60% homology thereto; ix) the amino acid sequence of Allochromatium vinosum membrane-bound hydrogenase (SEQ ID NO: 18 and / or 19) or an amino acid sequence having at least 60% homology thereto; x) the amino acid sequence of Salmonella typhimurium LT2 nickel-iron hydrogenase 5 (SEQ ID NO: 20 and / or 21) or an amino acid sequence having at least 60% homology thereto; xi) the amino acid sequence of Desulfovibrio vulgaris F hydrogenase from Miyazaki (SEQ ID NO: 23 and / or 24) or an amino acid sequence having at least 60% homology thereto; xii) the amino acid sequence of Clostridium beijerinckii SM10 (CbA5H) [FeFe]-hydrogenase (KX147468) (SEQ ID NO: 25), the amino acid sequence of Clostridium beijerinckii ATCC 51743 [FeFe]-hydrogenase (Cbei_4110) (SEQ ID NO: 26), the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_1773) (SEQ ID NO: 27), or the amino acid sequence of Clostridium beijerinckii [FeFe]-hydrogenase (Cbei_3796) (SEQ ID NO: 28), or an amino acid sequence that is at least 60% homologous thereto; xiii) the amino acid sequence of Clostridium pasteurianum [FeFe]-hydrogenase (hydA) (SEQ ID NO: 29) or an amino acid sequence at least 60% homologous thereto; xiv) the amino acid sequence of Chlamydomonas reinhardtii [FeFe]-hydrogenase (hyd1) (SEQ ID NO: 30) or an amino acid sequence at least 60% homologous thereto; xv) the amino acid sequence of Chlorella [FeFe]-hydrogenase (SEQ ID NO: 31 and / or 32) or an amino acid sequence having at least 60% homology thereto; xvi) an amino acid sequence of a portion of a soluble hydrogenase from Ralstonia eutropha (SEQ ID NO: 33 and / or 34) or an amino acid sequence having at least 60% homology thereto; xvii) an amino acid sequence of a portion of a soluble hydrogenase from Rhodococcus opacus (SEQ ID NO: 35 and / or 36) or an amino acid sequence having at least 60% homology thereto; xviii) the amino acid sequence of a membrane-bound hydrogenase of Desulfovibrio fructivorum (SEQ ID NO: 37 and / or 38) or an amino acid sequence having at least 60% homology thereto; xix) the amino acid sequence of Clostridium acetobutylicum iron-ferrohydrogenase (SEQ ID NO: 39) or an amino acid sequence having at least 60% homology thereto; xx) the amino acid sequence of a nickel-iron selenide hydrogenase from Desulfuromicrobium rod-shaped (SEQ ID NO: 40 and / or 41) or an amino acid sequence having at least 60% homology thereto; xxi) an amino acid sequence of a portion of a soluble hydrogenase from Hydrogenophilus thermoflavonoids (SEQ ID NO: 42 and / or 43) or an amino acid sequence having at least 60% homology thereto; xxii) the amino acid sequence of Desulfovibrio maxima periplasmic [NiFe] hydrogenase (SEQ ID NO: 44 and / or 45) or an amino acid sequence at least 60% homologous thereto; or xxiii) the amino acid sequence of the soluble alpha subunit of Pyrococcus furiosus (SEQ ID NO: 46) or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

10. A method according to any one of the preceding claims, wherein: The supported biocatalyst comprises carbon monoxide dehydrogenase or a functional fragment or derivative thereof.

11. The method according to claim 10, wherein: The carbon monoxide dehydrogenase is selected from or includes: i) the amino acid sequence of Desulfovibrio hildenborough carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 47) or an amino acid sequence having at least 60% homology thereto; ii) the amino acid sequence of Desulfovibrio miyazaki vulgaris carbon monoxide dehydrogenase (DvMF) (SEQ ID NO: 48) or an amino acid sequence having at least 60% homology thereto; iii) the amino acid sequence of Desulfovibrio psychrotrophicus carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 49) or an amino acid sequence having at least 60% homology thereto; iv) the amino acid sequence of Desulfurobacterium spongiosum carbon monoxide dehydrogenase (SAMN05216233) (SEQ ID NO: 50) or an amino acid sequence having at least 60% homology thereto; v) the amino acid sequence of Desulfovibrio halodesulfuri carbon monoxide dehydrogenase (SP90) (SEQ ID NO: 51) or an amino acid sequence having at least 60% homology thereto; vi) the amino acid sequence of Desulfovibrio desulfuricans carbon monoxide dehydrogenase (Ddes) (SEQ ID NO: 52) or an amino acid sequence having at least 60% homology thereto; vii) the amino acid sequence of Desulfurophilus alkali carbon monoxide dehydrogenase (DaAHT2) (SEQ ID NO: 53) or an amino acid sequence having at least 60% homology thereto; viii) the amino acid sequence of Pseudomonas aeruginosa carbon monoxide dehydrogenase (Daes) (SEQ ID NO: 54) or an amino acid sequence having at least 60% homology thereto; ix) the amino acid sequence of Desulfovibrio alaska carbon monoxide dehydrogenase (Dde_3028) (SEQ ID NO: 55) or an amino acid sequence having at least 60% homology thereto; x) the amino acid sequence of Desulfovibrio ferrochlore carbon monoxide dehydrogenase (DFE_2686) (SEQ ID NO: 56) or an amino acid sequence having at least 60% homology thereto; xi) the amino acid sequence of Thermohydrophilus hydrogenoformans carbon monoxide dehydrogenase 2 (cooS2) (SEQ ID NO: 57) or an amino acid sequence having at least 60% homology thereto; xii) the amino acid sequence of Desulfurizing Algae carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 58) or an amino acid sequence having at least 60% homology thereto; xiii) the amino acid sequence of a carbon monoxide dehydrogenase from Thermoanaerobacter subterranea subsp. tengchong (TTE1708) (SEQ ID NO: 59) or an amino acid sequence having at least 60% homology thereto; xiv) the amino acid sequence of Thermomonas toyota carbon monoxide dehydrogenase (SAMN00808754_0706) (SEQ ID NO: 60) or an amino acid sequence having at least 60% homology thereto; xv) the amino acid sequence of Desulfolobus carbon monoxide dehydrogenase (gene: JT06_17280) (SEQ ID NO: 61) or an amino acid sequence having at least 60% homology thereto; xvi) the amino acid sequence of Desulfotomaculum copahuensis carbon monoxide dehydrogenase (A6M21_00615) (SEQ ID NO: 62) or an amino acid sequence having at least 60% homology thereto; xvii) the amino acid sequence of a carbon monoxide dehydrogenase from Pseudomonas propionici (cooS2) (SEQ ID NO: 63) or an amino acid sequence having at least 60% homology thereto; xviii) the amino acid sequence of anaerobic methylbacterial carbon monoxide dehydrogenase (cooS2) (SEQ ID NO: 64) or an amino acid sequence having at least 60% homology thereto; xix) the amino acid sequence of Mycosporium sylvatum carbon monoxide dehydrogenase (cooS2) (SEQ ID NO: 65) or an amino acid sequence having at least 60% homology thereto; xx) the amino acid sequence of Helicobacter mobilis carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 66) or an amino acid sequence having at least 60% homology thereto; xxi) the amino acid sequence of Desulfurobacterium palustris carbon monoxide dehydrogenase (DCCM_2691) (SEQ ID NO: 67) or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

12. A method according to any one of the preceding claims, wherein: The supported biocatalyst comprises formate dehydrogenase or a functional fragment or derivative thereof.

13. The method according to claim 12, wherein: The formate dehydrogenase is selected from or includes: i) the amino acid sequence of the major subunit (fdnG) of the nitrate-inducible formate dehydrogenase from Escherichia coli (SEQ ID NO: 68) or an amino acid sequence having at least 60% homology thereto; ii) the amino acid sequence of Shigella flexneri formate dehydrogenase-N nitrate-inducible alpha subunit (fdnG) (SEQ ID NO: 69) or an amino acid sequence having at least 60% homology thereto; iii) the amino acid sequence of formate dehydrogenase-Nα subunit (fdnG) from the family Enterobacteriaceae (SEQ ID NO: 70) or an amino acid sequence having at least 60% homology thereto; iv) the amino acid sequence of Salmonella typhimurium molybdenum purine oxidoreductase (fdnG) (SEQ ID NO: 71) or an amino acid sequence having at least 60% homology thereto; v) the amino acid sequence of the major subunit (fdnG) of the nitrate-inducible formate dehydrogenase from Citrobacter rodentium (SEQ ID NO: 72), or an amino acid sequence having at least 60% homology thereto; vi) the amino acid sequence of Escherichia albicans formate dehydrogenase-Nα subunit (fdnG) (SEQ ID NO: 73) or an amino acid sequence having at least 60% homology thereto; vii) the amino acid sequence of formate dehydrogenase-Nα subunit (fdnG) from the family Enterobacteriaceae (SEQ ID NO: 74) or an amino acid sequence having at least 60% homology thereto; viii) the amino acid sequence of formate dehydrogenase-Nα subunit (fdnG) of Enterobacteriaceae 4M9 (SEQ ID NO: 75) or an amino acid sequence having at least 60% homology thereto; ix) the amino acid sequence of Erwinia formate dehydrogenase-Nα subunit (fdnG) (SEQ ID NO: 76) or an amino acid sequence having at least 60% homology thereto; x) the amino acid sequence of Jejubacter calystegiae formate dehydrogenase-Nα subunit (fdnG) (SEQ ID NO: 77) or an amino acid sequence having at least 60% homology thereto; xi) the amino acid sequence of the Millerella wisconsinensis formate dehydrogenase Nα subunit containing selenocysteine ​​(M992_0960) (SEQ ID NO: 78) or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

14. A method according to any one of the preceding claims, wherein: The oxidoreductase or a functional fragment or derivative thereof is immobilized on the carrier material.

15. A method according to any one of the preceding claims, wherein: The carrier material is conductive or semiconductive.

16. A method according to any one of the preceding claims, wherein: The support material includes: carbon, metal or metal alloy, metal oxide or mixed metal oxide, metal hydroxide, metal chalcogenide, semiconductor material or conductive polymer, or a mixture thereof.

17. A method according to any one of the preceding claims, wherein: The support material comprises a carbon material or consists of a carbon material.

18. The method according to claim 17, wherein: The carbon material includes graphite, carbon nanotubes, carbon black, activated carbon, carbon nanopowder, glassy carbon, carbon fiber, carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite, doped or surface-modified carbon or doped diamond.

19. The method according to claim 17 or 18, wherein: The carbon material comprises: - doped graphene, wherein the graphene is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals; - doped carbon nanotubes, wherein the carbon nanotubes are doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals; - doped diamond, wherein the diamond is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen and silicon; - doped carbon black, wherein the carbon black is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals; and / or - doped activated carbon, wherein the activated carbon is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanides and transition metals; And / or wherein the carbon material comprises carboxylic acid surface groups or is modified to comprise carboxylic acid surface groups.

20. A method according to any one of the preceding claims, wherein: The nitrogen-containing functional group is a nitro group, an azido group, a hydroxylamine group, a nitroso group, a nitrile group, a diazo group, a diazonium group, an isocyano group, an isothiocyanate group, an isocyanate group, a hydrazone group, a hydrazine group, an amidine group, an azo group or a guanidine group.

21. A method according to any one of the preceding claims, wherein: The target compound is a nitroaromatic compound; Preferably, the nitroaromatic compound comprises a hydrocarbyl aryl or heteroaryl group substituted by a nitro group, wherein the hydrocarbyl aryl or heteroaryl group is optionally further substituted.

22. A method according to any one of the preceding claims, wherein: The biocatalyst does not comprise an oxidoreductase or a functional fragment or derivative thereof comprising an active site capable of catalyzing the enzymatic reduction of a nitrogen-containing functional group; Preferably, the biocatalyst does not contain nitroreductase.

23. A method according to any one of the preceding claims, wherein: The method does not involve the transfer of electrons to the target compound via one or more cofactors.

24. A system comprising: i) a biocatalyst comprising an oxidoreductase or a functional fragment or derivative thereof supported on a carrier material; ii) molecular reducing agents; and iii) a target compound comprising a reducible nitrogen-containing functional group; wherein the system is configured such that, in use, (a) the molecular reductant is oxidized by an oxidoreductase or a functional fragment or derivative thereof; and (b) the nitrogen-containing functional group is reduced at the support material; Preferably, the oxidoreductase or functional fragment or derivative transfers electrons to the support material, and the reduction of the nitrogen-containing functional group comprises direct electron transfer from the support material to the target compound.

25. The system of claim 24, wherein: - the oxidoreductase or a functional fragment or derivative thereof is as defined in any one of claims 5, 6 or 8-14; and / or - the carrier material is as defined in any one of claims 15 to 19; and / or - the molecular reducing agent is as defined in claim 7; and / or - the target compound and / or the nitrogen-containing functional group are as defined in any one of claims 20-21; and / or - wherein the biocatalyst is as defined in claim 22.

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