Reduction of nitrogen-containing groups in target compounds using biocatalysts

Abstract

Provided herein is a method of creating a functional group in a target compound using a biocatalyst described in more detail herein. Also provided are related systems and compositions.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a method for reducing a functional group in a target compound using a biocatalyst. In particular, the present invention relates to a method for reducing a nitrogen-containing functional group in a target compound using a biocatalyst supported on a support material. The present invention further relates to a system and apparatus for performing such a method.

Background Art

[0002] Background Chemical manufacturing processes are typically associated with many environmental concerns. Reagents such as catalysts used are often non-renewable and / or toxic. Extreme operating conditions such as high temperature and pressure are typically required, and providing such conditions is energy inefficient. Toxic solvents are often required to achieve satisfactory yields. Furthermore, the reagents used are often non-selective and require complex synthetic strategies to selectively process only the desired functional groups within the molecule.

[0003] The non-selectivity of conventional processes is particularly problematic in the field of fine chemical manufacturing, including in the synthesis of pharmaceuticals where minimizing the production of impurities is particularly important. Such impurities typically need to be removed through purification strategies, but such purification methods are costly and typically inefficient. Thus, the formation of unwanted by-products in chemical reactions increases economic costs and also typically leads to environmental damage since the chemicals used in the purification process are also harmful.

[0004] One major class of chemical reactions that is particularly important in synthesis is the reduction of nitrogen-containing functional groups such as nitro (-NO 2 ) groups, azide (-N 3 ) groups, nitrile (-CN) groups, etc. The desired chemical result of such reactions is typically an amine (-NH 3 ) or quaternary ammonium (-NH 4 +)The formation of a base, while known methods of performing such reactions are typically inefficient and lead to significant by-products, and / or use expensive non-renewable reagents and environmentally harmful reaction conditions.

[0005] For example, some strategies for the industrial reduction of nitro groups to amine groups rely on the use of noble metal catalysts, such as palladium (Pd). However, such catalysts are extremely expensive, non-renewable, associated with significant environmental impacts during their manufacture, and typically have reduced activity in the presence of common contaminants in the feedstock. For example, carbon monoxide (CO) is a common contaminant in hydrogen sources obtained from natural gas, which is often used as a reducing agent in such reactions, meaning that high purity hydrogen (which has its own economic and environmental implications) is typically required in such reactions. Furthermore, the reaction conditions required when using such catalysts are typically harsh, using high temperatures and / or dangerous organic solvents. Additionally, such reactions often produce significant by-products resulting from the catalysis of unwanted side reactions, such as non-specific reduction, incomplete reduction, and bond rearrangement.

[0006] In attempts to avoid the problems associated with the use of noble metal catalysts for nitrogen group reduction, some recent efforts have focused on the use of biological catalysts, such as enzymes. The enzymatic treatment of chemical reagents offers advantages compared to conventional chemical treatment methods. Enzymes are renewable and biodegradable, and thus overcome environmental concerns regarding the manufacture and disposal of chemical catalysts. Enzymes are typically harmless and non-toxic, i.e., addressing safety concerns associated with chemical catalysts. Enzymes typically operate at moderate temperatures and atmospheric pressure, i.e., reducing the energy requirements associated with conventional chemical treatment. Enzymes are also typically highly selective with respect to their chemical substrates, and approaches such as rational enzyme engineering and directed mutagenesis continue to expand the range of reactions that can be undertaken. Enzymatic catalysis thus offers many advantages compared to conventional chemical approaches.

[0007] These advantages have led to research into the industrial use of enzymes that can catalyze the reduction of nitro groups, such as nitroreductase enzymes. Such enzymes have an active site that can catalyze the reduction of nitro groups. However, the use of such enzymes addresses at least partially the problems that occur with the noble metal catalysts described above, but significant problems remain.

[0008] For example, enzyme catalysts such as nitroreductase are typically used as homogeneous catalysts in solutions having reactant (and typically also product) molecules. However, this causes 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 that use harsh chemical conditions, and can even result in the product undergoing further unwanted reactions within the purification workflow.

[0009] Furthermore, nitroreductase enzymes that are being studied for use in the reduction of nitrogen-containing functional groups typically require cofactors that assist for their catalytic cycles to operate. As will be explained below, the need to recycle the cofactors required in their catalytic cycles is a particular difficulty in the use of such enzymes.

[0010] Cofactors play an important role in many enzyme-catalyzed biochemical reactions and are typically non-protein chemical compounds that act to transfer chemical groups between enzymes. Cofactors are also sometimes known as "cosubstrates" that reflect their handling in the process of that catalysis of their main reaction by the enzyme. By way of example, an oxidoreductase enzyme that catalyzes an oxidation reaction of a reagent to produce a product can couple that oxidation to the reduction of a cofactor as an electron sink. In this case, the overall reaction catalyzed by the enzyme is: Reduced reagent + Oxidized cofactor → Oxidized product + Reduced cofactor which can be represented as.

[0011] Similarly, enzymes that catalyze the reduction of a reagent to produce a product typically couple that reduction to the oxidation of a cofactor as an electron source or a reducing equivalent such as a hydride ion: Oxidized reagent + Reduced cofactor → Reduced product + Oxidized cofactor

[0012] The biological use of cofactors is not limited to the simple redox reactions represented above, but also involves more complex reactions such as atom insertion reactions and rearrangement reactions.

[0013] Important 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.

[0014] Enzymes that rely on cofactor regeneration are well-known, but on the other hand, their use in industry is limited by the difficulty in providing sufficient cofactors for the enzymes to use. One option is to provide a supra-stoichiometric amount of cofactor for the reagent in question. However, the high cost and typically low stability of reduced cofactor molecules mean that this is not a viable approach. Other approaches being considered include the use of systems that regenerate cofactor molecules in their desired form (i.e., recycle cofactor molecules). However, conventional cofactor regeneration systems have problems. For example, current industrial practice for enzymatic NAD(P)H recycling relies on supra-stoichiometric amounts of carbon-based (organic) sacrificial reducing agents such as glucose or isopropanol. However, this leads to additional costs, generates waste, and requires additional downstream processing steps for the desired product, and has poor atom efficiency. The industrial use of flavin-utilizing enzymes is almost completely hindered by the lack of suitable means to recycle flavin cofactors. Electrochemical reduction of oxidized flavin cofactors has been proposed, but such systems are difficult to incorporate into industrial-related situations, involve the use of expensive materials such as noble metals and highly processed carbon materials as the required electrode materials, and tend to generate unwanted side reaction products.

[0015] Accordingly, there is an urgent need for improved methods for catalyzing the reduction of reducible functional groups, particularly nitrogen-containing functional groups. In particular, methods that are atom-efficient; avoid the difficulties associated with electrochemical treatment of reagents; do not rely on the use of expensive sacrificial organic reducing agents; are selective; lead to complete reduction rather than only partial reduction; avoid the formation of by-products; and / or do not rely on complex cofactor regeneration methods; and avoid the requirements for highly reactive chemical reagents that are expensive or dangerous. The present invention aims to address some or all of these problems. SUMMARY OF THE INVENTION

[0016] In an attempt to address the above problems, the inventors have surprisingly found that a target compound containing a reducible nitrogen-containing functional group can be cleanly and efficiently reduced by using a biocatalyst containing a redox enzyme such as a hydrogenase enzyme supported on a support material such as carbon, in the presence of a molecular reducing agent such as hydrogen. Surprisingly, the inventors have found that by contacting the target compound with such a catalyst, the molecular reducing agent can be oxidized by the redox enzyme and the nitrogen-containing functional group can be reduced by the support material.

[0017] As will be described in more detail herein, the reduction is clean and atom efficient. The reaction occurs under mild conditions and avoids or minimizes the use of toxic reagents such as organic solvents associated with the use of noble metal catalysts as described above. Side reactions such as rearrangement can be avoided and there is no need for expensive sacrificial organic reducing agents. The enzymes used typically do not rely on cofactors that need to be regenerated (such enzymes are not necessarily excluded). The catalyst is heterogeneous, which leads to easier purification of the product from the reaction mixture. In particular, the reaction typically leads to a much more complete reduction (up to 100% complete reduction of the nitrogen-containing functional group) compared to the use of similar enzymes in solution.

[0018] Accordingly, provided herein is a method for reducing a nitrogen-containing functional group in a target compound, the method comprising contacting the target compound with a biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a support material, in the presence of a molecular reducing agent for oxidation by the redox enzyme or a functional fragment or derivative thereof, - the molecular reducing agent being oxidized by the redox enzyme or a functional fragment or derivative thereof; and - the nitrogen-containing functional group being reduced by the support material: under such conditions.

[0019] Preferably, the method comprises contacting the target compound with the support material.

[0020] Preferably, the reduction of the nitrogen-containing functional group involves direct electron transfer from the support material to the target compound. Preferably, the method involves reducing the nitrogen-containing functional group to form an amine group or a quaternary ammonium group.

[0021] Preferably, the redox enzyme or a functional fragment or derivative thereof is in electronic contact with the support material. Preferably, the redox enzyme or functional fragment or derivative transfers electrons to the support material via an intramolecular electron conduction pathway. Preferably, the intramolecular electron conduction pathway includes a series of [FeS] clusters.

[0022] Preferably, the molecular reducing agent is selected from hydrogen, carbon monoxide, formate, their isotopes, and mixtures thereof. Preferably, the molecular reducing agent comprises or consists of hydrogen or its isotope.

[0023] Preferably, in one embodiment, the supported biocatalyst comprises a hydrogenase enzyme or a functional fragment or derivative thereof. Preferably, the hydrogenase is selected from or comprises any one or more of the amino acid sequences of SEQ ID NOs: 1 to 46, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0024] Preferably, in another embodiment, the supported biocatalyst comprises a carbon monoxide dehydrogenase enzyme 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 to 67, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0025] Preferably, in yet another embodiment, the supported biocatalyst comprises formate dehydrogenase enzyme 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 to 78, or an amino acid sequence having at least 60% homology thereto; or a functional fragment, derivative or variant thereof.

[0026] Preferably, the oxidoreductase or a functional fragment or derivative thereof is immobilized on a support material. Preferably, the support material is electronically conductive or semiconductive.

[0027] 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.

[0028] Preferably, the support material comprises or consists of a carbon material.

[0029] Preferably, the carbon material comprises graphite, carbon nanotube(s), carbon black, activated carbon, carbon nano powder, vitreous carbon, carbon fiber(s), 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 is: - doped graphene, wherein the graphene is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements and transition metals; - doped carbon nanotube(s), wherein the carbon nanotube(s) is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements and transition metals; - Doped diamond, wherein the diamond is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen and silicon, doped diamond; - Doped carbon black, wherein the carbon black is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements and transition metals, doped carbon black; and / or - Doped activated carbon, wherein the activated carbon is doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements and transition metals, doped activated carbon comprising; and / or the carbon material comprises or is modified to comprise carboxylic acid surface groups.

[0030] Preferably, the nitrogen-containing functional group is a nitro, azide, hydroxylamine, nitroso, nitrile, diazo, diazonium, isocyanide, isothiocyanate, isocyanate, hydrazone, hydrazine, amidine, azo, or guanidine group.

[0031] Preferably, the target compound is a nitroaromatic compound. Preferably, the nitroaromatic compound comprises a hydrocarbyl aromatic group or a heteroaromatic group substituted with a nitro group, wherein the hydrocarbyl aromatic group or heteroaromatic group is optionally further substituted.

[0032] Preferably, the biocatalyst does not comprise a redox enzyme or a functional fragment or derivative thereof that can catalyze the enzymatic reduction of a nitrogen-containing functional group. Preferably, the biocatalyst does not comprise a nitroreductase enzyme. Preferably, the method does not involve the transfer of electrons to the target compound via one or more cofactors.

[0033] Also provided herein is a system, i) A biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a support material; ii) A molecular reducing agent; and iii) A target compound containing a reducible nitrogen-containing functional group comprising; wherein the system is configured such that, in use, (a) the molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and (b) the nitrogen-containing functional group is reduced by the support material, a system.

[0034] Preferably, in the said system, the redox enzyme or a 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.

[0035] Preferably, in the said system, the redox enzyme or a functional fragment or derivative thereof is as defined herein; and / or the support material is as defined herein; and / or the molecular reducing agent is as defined herein; and / or the target compound and / or the nitrogen-containing functional group is as defined herein; and / or the biocatalyst is as defined herein.

Brief Description of the Drawings

[0036]

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Mode for Carrying Out the Invention

[0037] Detailed Description of the Invention Method of the Invention As discussed above, provided herein is a method for reducing a functional group in a target compound, the method comprising contacting the target compound with a biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a support material, in the presence of a molecular reducing agent for oxidation by the redox enzyme or a functional fragment or derivative thereof. - The molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and - The functional group is reduced by the support material: A method comprising contacting under such conditions.

[0038] 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.

[0039] Accordingly, provided herein is a method for reducing a nitrogen-containing functional group in a target compound, the method comprising contacting the target compound with a biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a support material, in the presence of a molecular reducing agent for oxidation by the redox enzyme or a functional fragment or derivative thereof, - The molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and - The nitrogen-containing functional group is reduced by the support material: A method comprising contacting under such conditions.

[0040] The method provided herein comprises reducing a functional group of a target compound with a support material. That is, the method typically comprises contacting the target compound with the support material. Suitable support materials are described in more detail herein.

[0041] Typically, the methods provided herein involve the direct transfer of electrons and / or hydride ions from a support material to a target compound. More typically, the methods provided herein involve the direct transfer of electrons from a support material to a target compound. Typically, the method involves the direct transfer of electrons from a support material to a reducible functional group of a target compound. In some embodiments, the direct transfer of electrons involves electron tunnelling from a support material to an atom of a functional group of a target compound. In some embodiments, the reducible functional group of the target compound is in contact with the surface of the support material. The direct electron transfer from a support material to a functional group of a target compound occurs, namely, at the solvent-accessible surface of the support material, i.e., at the interface between the support material and a solvent (e.g., an aqueous reaction solvent). The direct electron transfer from a support material to a functional group of a target compound typically does not involve contacting the target compound with a reductase (e.g., nitroreductase) immobilized on the support material. In other words, the disclosed methods typically do not involve contacting a target compound or a reducible functional group thereof with an enzyme immobilized on a support material. As such, the support material can be considered to provide an "active site" (using a term analogous to enzymology) for the reduction of the reducible functional group.

[0042] The electron transfer from a support material to a functional group typically is not mediated by an enzyme or its cofactor. The cofactor is as described above. In other words, the disclosed methods typically do not involve the transfer of electrons to a target compound via one or more cofactors.

[0043] In particular, the electron transfer from a support material to a functional group typically is not catalyzed by an enzyme for reducing the functional group of a target compound. That is, for example, a supported biocatalyst typically does not include a reductase capable of catalyzing (e.g., fully reducing) the reduction of a functional group. In other words, a supported biocatalyst typically does not include a redox enzyme 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 nitroreductase.

[0044] That is, it will be apparent that the direct electron transfer from the support material to the functional group as used herein does not include the enzymatic reduction of the functional group using, for example, nitroreductase, cytochrome P450 monooxygenase, nitrile reductase, azoreductase, etc. Thus, typically, the biocatalyst does not contain nitroreductase. Similarly, the biocatalyst typically does not contain azoreductase or nitrile reductase or other enzymes containing an active site (e.g., substrate-binding active site) capable of reducing a reducible functional group. In some embodiments, the supported biocatalyst consists of the redox enzyme described herein supported on a support.

[0045] That is, it is important to distinguish between a redox enzyme that catalyzes the oxidation of a molecular reducing agent and a reductase that typically has an active site (e.g., substrate-binding active site) that catalyzes the reduction of a reducible functional group of a target compound. The redox enzyme that catalyzes the oxidation of a molecular reducing agent as used herein is not an example of a reductase for reducing a reducible functional group of a target compound. For example, a redox enzyme that catalyzes the oxidation of a molecular reducing agent typically does not contain an active site (e.g., substrate-binding active site) that can (e.g., inherently can) accommodate the target compound or its reducible functional group. A redox enzyme that catalyzes the oxidation of a molecular reducing agent can operate only at a potential or under reaction conditions that are incompatible with the enzymatic reduction of a reducible functional group of a target compound by a reductase such as nitroreductase, azoreductase, or nitrile reductase. For example, reductases for reducing reactive functional groups typically require cofactors (e.g., NADH, NAD(P)H, or flavins such as FAD, FMN, or riboflavin) described herein to mediate electron transfer to their oxidized substrates. Typically, in the methods herein, no such cofactors are present. That is, typically, the disclosed methods do not involve the use of cofactors in the reaction medium, e.g., in the reaction solution.

[0046] As noted 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. As will be apparent from the discussion herein, such groups include nitro groups (R-NO 2 ), azide groups (R-N 3 ), hydroxylamine groups (R-NR’OH), nitroso groups (R-NO), nitrile groups (R-CN), diazo groups (R-CR’=N 2 ), diazonium groups (R-N 2 + ), isocyanide groups (R-NC); isothiocyanate groups (R-NCS), isocyanate groups (R-N=C=O), hydrazone groups (R-CR’=N-NR’), hydrazine groups (R-NR’-NR’ 2 ), amidine groups (R-C(NR’)NR’ 2 ), azo groups (R-N=N=R’), and guanidine groups (R-NR’-C(NR’)-NR’ 2 ), etc. (wherein R represents the remainder of the target compound, and each R’ is independently H or a hydrocarbyl, such as an alkyl, e.g., a C 1-6 or C 1-4 alkyl, etc.; typically represents 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). Even 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).

[0047] Typically, the methods disclosed herein involve substantially complete or complete reduction of the reducible functional group. As used herein, the term “complete reduction” means that the reducible functional group (e.g., the nitrogen atom in the reducible functional group) is reduced to its lowest stable oxidation state. For example, complete reduction of a nitro group is the amine (R-NH 2 ) or quaternary ammonium (R-NH 2 ) of the nitro group (R-NO3 + ) corresponding to a six-electron reduction to the base, while partial reduction of the nitro group can result in, for example, the formation of hydroxylamine (R-NHOH). That is, for example, when the reducible functional group is a nitro group (R-NO 2 )(e.g., an aromatic nitro group), the method typically involves reducing the nitro group to an amine or quaternary ammonium (R-NH 3 + ) group, where R is the remainder of the target compound. When the reducible functional group is an azide group (R-N 3 )(e.g., an aromatic nitrile group), the method typically involves reducing the azide group to an amine or quaternary ammonium (R-NH 3 + ) group, where 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 typically involves reducing the nitrile group to an amine or quaternary ammonium (e.g., R-CR’ 2 NH 3 + ) group, where R is the remainder of the target compound.

[0048] The method can include contacting a sample containing a plurality of target compounds with a biocatalyst in the presence of a molecular reducing agent, as defined in more detail herein. Typically, at least 60% of the target compounds in the sample, such as at least 50%, 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, can be completely reduced to nitrogen-containing functional groups.

[0049] The methods disclosed herein are typically highly selective. Typically, 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 can include contacting a target compound comprising a nitrogen-containing reducible functional group and a C=C double bond with a biocatalyst described herein. In such embodiments, typically, 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. Since such selective reduction is typically not possible with noble metal catalysts such as palladium, this can represent a particular advantage of the disclosed methods.

[0050] Reducing agent As will be apparent from the above discussion, the disclosed methods involve the oxidation of a molecular reducing agent by an oxidoreductase or a functional fragment or derivative thereof. Suitable enzymes are described in more detail herein.

[0051] Typically, the molecular reducing agent is selected from hydrogen (H 2 ), carbon monoxide (CO), formate (CH(O)O - deprotonated as or the protonated HC(O)OH also known as formic acid), their isotopes, and mixtures thereof. Particular advantages can result from the use of such reducing agents since the products of their oxidation can be readily removed from the reaction mixture and / or do not require removal. That is, the protons formed by the oxidation of molecular hydrogen typically do not need to be removed from the reaction mixture. Carbon dioxide resulting from the oxidation of CO or formate can be readily removed, for example, as a gas. That is, reactions using such reducing agents can be clean and can avoid the need for large-scale purification. Typically, the molecular reducing agent is selected from hydrogen and carbon monoxide, their isotopes, and mixtures thereof. More typically, the molecular reducing agent is hydrogen (i.e., molecular hydrogen) or an isotope thereof.

[0052] In some embodiments, the reducing agent used in the disclosed method is determined by the redox enzyme contained in the biocatalyst. In other embodiments, the redox enzyme 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 a compatible redox enzyme / reducing agent pair. Exemplary combinations of redox enzymes and reducing agents are shown in the following table.

[0053] JPEG2025517686000001.jpg56158

[0054] A mixture of a redox enzyme and a corresponding molecular reducing agent can be used. For example, the biocatalyst can include 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 reducing agent can include a mixture of molecular hydrogen (or its isotopes) and carbon monoxide (or its isotopes). The biocatalyst can include 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 reducing agent can include a mixture of molecular hydrogen (or its isotopes) and formate (or its isotopes). The biocatalyst can include 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 reducing agent can include a mixture of carbon monoxide (or its isotopes) and formate (or its isotopes).

[0055] When the molecular reducing agent is hydrogen, its suitable isotopes include 1 H 2 、 2 H 2 and 3 H 2 including. Mixed isotopes (e.g. 1 H 2 H and 1 H3 H) is also included. Preferably, hydrogen is 1 H 2 It will be apparent that organic molecules such as glucose, formate, and ethanol, isopropanol, etc., used herein are not sources of molecular hydrogen. However, in some embodiments, such compounds can be used as molecular reducing agents described in more detail herein.

[0056] Typically, when the reducing agent is molecular hydrogen, the molecular hydrogen is provided in gaseous form. The gas can be mixed with the aqueous solution in which the biocatalyst and the target compound and optionally other reaction components are present. At 1 bar of H 2 in water, the solubility of H in water 2 is 0.8 mM. In other words, when the reducing agent 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 redox enzyme operates) is 0.8 mM of hydrogen. Other pressures can also be used. For example, the gas pressure in the reaction vessel can be from 0.1 to 10 bar, such as about 1 bar, for example from about 0.2 to about 5 bar, for example from 0.5 to 2 bar, etc., from 0.01 to about 100 bar. Increasing the gas pressure will increase the concentration of hydrogen in the reaction solution. Decreasing the gas pressure will decrease the concentration of hydrogen in the reaction solution.

[0057] To avoid doubt, molecular hydrogen can also be provided in the form of a solution in which molecular hydrogen is dissolved (e.g., an aqueous solution containing buffer salts, for example, as described in more detail herein).

[0058] When the reducing agent is molecular hydrogen, hydrogen is hydrogen and CO, CO 2 air, O 2 N 2 Ar, etc. It can be provided as a mixture with other gases. When provided as a mixture, the mixture is from about 5% to about 50% H 2 etc., from 1% to about 95%, for example from about 2% to about 80% H 2can contain from about 0.1% to about 99% hydrogen, such as. Exemplary mixtures can include, for example, 50% hydrogen and 50% N 2 can be included.

[0059] When the reducing agent is molecular hydrogen, the hydrogen can be of any suitable purity. For example, when it is important to control the impurity level in the final product mixture, hydrogen with a purity of 99% or higher (e.g., 99.9%, 99.99% or 99.999%) can be used. In other embodiments, when it is not so important to control the impurity level 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 "syngas". Syngas produced by gasification of coal is generally a mixture of 30 to 60% carbon monoxide, 25 to 30% hydrogen, 5 to 15% carbon dioxide, and 0 to 5% methane, and can optionally also contain smaller amounts of other gases. Hydrogen waste off-gases from other industrial processes can also typically be used. Thus, the methods of the present disclosure are ideally suited for use as part of a larger reaction sequence that includes a reaction step involving hydrogen either as a reactant or as a product, and the waste hydrogen from such a reaction step is used as the molecular reducing agent in the disclosed methods.

[0060] When the reducing agent is molecular hydrogen or an isotope thereof, the molecular hydrogen or isotope thereof can be provided from any suitable source, such as a gas cylinder. Alternatively, the molecular hydrogen or isotope thereof can be produced on-site, for example, by electrolysis of water.

[0061] When the molecular reducing agent is carbon monoxide, suitable isotopes thereof are 12 C 16 O, 13 C 16 O, 14 C 16 O, 12 C 17 O, 13 C 17 O, 14 C 17 O, 12 C 18O, 13 C 18 O and 14 C 18 O. Preferably, carbon monoxide is 12 C 16 O.

[0062] Typically, when the reducing agent is carbon monoxide, the carbon monoxide is provided in gaseous form. The gas can be mixed with an aqueous solution in which the biocatalyst and the target compound and any other reaction components are present. At 1 bar of CO, the solubility of CO in water is 0.95 mM. In other words, when the reducing agent 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 redox enzyme operates) is 0.95 mM of carbon monoxide. Other pressures can also be used. For example, the gas pressure in the reaction vessel can be from 0.1 to 10 bar, such as about 1 bar, for example from about 0.2 to about 5 bar, for example from 0.5 to 2 bar, such as from 0.01 to about 100 bar. Increasing the gas pressure will increase the concentration of carbon monoxide in the reaction solution. Decreasing the gas pressure will decrease the concentration of carbon monoxide in the reaction solution.

[0063] To avoid doubt, carbon monoxide can also be provided in the form of a solution in which carbon monoxide is dissolved (e.g., an aqueous solution containing buffer salts as described in more detail herein).

[0064] When the reducing agent is carbon monoxide, the carbon monoxide can be provided as a mixture with other gases such as carbon monoxide and H 2 , CO 2 , air, O 2 , N 2 , Ar. When provided as a mixture, the mixture can contain from about 1% to about 95%, such as from about 5% to about 50% of CO, for example from about 2% to about 80% of H 2 , etc., from about 0.1% to about 99% of CO. An exemplary mixture can contain, for example, 50% CO and 50% N 2 .

[0065] When the reducing agent is CO, the CO can be of any suitable purity. For example, when it is important to control the impurity level in the final product mixture, CO with a purity of 99% or higher (e.g., 99.9%, 99.99% or 99.999%) can be used. In other embodiments, when it is not so important to control the impurity level in the final product mixture, CO with a lower purity can be used. For example, relatively low purity CO can be provided in the form of the "synthesis gas" described above. CO waste off-gas from other industrial processes can also typically be used. Thus, the disclosed method is ideally suited to be utilized as part of a larger reaction sequence that includes a reaction step involving CO either as a reactant or as a product, and the waste CO from such a reaction step is used as the molecular reducing agent in the disclosed method. When the reducing agent is CO or an isotope thereof, the CO or its isotope can be provided from any suitable source, such as a gas cylinder.

[0066] When the molecular reducing agent is formate, suitable isotopes thereof are 12 C-formate, 13 C-formate, and 14 carbon isotopes such as C-formate; 16 O-formate, 17 O-formate, and 18 oxygen isotopes such as O-formate; and 1 H-formate, 2 H-formate and 3 hydrogen isotopes such as H-formate. Preferably, the formate consists of 12 C, 16 O and 1 H.

[0067] Typically, when the reducing agent is formate, the formate is provided as a metal formate or formic acid. Examples of formates include lithium formate, sodium formate, potassium formate, magnesium formate, calcium formate, ammonium formate, etc. Formic acid can be provided as an aqueous solution containing from about 1% to about 99% formic acid, or 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% pure).

[0068] When the reducing agent is formate (or formic acid), the formate (or formic acid) can be present in the reaction solution at a concentration of from about 1 μM to about 1 M, such as from about 10 μM to about 100 mM, such as from about 100 μM to about 10 mM, such as about 1 mM. Other components such as buffer salts can also be present.

[0069] Oxidoreductase 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 electronically conductive or semiconductive. Suitable support materials are described in more detail herein.

[0070] The oxidoreductase is typically in electronic 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 such that electrons can move from the active site of the oxidoreductase (i.e., the active site where the oxidation of the molecular reductant is catalyzed) to the support material.

[0071] Typically, electrons can move from a redox enzyme (e.g., from the active site of a redox enzyme) 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 via a redox enzyme is typically direct electron transfer. The present invention encompasses the use of an electron mediator (e.g., a redox-active dye such as methyl or benzyl viologen) to mediate electron transfer from a molecular reducing agent to a support material, but the electron transfer is typically not mediated by an electron transfer agent such as a mediator, e.g., typically not mediated by a redox-active dye such as methyl or benzyl viologen.

[0072] Typically, the electron transfer from a molecular reducing agent to a support material involves direct electron transfer through a redox enzyme. Typically, the direct electron transfer is via an intramolecular electron conduction pathway. Typically, the intramolecular electron conduction pathway includes a series of [FeS] clusters. As will be recognized by those skilled in the art, [FeS] clusters include [3Fe4S] clusters and [4Fe4S] clusters. The [FeS] clusters can be described as distal clusters, proximal clusters, or intermediate clusters. The notations "distal" and "proximal" in this context are routine in the art. For a protein containing an active site and a chain of or a 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 thus the farthest from the active site. The [FeS] clusters between the proximal cluster and the distal cluster are called intermediate clusters. The distal cluster is often solvent-accessible. That is, in some embodiments, the electron transfer is via a chain 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 tunnelling step.

[0073] In some embodiments, the redox enzyme includes a natural electron transfer partner such as cytochrome (such as the cytochrome of SEQ ID NO: 22 or its functional fragment, derivative or variant). The cytochrome can be included in the redox enzyme at a position for electron transfer to the distal [FeS] cluster. In other words, the cytochrome can be located at the distal end of a series of [FeS] clusters.

[0074] Typically, the redox enzyme is a hydrogenase enzyme, a carbon monoxide dehydrogenase enzyme and / or a formate dehydrogenase enzyme, or a functional fragment or derivative thereof.

[0075] The redox enzyme is preferably selected or modified to catalyze the oxidation of the reducing agent close to the thermodynamic reduction potential E 0 of the molecular reducing agent. For example, when the reducing agent is molecular hydrogen, the redox enzyme is preferably a hydrogenase and preferably catalyzes the oxidation of hydrogen close to the thermodynamic reduction potential E + of the 2H 2 / H o couple (``E o (2H + / H 2 )''). (One of ordinary skill in the art will recognize that at 25 ° C, pH 7.0 and 1 bar of H 2 , E o (2H + / H 2 ) = -0.413 V and changes according to the Nernst equation.) Preferably, the redox enzyme is at an applied potential less than 100 mV positive than E o (2H + / H 2 ); more preferably at an applied potential less than 50 mV positive than E o (2H + / H 2 ) to catalyze the oxidation of H 2 or x H 2 . Selected or modified to catalyze oxidation under the experimental conditions in question E o(2H + / H 2 ) close to H 2 Methods for determining the ability of an enzyme to catalyze oxidation are routine for those skilled in the art and are described, for example, in Vincent et al, J. Am. Chem. Soc. (2005) 127, 18179-18189.

[0076] When the reducing agent is CO, the redox enzyme is preferably carbon monoxide dehydrogenase (CODH), and preferably CO under experimental conditions 2 / CO pair thermodynamic reduction potential E o (“E o (CO 2 / CO)”) is selected or modified to catalyze the oxidation of CO close to it. (Those skilled in the art will recognize that at 25 °C, E o (CO 2 / CO) = -0.53V). Preferably, the redox enzyme is at an applied potential less than 100 mV positive than E o (CO 2 / CO); more preferably, it is selected or modified to catalyze CO oxidation at an applied potential less than 50 mV positive than E o (CO 2 / CO). Similarly, when the reducing agent is formate, the redox enzyme is preferably formate dehydrogenase, and preferably CO under experimental conditions 2 / formate pair thermodynamic reduction potential E o (“E o (CO 2 / formate)”) is selected or modified to catalyze the oxidation of formate close to it. (Those skilled in the art will recognize that at 25 °C, E o (CO 2 / formate) = -0.42V). Preferably, the redox enzyme is at an applied potential less than 100 mV positive than E o (CO 2 / formate); more preferably, it is selected or modified to catalyze formate oxidation at an applied potential less than 50 mV positive than E o (CO 2 / formate).

[0077] In some embodiments, the redox enzyme is selected or modified to oxidize the reducing agent at a potential more negative than the reduction potential of the target compound.

[0078] The redox enzyme is typically selected or modified to be more highly active. For example, the redox enzyme can be selected or modified to have efficient substrate turnover.

[0079] Enzyme turnover can be calculated in many ways. The total turnover number (TTN, also known as TON) is a measure of the number of moles of product per mole of enzyme. As those skilled in the art will recognize, the TTN thus indicates the number of times the enzyme has turned over (i.e., oxidized the molecules of the molecular reductant). Preferably, the TTN of the redox enzyme is at least 1,000,000, preferably at least 10 8 e.g., at least 10 9 such as at least 10 7 such as at least 100, more preferably at least 1000, e.g., at least 10,000 or at least 100,000, etc., at least 10.

[0080] The turnover frequency (TOF) is a measure of the number of moles of product produced per mole of enzyme present per second. Thus, in the methods provided herein, the TOF indicates the number of moles of oxidized reductant produced per mole of redox enzyme per second. Thus, the TOF is identified by the number of catalytic cycles undertaken per second by each enzyme molecule. Preferably, in the method of the present invention, the first polypeptide has a TOF of from 0.1 to 1000 s -1 more preferably from about 10 to about 50 s -1 such as from 1 to 100 s -1 of.

[0081] When the supported biocatalyst contains a hydrogenase enzyme, any suitable hydrogenase can be used. The hydrogenase can contain an active site that includes iron atoms (such as in [FeFe]-hydrogenase) or both nickel and iron atoms (such as in [NiFe]- and [NiFeSe]-hydrogenases). Preferably, the hydrogenase contains an active site that includes both nickel and iron atoms. Suitable proteins are described below.

[0082] When the supported biocatalyst contains a hydrogenase enzyme, the hydrogenase enzyme is typically an uptake hydrogenase or a hydrogen-sensing hydrogenase. Uptake hydrogenases are used in vivo by organisms to generate energy by the oxidation of molecular hydrogen in their environment. In vivo, they couple the oxidation of H 2 to the reduction of anaerobic acceptors such as nitrate and sulfate, or O 2 . Typically, uptake hydrogenases contain a signal peptide (often about 30 to about 60 amino acid residues in length) at the N-terminus of the small subunit. Typically, the signal peptide contains the [DENST]RRxFxK motif. Hydrogen-sensing hydrogenases (also known as regulatory hydrogenases) are used in vivo by organisms to sense hydrogen levels in order to regulate the biosynthesis of uptake hydrogenase in response to H 2 . Regulatory hydrogenases typically do not contain a signal peptide characteristic of uptake hydrogenases. Regulatory hydrogenases are often insensitive to O 2 .

[0083] Typically, the hydrogenases for use in the disclosed methods are selected or modified to be oxygen-tolerant. Oxygen-tolerant hydrogenases catalyze their H 2While retaining at least 1%, preferably at least 5%, more preferably at least 50%, etc., of at least 10%, preferably at least 20%, more preferably at least 50%, etc., of the acidification activity, such as at least 80%, for example at least 90%, preferably at least 95%, for example at least 99%, etc., in the presence of at least 0.01%, preferably at least 0.1%, more preferably at least 1%, etc., of O 2 such as at least 5% of O 2 , for example at least 10% of O 2 such as, etc., at least 0.01% of O 2 , preferably at least 0.1% of O 2 , more preferably at least 1% of O 2 in the presence of oxygen, etc., such as in the presence of oxygen, H 2 or x H 2 can be oxidized. Various oxygen-tolerant hydrogenases are known to those skilled in the art.

[0084] Typically, hydrogenases for use in the disclosed methods do not contain a native flavin active site (also known as a prosthetic group) for NAD(P) + reduction, but such hydrogenases are not excluded. Some known hydrogenases, such as soluble hydrogenase (SH) enzymes from R. eutropha, Rhodococcus opacus, Hydrogenophilus thermoluteolus, and Pyrococcus furiosus, actually contain such active sites. However, without being bound by theory, hydrogenases lacking such prosthetic groups are typically thought to have increased stability compared to hydrogenases containing such prosthetic groups. Examples of hydrogenases lacking the 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).

[0085] When the supported biocatalyst contains a hydrogenase, the hydrogenase is often a class 1 or 2b hydrogenase. References to hydrogenase classes such as class 1 and class 2b refer to the established Vignais classification scheme described in Vignais and Billoud, Chem. Rev. 2007, 107, 4206-4272, which is known to those skilled in the art. The hydrogenase can be any of the class 1 or class 2b hydrogenases listed in Vignais and Billoud, Chem. Rev. 2007, 107, 4206-4272, the content of which is incorporated by reference.

[0086] Preferably, when the supported biocatalyst contains a hydrogenase, the hydrogenase is: 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) the amino acid sequence of the Ralstonia eutropha membrane-bound hydrogenase moiety (SEQ ID NO: 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto; iv) the amino acid sequence of the Ralstonia eutropha regulatory hydrogenase moiety (SEQ ID NO: 8 and / or 9) or an amino acid sequence having at least 60% homology thereto; v) the amino acid sequence of Aquifex aeolicus 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 Hydrogenovibrio marinus hydrogenase (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of Thiocapsa roseopersicina hydrogenase (SEQ ID NO: 14 and 15) or an amino acid sequence having at least 60% homology thereto; viii) The amino acid sequence of Alteromonas macleodii 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 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; 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; 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 having at least 60% homology thereto; xiii) The amino acid sequence of Clostridium pasteurianum [FeFe]-hydrogenase (hydA) (SEQ ID NO: 29) or an amino acid sequence having at least 60% homology thereto; xiv) The amino acid sequence of [FeFe]-hydrogenase (hyd1) from Chlamydomonas reinhardtii (SEQ ID NO: 30) or an amino acid sequence having at least 60% homology thereto; xv) The amino acid sequence of [FeFe]-hydrogenase from Chlorella variabilis (SEQ ID NO: 31 and / or 32) or an amino acid sequence having at least 60% homology thereto; xvi) The amino acid sequence of the soluble hydrogenase portion of Ralstonia eutropha (SEQ ID NO: 33 and / or 34) or an amino acid sequence having at least 60% homology thereto; xvii) The amino acid sequence of the soluble hydrogenase portion of 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 the membrane-bound hydrogenase of Desulfovibrio fructosovorans (SEQ ID NO: 37 and / or 38) or an amino acid sequence having at least 60% homology thereto; xix) The amino acid sequence of the iron-iron hydrogenase of Clostridium acetobutylicum (SEQ ID NO: 39) or an amino acid sequence having at least 60% homology thereto; xx) The amino acid sequence of the nickel-iron-selenium hydrogenase of Desulfomicrobium baculatum (SEQ ID NO: 40 and / or 41) or an amino acid sequence having at least 60% homology thereto; xxi) The amino acid sequence of the soluble hydrogenase portion of Hydrogenophilus thermoluteolus (SEQ ID NO: 42 and / or 43) or an amino acid sequence having at least 60% homology thereto; xxii) The amino acid sequence of the Periplasmic [NiFe] hydrogenase of Desulfovibrio gigas (SEQ ID NO: 44 and / or 45) or an amino acid sequence having at least 60% homology 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 selected from or comprising a functional fragment, derivative or variant thereof.

[0087] More preferably, when the supported biocatalyst contains hydrogenase, the hydrogenase is: 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) The amino acid sequence of the membrane-bound hydrogenase moiety 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) The amino acid sequence of the regulatory hydrogenase moiety 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 Aquifex aeolicus 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 Hydrogenovibrio marinus hydrogenase (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of Thiocapsa roseopersicina hydrogenase (SEQ ID NO: 14 and 15) or an amino acid sequence having at least 60% homology thereto; viii) The amino acid sequence of Alteromonas macleodii 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 the membrane-bound hydrogenase of Allochromatium vinosum (SEQ ID NO: 18 and / or 19), or an amino acid sequence having at least 60% homology thereto; x) The amino acid sequence of the nickel-iron hydrogenase 5 of Salmonella enterica serovar Typhimurium LT2 (SEQ ID NO: 20 and / or 21), or an amino acid sequence having at least 60% homology thereto; xi) The amino acid sequence of the hydrogenase of Desulfovibrio vulgaris Miyazaki F (SEQ ID NO: 23 and / or 24), or an amino acid sequence having at least 60% homology thereto; xii) The amino acid sequence of the [FeFe]-hydrogenase (KX147468) of Clostridium beijerinckii SM10 (CbA5H) (SEQ ID NO: 25); the amino acid sequence of the [FeFe]-hydrogenase (Cbei_4110) of Clostridium beijerinckii ATCC 51743 (SEQ ID NO: 26); the amino acid sequence of the [FeFe]-hydrogenase (Cbei_1773) of Clostridium beijerinckii (SEQ ID NO: 27); or the amino acid sequence of the [FeFe]-hydrogenase (Cbei_3796) of Clostridium beijerinckii (SEQ ID NO: 28), or an amino acid sequence having at least 60% homology thereto; or xiii) The amino acid sequence of the iron-iron hydrogenase of Clostridium acetobutylicum (SEQ ID NO: 39), or an amino acid sequence having at least 60% homology thereto selected from or including the above.

[0088] Even more preferably, when the supported biocatalyst contains a hydrogenase, the hydrogenase is: 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) The amino acid sequence of the membrane-bound hydrogenase moiety 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) The amino acid sequence of Aquifex aeolicus hydrogenase 1 (SEQ ID NO: 10 and / or 11) or an amino acid sequence having at least 60% homology thereto; v) The amino acid sequence of Hydrogenovibrio marinus hydrogenase (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto; vi) The amino acid sequence of Alteromonas macleodii hydrogenase (SEQ ID NO: 16 and / or 17) or an amino acid sequence having at least 60% homology thereto; or vii) 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; or is selected from or comprises a functional fragment, derivative or variant thereof.

[0089] Most preferably, when the supported biocatalyst contains 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.

[0090] Most preferably, in another embodiment, when the supported biocatalyst contains 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.

[0091] Preferably, when the supported biocatalyst contains carbon monoxide hydrogenase, the carbon monoxide hydrogenase is: i) the amino acid sequence of Desulfovibrio vulgaris 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 vulgaris Miyazaki 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 psychrotolerans 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 Desulfoluna spongiiphila 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 carbon monoxide dehydrogenase (SP90) of Halodesulfovibrio spirochaetisodalis (SEQ ID NO: 51) or an amino acid sequence having at least 60% homology thereto; vi) The amino acid sequence of carbon monoxide dehydrogenase (Ddes) of Desulfovibrio desulfuricans (SEQ ID NO: 52) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of carbon monoxide dehydrogenase (DaAHT2) of Desulfurivibrio alkaliphilus (SEQ ID NO: 53) or an amino acid sequence having at least 60% homology thereto; viii) The amino acid sequence of carbon monoxide dehydrogenase (Daes) of Pseudodesulfovibrio aespoeensis (SEQ ID NO: 54) or an amino acid sequence having at least 60% homology thereto; ix) The amino acid sequence of carbon monoxide dehydrogenase (Dde_3028) of Desulfovibrio alaskensis (SEQ ID NO: 55) or an amino acid sequence having at least 60% homology thereto; x) The amino acid sequence of carbon monoxide dehydrogenase (DFE_2686) of Desulfovibrio ferrophilus (SEQ ID NO: 56) or an amino acid sequence having at least 60% homology thereto; xi) The amino acid sequence of carbon monoxide dehydrogenase 2 (cooS2) of Carboxydothermus hydrogenoformans (SEQ ID NO: 57) or an amino acid sequence having at least 60% homology thereto; xii) The amino acid sequence of carbon monoxide dehydrogenase (cooS) of Desulfofundulus salinum (SEQ ID NO: 58) or an amino acid sequence having at least 60% homology thereto; xiii) The amino acid sequence of carbon monoxide dehydrogenase tengcongensis (TTE1708) of Caldanaerobacter subterraneus (SEQ ID NO: 59) or an amino acid sequence having at least 60% homology thereto; xiv) The amino acid sequence of carbon monoxide dehydrogenase (SAMN00808754_0706) of Thermanaeromonas toyohensis (SEQ ID NO: 60) or an amino acid sequence having at least 60% homology thereto; xv) The amino acid sequence of carbon monoxide dehydrogenase (gene: JT06_17280) of Desulfobulbus sp. (SEQ ID NO: 61) or an amino acid sequence having at least 60% homology thereto; xvi) The amino acid sequence of carbon monoxide dehydrogenase (A6M21_00615) of Desulfotomaculum copahuensis (SEQ ID NO: 62) or an amino acid sequence having at least 60% homology thereto; xvii) The amino acid sequence of carbon monoxide dehydrogenase (cooS2) of Pelotomaculum propionicum (SEQ ID NO: 63) or an amino acid sequence having at least 60% homology thereto; xviii) The amino acid sequence of carbon monoxide dehydrogenase (cooS2) of Methylomusa anaerophila (SEQ ID NO: 64) or an amino acid sequence having at least 60% homology thereto; xix) The amino acid sequence of carbon monoxide dehydrogenase (cooS2) of Sporomusa silvacetica (SEQ ID NO: 65) or an amino acid sequence having at least 60% homology thereto; xx) The amino acid sequence of carbon monoxide dehydrogenase (cooS) of Heliobacillus mobilis (SEQ ID NO: 66) or an amino acid sequence having at least 60% homology thereto; xxi) The amino acid sequence of carbon monoxide dehydrogenase (DCCM_2691) of Desulfocumis palustris (SEQ ID NO: 67) or an amino acid sequence having at least 60% homology thereto; Or is selected from or includes a functional fragment, derivative or variant thereof.

[0092] Preferably, when the supported biocatalyst contains formate dehydrogenase, the formate dehydrogenase is: i) The amino acid sequence of Escherichia coli formate dehydrogenase, nitrate-inducible, major subunit (fdnG) (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 Enterobacteriaceae bacterium formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 70) or an amino acid sequence having at least 60% homology thereto; iv) The amino acid sequence of Salmonella typhimurium molybdopterin oxidoreductase (fdnG) (SEQ ID NO: 71) or an amino acid sequence having at least 60% homology thereto; v) The amino acid sequence of Citrobacter rodentium formate dehydrogenase, nitrate-inducible, major subunit (fdnG) (SEQ ID NO: 72) or an amino acid sequence having at least 60% homology thereto; vi) The amino acid sequence of Escherichia alba formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 73) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of Enterobacteriaceae bacterium formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 74) or an amino acid sequence having at least 60% homology thereto; viii) The amino acid sequence of Enterobacteriaceae bacterium 4M9 formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 75) or an amino acid sequence having at least 60% homology thereto; ix) The amino acid sequence of Erwinia sp. formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 76) or an amino acid sequence having at least 60% homology thereto; x) The amino acid sequence of formate dehydrogenase-N subunit alpha (fdnG) of Jejubacter calystegiae (SEQ ID NO: 77) or an amino acid sequence having at least 60% homology thereto; xi) The amino acid sequence of selenocysteine-containing formate dehydrogenase N alpha subunit (M992_0960) of Moellerella wisconsensis (SEQ ID NO: 78) or an amino acid sequence having at least 60% homology thereto; Or is selected from or comprises a functional fragment, derivative or variant thereof.

[0093] Preferably, when the supported biocatalyst comprises or consists of an oxidoreductase comprising one or more amino acid sequences having at least 60% homology to a specific sequence, each amino acid sequence independently has at least 70% homology to the specific sequence, 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%. More preferably, each amino acid sequence independently has at least 70% homology to the specific sequence, 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%. To avoid doubt, in the case of an oxidoreductase having two or more amino acid sequences, the respective percentage homologies of the two or more sequences to their respective specific sequences can be the same or different, preferably the same. The percentage homology and / or percentage identity are each preferably determined over the full length of the specific reference sequence described herein.

[0094] It will be apparent to those skilled in the art that the redox enzyme can be either a single polypeptide or can comprise multiple polypeptides. The redox enzyme can be a portion, such as one or more domains of a multi-domain polypeptide. For example, those skilled in the art will recognize that redox enzymes such as hydrogenase and carbon monoxide dehydrogenase typically contain two or more subunits. As used herein, the term "redox enzyme" refers to one or more of the subunits of the relevant protein. For example, when the redox enzyme is Escherichia coli hydrogenase 1 (SEQ ID NO: 1 and / or 2), the redox enzyme can (i) contain SEQ ID NO: 1 but not SEQ ID NO: 2; (ii) contain SEQ ID NO: 2 but not SEQ ID NO: 1; or (iii) contain both SEQ ID NO: 1 and SEQ ID NO: 2. When the redox enzyme is Escherichia coli hydrogenase 2 (SEQ ID NO: 3 and / or 4), the redox enzyme can (i) contain SEQ ID NO: 3 but not SEQ ID NO: 4; (ii) contain SEQ ID NO: 4 but not SEQ ID NO: 3; or (iii) contain both SEQ ID NO: 3 and SEQ ID NO: 4. Typically, when the redox enzyme is a hydrogenase enzyme having two or more subunits, the redox enzyme contains the two or more subunits.

[0095] The redox enzyme can be used in monomeric or multimeric forms. For example, when the redox enzyme includes a hydrogenase that can exist in monomeric or dimeric forms, the redox enzyme can be provided in monomeric or dimeric forms. For example, Escherichia coli hydrogenase 1 can be purified as either a dimer, a monomer, or a mixture thereof. When the redox enzyme includes Escherichia coli hydrogenase 1 (i.e., SEQ ID NO: 1 and / or 2), the first polypeptide can be provided as a monomer (1×SEQ ID NO: 1 and / or 1×SEQ ID NO: 2), as a dimer (2×SEQ ID NO: 1 and / or 2×SEQ ID NO: 2), or as a mixture thereof. When providing the redox enzyme as a mixture of monomers and dimers, the mixture typically contains from about 1% to about 99% monomers and from about 99% to about 1% dimers. Sometimes, the amounts of monomers and dimers can be substantially the same, and the first polypeptide can contain, for example, from about 30% to about 70% monomers and from about 70% to about 30% dimers, such as from about 40% to about 60% monomers and from about 60% to about 40% dimers. Sometimes, the redox enzyme contains from about 1 to about 10% monomers / from about 90% to about 99% dimers, for example from about 1% to about 5% monomers / from about 95% to about 99% dimers. Sometimes, the redox enzyme contains from about 1 to about 10% dimers / from about 90% to about 99% monomers, for example from about 1% to about 5% dimers / from about 95% to about 99% monomers.

[0096] It will also be apparent to those skilled in the art that the supported biocatalyst can contain a related protein that can be co-purified with, for example, a redox enzyme. For example, when the supported biocatalyst includes a hydrogenase having the amino acid sequence of SEQ ID NO: 1 and / or 2 (or a functional fragment, derivative, or variant thereof), the supported biocatalyst can further contain a natural cytochrome electron transfer partner such as the cytochrome of SEQ ID NO: 22 (or a functional fragment, derivative, or variant thereof). That is, in embodiments where the redox enzyme includes SEQ ID NO: 1 and / or 2 (or a functional fragment, derivative, or variant thereof), the supported biocatalyst can also contain SEQ ID NO: 22 (or a functional fragment, derivative, or variant thereof).

[0097] The polypeptides used in the present invention Methods for the expression of proteins in cell (e.g., microbial) expression systems are well known and routine to those skilled in the art. For example, oxidoreductases can be isolated from their host organisms independently using routine purification methods. For example, host cells can be grown in an appropriate medium. Cell lysis allows access to the internal components of the cell. Membrane proteins can be solubilized with detergents such as Triton X (e.g., Triton X-114, (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, available from Sigma Aldrich). Soluble or solubilized proteins can be isolated and purified using standard chromatography techniques such as size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography. Alternatively, oxidoreductases can be encoded in one or more nucleotide vectors and subsequently expressed in appropriate host cells (e.g., microbial cells such as Escherichia coli). A purification tag such as HIS (hexahistidine) tag can be encoded (typically at the C-terminus or N-terminus of the relevant polypeptide), and this can be used to isolate the tagged protein using affinity chromatography, for example using nickel or cobalt NTA chromatography. Optionally, a protease recognition sequence can be incorporated between the oxidoreductase and the affinity purification tag to allow removal of the tag 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.

[0098] As described herein, the redox enzyme can be an enzyme or a functional fragment, derivative or variant of an amino acid sequence. As will be recognized by those skilled in the art, fragments of an amino acid sequence include deletion mutants of such sequences in which one or more amino acids, such as at least 1, 2, 5, 10, 20, 50 or 100, are deleted. The deletions can occur at the C-terminus or N-terminus of the native sequence or within the native sequence. Typically, the deletion of one or more amino acids does not affect the residues surrounding the active site of the enzyme. Derivatives of an amino acid sequence include post-translationally modified sequences that are modified in vivo or ex vivo. Many different protein modifications are known to those skilled in the art and include modifications for introducing new functionality to an amino acid residue, modifications for protecting a reactive amino acid residue, or modifications for linking an amino acid residue to a chemical moiety such as a linker or a reactive functional group on a substrate (surface) for attachment to such an amino acid residue.

[0099] Derivatives of an amino acid sequence include addition mutants of such sequences in which one or more amino acids, such as at least 1, 2, 5, 10, 20, 50 or 100, are added to or introduced into the native sequence. The additions can 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 does not affect the residues surrounding the active site of the enzyme.

[0100] Variants of amino acid sequences include sequences 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 with one or more non-native residues. Such variants can, i.e., include point mutations or be more substantial and, for example, using native chemical ligation, splice a non-native amino acid sequence into a partial native sequence to produce variants of native enzymes. Variants of amino acid sequences include sequences having native and / or non-native amino acids. The aforementioned variants, derivatives and functional fragments of amino acid sequences retain at least some of the activity / functionality of the native / wild-type sequence. Preferably, the aforementioned variants, derivatives and functional fragments of the sequences increase / improve the activity / functionality when compared to the native / wild-type sequence.

[0101] Variants of enzymes, such as the redox enzymes described herein, can preferably be modified to have increased catalytic activity for 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, at least 100-fold, preferably at least 1000-fold. The catalytic activity can be determined by any suitable method. For example, the catalytic activity is the Michaelis constant K M (Increased activity is typically associated with a decreased K M value) and / or the catalytic rate constant, k cat (Increased activity is typically associated with an increased k cat value).

[0102] K M and k cat Measurement of is routine for those skilled in the art. For example, the K M of a polypeptide for a substrate can be determined spectrophotometrically, e.g., with 1 mM substrate, 5 mM benzyl viologen (oxidized form; ε = 8.9 mM -1 cm -1) It can be determined by measuring the absorbance at 578 nm at 30 °C under anaerobic conditions in a 50 mM Tris-HCl buffer, pH 8.0, containing 90 μM dithionite and 10 to 30 pmol of the enzyme. Examples of solution assays for determining the absorbance of oxidized and reduced flavin are described in the Examples.

[0103] Homology can be determined using standard methods in the art. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology, for example using its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or align sequences (such as identifying equivalent residues or corresponding sequences (typically using their default settings)), as described, for example, in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0104] Similarity can be measured using pairwise identity or by applying a scoring matrix such as BLOSUM62 and converting to an equivalent identity. Positions that have been deliberately mutated will be masked when determining homology as they represent functional rather than evolutionary changes. Similarity can be determined more sensitively, for example, using PSIBLAST against a comprehensive database of protein sequences and by applying a position-specific scoring matrix. Different scoring matrices can be used that reflect the chemical and physical properties of the amino acids rather than the frequency of substitutions over the evolutionary time scale (e.g., charge). Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, conservative substitutions can introduce another amino acid that is aromatic or aliphatic in place of an 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 major amino acids defined in Table A below. When amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table B.

[0105]

Table A

[0106]

Table B

[0107] Preferably, sequence homology can be evaluated in terms of sequence identity. Without limitation, any of a variety of sequence alignment methods, including global methods, local methods, and hybrid methods such as segment approach methods, can be used to determine the percent identity. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from beginning to end of the molecule and determine the best alignment by summing the scores of individual residue pairs and imposing a gap penalty. Preferred methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680(1994)) and iterative refinements (Gotoh, J. Mol. Biol. 264(4) 823-838(1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Preferred methods include Match-box, (Depiereux and Feytmans, CABIOS 8(5) 501 -509(1992)); Gibbs sampling, (Lawrence et al., Science 262(5131) 208-214(1993)); and Align-M (Van Walle et al., Bioinformatics, 20(9) 1428-1435(2004)). That is, percent sequence identity is determined by conventional methods. See, e.g., 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 to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) shown below (amino acids are indicated by the standard single letter code).

[0108] JPEG2025517686000004.jpg102150

[0109] The identity percentage is then: 100×(T / L) (wherein, T = the total number of identical matches L = the length of the longer sequence plus the number of gaps introduced into the longer sequence to align the two sequences) is calculated as.

[0110] Supporting material As discussed, the disclosed method involves the use of a supported biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a supporting material.

[0111] An advantage resulting from the support of the redox enzyme is that the supported biocatalyst can be easily removed from the reaction mixture. For example, the support(s) can be removed by sedimentation, filtration, centrifugation, etc. Many such methods are known to those skilled in the art; for example, filtration can be achieved using a simple filter paper to remove solid components from a liquid composition; or, a solid / liquid mixed composition can be sedimented and the liquid then decanted from the sedimented solid.

[0112] An oxidoreductase (or a functional fragment or derivative thereof) is typically immobilized on a support material. As used herein, the term "immobilized" encompasses adsorption, capture, and / or crosslinking between the support and the polypeptide. Adsorption encompasses non-covalent interactions including electrostatic interactions, hydrophobic interactions, etc. A charged adsorption enhancer such as polymyxin B sulfate can be used to enhance adsorption. Capture encompasses containment of the enzyme on the surface of the support, e.g., within a polymer film or in a hydrogel. Crosslinking encompasses covalent attachment either directly between enzymes (e.g., via amide coupling such as via EDC / NHS and / or other coupling agents routine to those skilled in the art) or using one or more covalent crosslinking agents such as thiol-terminated linkers or crosslinking reagents. Immobilization means including or consisting of adsorption are preferred. Combinations of some or all of the above-described immobilization means can be used. For example, the enzyme can be linked (e.g., covalently) to a binding group for non-covalent adsorption onto the support material. For example, the enzyme can be covalently attached to a hydrophobic group (e.g., an aromatic or heteroaromatic group) for non-covalent interactions (e.g., via hydrophobic interactions, pi-pi stacking, etc.) with the support material. Suitable binding groups are known in the art and include molecules such as pyrene and its derivatives.

[0113] In the disclosed method, the support material is typically electrically conductive or semiconductive.

[0114] Preferably, the or each support is independently carbon (including doped carbon materials), metal or metal alloy, metal oxide (including mixed metal oxides), metal hydroxide (including layered double hydroxides), metal chalcogenide, semiconductor material (including carbon nitride, silicate, germanium compounds and gallium compounds such as silicon carbide, doped silicon and / or doped germanium), or electronically conductive polymer; or mixtures thereof. As will be recognized by those skilled in the art, suitable support materials may include mixtures of 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 silica, alumina, etc.). The term "mixture" as used herein encompasses both atomic mixtures such as alloys and heterogeneous mixtures such as mixtures of particles of one or more materials disclosed herein.

[0115] Any suitable support material can be used.

[0116] More preferably, the or each support material is independently: i) carbon; and / or ii) a metal or metal alloy selected from gold, silver, tungsten, iridium, platinum, palladium, copper, titanium, brass, and steel; and / or iii) a material selected from titanium oxide, indium oxide, tin oxide, and indium tin oxide including.

[0117] Even more preferably, the or each support material contains a carbon material. Even more preferably, the or each support material is independently graphite, carbon nanotube(s), carbon black, activated carbon, carbon nanopowder, vitreous carbon, carbon fiber(s), carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite, doped or surface-modified carbon, or a carbon material including doped diamond.

[0118] Examples of doped carbon materials include carbon doped with boron, nitrogen, oxygen, phosphorus, silicon, sulfur, transition metals of Group 1 or Group 2 (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), post-transition metals (e.g., aluminum, gallium, indium, and tin) and / or lanthanide elements (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 at% to about 10 at%, such as from 0.01 at% to about 1 at%, for example about 0.1 at%. 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 nano powder, vitreous carbon, carbon fibers, carbon cloth, carbon felt, carbon paper, graphene, highly oriented pyrolytic graphite, pyrolytic graphite, and diamond (optionally surface-modified diamond).

[0119] For example, when the carbon material includes graphene doped with a dopant, the graphene may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements, and transition metals. When the carbon material includes carbon nanotube(s) doped with a dopant, the carbon nanotube(s) may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements, and transition metals. When the carbon material includes diamond doped with a dopant, the diamond may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, and silicon. When the carbon material includes carbon black doped with a dopant, the carbon black may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements, and transition metals. When the carbon material includes activated carbon doped with a dopant, the activated carbon may preferably be doped with one or more dopants selected from nitrogen, boron, sulfur, oxygen, silicon, lanthanide elements, and transition metals.

[0120] Examples of surface-modified carbon materials include carbon materials containing acidic surface groups (such as carboxylic acids, carboxylic anhydrides, lactones, hydroxyls); basic surface groups (such as amides, imides, lactams, carbonyls, amines, imines, pyrrole groups and pyridine groups), and surface groups such as nitro, diazonium, nitroso, fluoro, chloro, bromo, and iodo groups. Functional groups on the carbon material can be introduced by methods such as oxidation (using reagents such as nitric acid, sulfuric acid, hydrogen peroxide, potassium permanganate, air, ozone, plasma, etc.), amidation, silanization, silylation, polymer grafting, polymer wrapping, surfactant adsorption, and encapsulation. Such modifications can generally be applied to the carbon materials disclosed herein. In some embodiments, acid oxidation to produce carboxylic acid groups on the carbon surface is preferred. That is, in some embodiments, preferred support materials include carbon black having carboxylic acid surface groups, activated carbon having carboxylic acid surface groups, and carbon nanotubes (s) having carboxylic acid surface groups.

[0121] Most preferably, the or each support material independently comprises graphite (or highly oriented pyrolytic graphite or pyrolytic graphite), activated carbon or carbon black; most preferably an activated carbon or a carbon material comprising carbon black.

[0122] Preferably, the or each support is an electronically conductive particle. Preferred electronically conductive particles include the materials described herein. Preferably, when the or each support comprises particles, the particles have a particle size of 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. Methods for determining particle size are routine in the art and include, for example, dynamic light scattering. Suitable electronically conductive particles for use in the methods of the present invention include "Black Pearls 2000" particles available from Cabot corp (Boston, Mass., USA); and conductive carbon black particles such as activated carbon "DARCO" (~100 mesh).

[0123] Target compound As discussed herein, the disclosed methods involve reducing reducible functional groups (such as nitrogen-containing functional groups) in the target compound.

[0124] Exemplary nitrogen-containing functional groups include nitro group (R-NO 2 ), azide group (R-N 3 ), hydroxylamine group (R-NR’OH), nitroso group (R-NO), nitrile group (R-CN), diazo group (R-CR’=N 2 ), diazonium group (R-N 2 + ), isocyanide group (R-NC); isothiocyanate group (R-NCS), isocyanate group (R-N=C=O), hydrazone group (R-CR’=N-NR’), hydrazine group (R-NR’-NR’ 2 ), amidine group (R-C(NR’)NR’ 2 ), azo group (R-N=N=R’), and guanidine group (R-NR’-C(NR’)-NR’ 2 ) etc. (wherein, R represents the remainder of the target compound, and each R’ is independently H or hydrocarbyl, such as alkyl, e.g., C 1-6 or C 1-4 alkyl etc.; typically represents H). More typically, the nitrogen-containing functional group is a nitro group (R-NO 2 ), azide group (R-N 3 ), hydroxylamine group (R-NR’OH), nitroso group (R-NO), nitrile group (R-CN), diazo group (R-CR’=N 2 ), diazonium group (R-N 2 + ), isocyanide group (R-NC); isothiocyanate group (R-NCS), isocyanate group (R-N=C=O), hydrazone group (R-CR’=N-NR’), and hydrazine group (R-NR’-NR’ 2 ). Even more typically, the nitrogen-containing functional group is a nitro group (R-NO 2 ), azide group (R-N 3)、a hydroxylamine group (R-NR’OH), a nitrile group (R-CN), a diazo group (R-N 2 + ) and is selected from an isocyanide group (R-NC). Even more typically, the nitrogen-containing functional group is a nitro group (R-NO 2 ) and an azide group (R-N 3 ). Most typically, the nitrogen-containing functional group is a nitro group.

[0125] The target compound can be any target compound, and the selection of an appropriate target compound for use according to the disclosed method is an operating parameter of the disclosed method and is well within the ability of one skilled in the art.

[0126] The method of the present invention finds utility particularly in the production of complex products such as in the synthesis or derivatization of natural products and in the production of pharmaceuticals. Thus, the target compound can be a pharmaceutical intermediate such as an oxidized form of a therapeutic agent, where the drug contains an amine group and the oxidized form of the drug contains an oxidized form of the amine group such as a nitro group. The characterization of the products obtained from the method of the present invention is well within the ability of one skilled in the art. For example, the products 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 examples.

[0127] The target compound is typically an organic compound and typically has a molecular weight of from about 20 to about 2000 g / mol, for example from about 50 to about 1000 g / mol such as from about 100 to about 700 g / mol.

[0128] Often, the nitrogen-containing functional group is attached to an aromatic group (e.g., covalently attached as a substituent) contained in the target compound (R), although non-aromatic groups are also encompassed by the methods of the present invention. Aromatic groups include hydrocarbyl aromatic groups (such as benzene) and heteroaromatic groups (such as pyridine), which may be further substituted. Exemplary non-aromatic groups include alkyl, alkenyl, carbocyclic, and heterocyclic groups, which may be further substituted. Conversions of both aromatic and aliphatic compounds have been demonstrated in examples using a wide range of oxidoreductases, demonstrating the broad applicability of the disclosed methods to a wide variety of target compounds.

[0129] Target compounds typically contain a plurality of such groups bonded together, with any additional substitutions. Substituents that may be present on the target compound (e.g., on the groups in the target compounds disclosed herein) are not limited and include, for example, H, halo (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 . Further substituents that may be present on the target compound (e.g., on the groups in the target compounds disclosed herein) include optionally substituted alkyl groups, e.g., C 1 to C 6 (e.g., C 1 to C 4 ) alkyl groups that are unsubstituted or substituted with OR' or halogen (e.g., F); CN; optionally substituted alkenyl groups, e.g., C 3 to C 1 to C 6 (e.g., C 1 to C 4 ) alkyl groups that are unsubstituted or substituted with OR' or halogen (e.g., F, e.g., CF 3 ). Typically, the groups in the target compounds disclosed herein may contain 1, 2, 3, or more substituents in addition to the nitrogen-containing functional group.

[0130] As used herein, R can comprise or consist of any suitable aromatic or aliphatic group or combination of groups. Suitable groups are defined herein.

[0131] To avoid doubt, multiple nitrogen-containing functional groups can be included in the target molecule and reduced according to the methods disclosed herein.

[0132] C 1 to C 20 An alkyl group such as a C1 to C20 alkyl group is a straight-chain or branched-chain alkyl group containing 1 to 20 carbon atoms. Similarly, a C1 to C10 alkyl group is a straight-chain or branched-chain alkyl group containing 1 to 10 carbon atoms. A C1 to C6 alkyl group is often a C1 to C4 alkyl group or a C1 to C3 alkyl group. Examples of C1 to C6 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. The term "alkyl" as used herein encompasses "alkylene", which is defined as a divalent moiety derived by removing two hydrogen atoms from an alkane. 1 to C 10 An alkyl group such as a C1 to C10 alkyl group is a straight-chain or branched-chain alkyl group containing 1 to 10 carbon atoms. A C1 to C6 alkyl group is often a C1 to C4 alkyl group or a C1 to C3 alkyl group. Examples of C1 to C6 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. The term "alkyl" as used herein encompasses "alkylene", which is defined as a divalent moiety derived by removing two hydrogen atoms from an alkane. 1 to C 10 A C1 to C6 alkyl group is often a C1 to C4 alkyl group or a C1 to C3 alkyl group. 1 to C 6 A C1 to C6 alkyl group is often a C1 to C4 alkyl group or a C1 to C3 alkyl group. 1 to C 4 A C1 to C6 alkyl group is often a C1 to C4 alkyl group or a C1 to C3 alkyl group. 1 to C 4 Examples of C1 to C6 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. The term "alkyl" as used herein encompasses "alkylene", which is defined as a divalent moiety derived by removing two hydrogen atoms from an alkane.

[0133] C 2 to C 20 An alkenyl group such as a C2 to C20 alkenyl group is a straight-chain or branched-chain alkenyl group containing 2 to 20 carbon atoms. Similarly, a C2 to C10 alkenyl group is a straight-chain or branched-chain alkenyl group containing 2 to 10 carbon atoms and having one or more, for example one or two, typically one double bond. A C2 to C6 alkenyl group is often a C2 to C4 alkenyl group or a C2 to C3 alkenyl group. 2 to C 10 An alkenyl group such as a C2 to C10 alkenyl group is a straight-chain or branched-chain alkenyl group containing 2 to 10 carbon atoms and having one or more, for example one or two, typically one double bond. A C2 to C6 alkenyl group is often a C2 to C4 alkenyl group or a C2 to C3 alkenyl group. 2 to C 10 A C2 to C6 alkenyl group is often a C2 to C4 alkenyl group or a C2 to C3 alkenyl group. 2 to C 6 A C2 to C6 alkenyl group is often a C2 to C4 alkenyl group or a C2 to C3 alkenyl group. 2 to C4 It is an alkenyl group. C 2 from C 4 Examples of alkenyl groups include ethenyl, propenyl, and butenyl. As used herein, the term "alkenyl" is defined as a bidentate moiety derived by removing two hydrogen atoms from an alkene and includes "alkenylene".

[0134] A carbocyclic group is typically a cyclic hydrocarbon containing from 3 to 20 carbon atoms, such as from 3 to 10 carbon atoms. The carbocyclic group can be saturated or partially unsaturated, but is typically saturated; when unsaturated, the group typically contains 1 or 2 double bonds, such as 1 double bond. Examples of saturated carbocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0135] The heterocyclic group typically contains from 3 to 20, for example from 3 to 10, atoms selected from C, O, N and S in the ring, is a cyclic group containing at least one heteroatom, and typically one or two heteroatoms. The heterocyclic group can be saturated or partially unsaturated, but is typically saturated; when unsaturated, the group typically contains 1 or 2, for example 1, double bond. 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, indoline, 2,3-dihydrobenzofuran, 2,3-dihydrobenzothiophene, 2,3-dihydro-1H-benzimidazole, 2,3-dihydrobenzoxazole, 2,3-dihydrobenzothiazole, benzodioxole, 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]dioxine and 2,3-dihydrobenzo[b][1,4]dioxine, including their quaternized derivatives.

[0136] The hydrocarbyl aromatic group is typically a substituted or unsubstituted, monocyclic or fused polycyclic aromatic group containing from 6 to 20 (e.g., from 6 to 10) carbon atoms in the ring portion, C 6 from C 20 aryl group, more typically C 6 from C 10It is an aryl group. Examples include monocyclic groups such as phenyl and fused bicyclic groups such as naphthyl and indenyl. Phenyl (benzene) is preferred.

[0137] A heteroaromatic group is typically a saturated or unsaturated monocyclic or fused polycyclic aromatic group containing 5 to 10 atoms, typically containing at least 1 heteroatom selected from O, S, and N, e.g., 1, 2, or 3 heteroatoms in the ring portion, which is a 5- to 10-membered heteroaryl group. Examples of heteroaryl groups 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 group can be a fused polycyclic system, for example, including a fused bicyclic system where the heteroaryl group is fused to an aryl group as defined herein.

[0138] It is not only substituted with a reducible nitrogen-containing functional group as defined herein, but additional substituents can be present as discussed above.

[0139] In some embodiments, the selection of the redox enzyme is determined by the reduction potential of the target compound. In some embodiments, the redox enzyme is capable of oxidizing a molecular reducing agent at a potential more negative than the reduction potential of the target compound. Determining the reduction potential of the target compound is straightforward, for example, by using electrochemistry as described in the examples. Similarly, it is straightforward to determine the potential at which a redox enzyme is capable of oxidizing a given molecular reducing agent. For example, protein film voltammetry can be used. Briefly, the redox enzyme is immobilized on an electrode optionally containing the support material described herein in the presence of the molecular reducing agent, and the potential applied to the electrode is varied and the current corresponding to the oxidation of the reducing agent is observed at the potential at which the redox enzyme is capable of oxidizing the molecular reducing agent.

[0140] Typically, the nitrogen-containing functional group is bonded to an unsaturated carbon atom. Often, the target compound contains an aromatic nitrogen-containing functional group. In other words, the nitrogen-containing functional group is typically bonded to an aromatic (hydrocarbyl aromatic or heteroaromatic) group. More typically, the target compound contains an aromatic nitro group; an aromatic azide 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 bonded to an aromatic group that is further substituted at the meta or para position relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently bonded to an aromatic group that is substituted at the ortho position relative to the nitrogen-containing functional group. In some embodiments, the nitrogen-containing functional group is covalently bonded 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 bonded to an aromatic group that is not substituted at the ortho position with a Bu group relative to the nitrogen-containing functional group. t with an aromatic group not substituted with a Bu group.

[0141] In some embodiments, the target compound comprises a plurality of 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. Typically, the methods provided herein can selectively reduce the nitrogen-containing functional group without reducing the non-nitrogen functional group. Without being bound by theory, the inventors believe that the reducible nitrogen-containing functional group may have a suitable reduction potential to be reduced by the supported biocatalyst comprising the redox enzyme described herein.

[0142] That is, as will be apparent, the disclosed methods include the formation of a reaction product comprising a reduced functional group, for example, a reduced nitrogen-containing functional group as described herein. Accordingly, provided herein is also 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 comprising a redox enzyme supported on a support material as described herein, in the presence of a molecular reducing agent as described herein, - the molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and - the nitrogen-containing functional group is reduced by the support material; - thereby forming a reaction product: under such conditions.

[0143] Typically, the reaction product comprises an aromatic amine, i.e., an amine or quaternary ammonium group bonded to a hydrocarbyl aromatic group or heteroaromatic group as defined herein.

[0144] Preferred Embodiments 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 nitro group, azide group, hydroxylamine group, nitrile group, diazo group and isocyanide group; and the reduction converts the nitrogen-containing functional group to an amine group; The method comprises contacting the target compound with a biocatalyst, where the biocatalyst comprises a redox enzyme selected from hydrogenase and / or carbon monoxide dehydrogenase and / or formate dehydrogenase supported on a conductive or semiconductive support material, or a functional fragment or derivative thereof; in the presence of a molecular reducing agent selected from molecular hydrogen and / or CO and / or formate; - the molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and - the nitrogen-containing functional group is reduced to an amine group by the support material: The method comprises contacting under such conditions.

[0145] In some further preferred embodiments, the disclosed method is a method for reducing a reducible nitrogen-containing functional group in a target compound, where the nitrogen-containing functional group is selected from a nitro group, an azide group, or a nitrile group; and the reduction converts the nitrogen-containing functional group to an amine group; The method comprises contacting the target compound with a biocatalyst, where the biocatalyst comprises a hydrogenase enzyme or a functional fragment or derivative thereof supported on a conductive or semiconductive support material, and the support material comprises carbon, a metal or metal alloy, a metal oxide or mixed metal oxide, a metal hydroxide, a metal chalcogenide, a semiconductor material, or an electronically conductive polymer, or a mixture thereof; in the presence of molecular hydrogen or an isotope thereof; - the hydrogen or its isotope is oxidized by the hydrogenase or a functional fragment or derivative thereof, and - the nitrogen-containing functional group is reduced to an amine group by the support material: The method comprises contacting under such conditions.

[0146] In still further preferred embodiments, the disclosed method is a method for reducing a nitro group in a nitroaromatic target compound; where the reduction converts the nitro group to an amine group; The method involves contacting the nitroaromatic target compound with a biocatalyst (wherein the biocatalyst comprises a hydrogenase enzyme or a functional fragment or derivative thereof supported on a carbon-based support material), in the presence of molecular hydrogen or an isotope thereof, - the hydrogen or its isotope is oxidized by the hydrogenase or a functional fragment or derivative thereof; and - the nitrogen-containing functional group is reduced to an amine group by the support material: under such conditions.

[0147] Reaction conditions In the disclosed method, the target compound is preferably initially added to the reaction medium or present in the reaction medium at a concentration of from 1 μM to 1 M, such as from about 500 μM to about 1 mM, for example from 100 μM to about 100 mM, such as from about 250 μM to about 10 mM, for example from 25 μM to 400 mM, such as from 50 μM to 200 mM, for example from 5 μM to 800 mM, such as from 10 μM to 600 mM.

[0148] As described above, when the molecular reducing agent is a gas such as hydrogen or CO, the disclosed method is typically carried out under a gas atmosphere; that is, in the presence of the gas (e.g., in the headspace of the reactor). Preferably, the gas atmosphere comprises hydrogen or an isotope thereof and / or CO or an isotope thereof and optionally an inert gas. O 2 Or its isotope may be present. Preferred inert gases are nitrogen, argon, helium, neon, krypton, xenon, radon, and sulfur hexafluoride (SF 6 ) and mixtures thereof, more preferably nitrogen and / or argon, most preferably nitrogen.

[0149] When the gas atmosphere comprises a mixture of hydrogen and an inert gas and / or O 2 the hydrogen is preferably present at a concentration of 1 - 100%, and the remaining gas is the inert gas and / or O as defined herein 2including. A preferred gas atmosphere is 80 - 100% H where the remaining gas contains one or more inert gases; and 0 - 20% H where the remaining gas contains one or more inert gases and / or O 2 (such as 1 - 4% H in air). When the gas atmosphere contains a mixture of CO and inert gas and / or O 2 including. A preferred gas atmosphere is 80 - 100% CO where the remaining gas contains one or more inert gases; and 0 - 20% CO (such as 1 - 4% CO in air) where the remaining gas contains one or more inert gases and / or O 2 (such as 1 - 4% H in air). When the gas atmosphere contains a mixture of CO and inert gas and / or O 2 including. When the gas atmosphere contains a mixture of CO and inert gas and / or O 2 CO is preferably present at a concentration of 1 - 100%, and the remaining gas contains the inert gas and / or O 2 as defined herein. A preferred gas atmosphere is 80 - 100% CO where the remaining gas contains one or more inert gases; and 0 - 20% CO (such as 1 - 4% CO in air) where the remaining gas contains one or more inert gases and / or O 2 including. The gas atmosphere may optionally also contain non - inert gases such as ammonia, carbon dioxide, and hydrogen sulfide. Preferably, however, 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 that 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 under reduced pressure (e.g., less than 1 bar) or high pressure (e.g., more 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 from about 1 bar to about 10 bar, for example from about 2 bar to about 8 bar, such as from about 4 bar to about 6 bar, for example about 5 bar.

[0150] The disclosed method can be carried out under aerobic or anaerobic conditions. As used herein, "aerobic conditions" strictly refers to a gas atmosphere that is not strictly anaerobic, for example, containing at least a trace amount of O

[0151] The appropriate O 2 level is typically more than 1% O 2 including. The appropriate O 2, for example, more than 2% O 2 and the like, more than 100 ppm, for example more than 1000 ppm (0.1%). Usually, O 2 levels are those in the atmosphere of O 2 levels, i.e., 21% O 2 but do not exceed; however, higher O 2 levels are not excluded.

[0152] The method provided is typically carried out in an aqueous composition which may optionally contain, for example, a buffer salt. For some applications a buffer is not required and the method of the invention can be carried out without any buffer. Preferred buffer salts that can be used in the method of the invention are Tris; phosphate; citrate / Na 2 HPO 4 ; citrate / 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; Trisma; HEPPSO; POPSO; TEA; EPPS; tricine; Gly-Gly; bicine; HEPBS; TAPS; AMPD; TABS; AMPSO; CHES; CAPSO; AMP; CAPS and CABS. The selection of the appropriate buffer for the desired pH is routine to those skilled in the art and guidance is available, for example, at http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html. The buffer salt is preferably used at a concentration in solution of from 1 mM to 1 M, preferably from 10 mM to 100 mM such as about 50 mM. The most preferred buffer for use in the method of the invention comprises 50 mM phosphate, pH 8.0.

[0153] The provided method is typically carried out in an aqueous composition. However, non-aqueous components can optionally be used in the composition used in the disclosed method, instead of or in addition to water. For example, one or more organic solvents (such as alcohols, DMSO, acetonitrile, etc.) or one or more ionic liquids can be used or included in the composition.

[0154] The disclosed method can be carried out in a mixed solvent system containing a plurality (e.g., 2, 3, 4, 5 or more) of aqueous or non-aqueous solvents, such as the solvents described herein. In some embodiments, the disclosed method is carried out in a single-phase solvent system. In some embodiments, the disclosed method is carried out in an immiscible solvent mixture such as a two-phase or three-phase solvent system.

[0155] The disclosed method is typically carried out at a temperature of from about 10°C to about 80°C, such as from about 20°C to about 60°C, for example from about 30°C to about 50°C.

[0156] The disclosed method is typically carried out at a pH of from about pH4 to about pH10, such as from about pH5 to about pH9, for example from about pH6 to about pH8, for example about pH7.

[0157] The disclosed method can be implemented in the apparatus provided herein. The apparatus typically includes a reaction vessel. The reaction vessel typically includes one or more inlets for molecular hydrogen gas or a hydrogen-containing liquid (e.g., a liquid such as a hydrogen-saturated buffer solution described herein); and / or one or more inlets for reagents; and / or one or more outlets for products. Additional equipment such as pressure control, temperature control, mixing devices, flow control, etc. can be incorporated. The apparatus can include as part of the apparatus for converting an initial reagent to a final product, i.e., can be configured to perform intermediate reaction steps. The apparatus can be controlled by equipment such as a computer controller. The apparatus can include means for detecting, e.g., spectrophotometric means, the metabolic turnover of cofactors, the utilization of reagents and / or the production of products. The apparatus can be configured to operate in a flow mode (i.e., continuous mode) or in a batch mode.

[0158] Accordingly, the method provided herein can be implemented in a flow setting, e.g., in a flow reaction cell. Alternatively, the method provided herein can be implemented in a batch setting, e.g., in a batch reaction cell.

[0159] System Also provided herein is a system for implementing the method disclosed herein.

[0160] Preferably, the system comprises: i) a biocatalyst comprising a redox enzyme or a functional fragment or derivative thereof supported on a support material; ii) a molecular reducing agent; and iii) a target compound comprising a reducible nitrogen-containing functional group and; wherein the system is configured such that, in use, (a) the molecular reducing agent is oxidized by the redox enzyme or a functional fragment or derivative thereof; and (b) the nitrogen-containing functional group is reduced by the support material.

[0161] Typically, the system is designed or configured such that the redox enzyme or functional fragment or derivative transfers electrons to a support material and the reduction of the nitrogen-containing functional group involves direct electron transfer from the support material to the target compound, e.g., to a reducible functional group of the target compound.

[0162] In the systems provided herein, the redox enzyme is typically as defined herein. The support material is typically as defined herein. The molecular reductant is typically as defined herein. The target compound and the functional groups contained therein are typically as defined herein. The system can be configured to operate as described for the methods provided herein. The system can be configured to operate under the reaction conditions defined herein.

[0163] Typically, the system further includes means for controlling the concentration of the molecular reductant 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 often configured as a flow cell containing the reagents described herein. The system (e.g., a flow cell) includes one or more inlets for the molecular reductant (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 products; and / or one or more of the pressure control, temperature control, mixing devices, flow control, etc., features of the devices provided herein.

[0164] The following examples illustrate the invention. However, they in no way limit the invention. In this regard, it is important to understand that the specific assays used in the Examples section are designed only to provide an indication of the effectiveness of the methods of the invention. There are many assays available for determining the effectiveness of a reaction, and a negative result in any one particular assay is thus not conclusive.

Example

[0165] Example 1 This example demonstrates the complete reduction of reducible nitrogen-containing functional groups by the disclosed method.

[0166] Escherichia coli hydrogenase 1 (Hyd1), a hydrogen-oxidizing redox enzyme, was expressed, purified, and immobilized on the support material described herein. The support material used was carbon black nanopowder (BP2000, Cabot corp). Hyd1 is a good H 2 oxidizing agent (H 2 →2H + +2e - ) and is O 2 tolerant. 1 For the control experiment, an equal amount of Hyd1 in solution without the support material was used. The reduction of an exemplary nitrogen-containing functional group (2-methyl-5-nitropyridine, the nitro group of compound A was quantified by 1H NMR spectroscopy (Figure 10)). Reaction conditions: A 0.25 mL reaction contained 5 mM of A in Tris-HCl (50 mM, pH 8.0) with 2% (v / v) DMSO under 1 bar of H 2 .

[0167]

[0168]

Chemical formula

[0169] ​

Table 1

[0170] This example demonstrates that the presence of the support material described herein leads to an improvement in nitrogen group reduction in that it leads to both more complete reduction and higher reduction efficiency. Without being bound by theory, the inventors believe that the support material serves as an "active site" to promote the complete reduction of nitrogen-containing functional groups when modified with redox enzymes such as hydrogenase, such as Hyd1. Without being bound by theory, the inventors propose that the mechanism for nitro reduction occurs via a series of electron and / or hydride transfers from the carbon material to the nitro group.

[0171] Example 2 Example 1 was repeated using 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 effect efficient and complete reduction of the nitro group, demonstrating the chemical versatility of the disclosed method.

[0172]

Chemical formula

[0173] The reaction efficiency was 1 determined by 1H NMR (Figure 11). Optimal reduction efficiency was observed for reaction (i) at pH 7 and the reaction proceeded to completion after 21 hours.

[0174] Example 3 E. coli Hyd-1 modified carbon particles were packed in a packed bed flow column as follows. A stainless steel flow cartridge (30×4 mm) was packed with 50 mg of activated carbon using a cartridge packer (ThalesNano). A 0.2 mL (1.9 mg) solution of Hyd1 was injected into the cartridge and it was sealed. The cartridge was sealed using the cartridge packer, and stirred on a vortex mixer and then stored at 4 °C for 1 hour. The cartridge was then stored at 4 °C for 1 hour, then installed in a reactor (H-Cube) and flushed with sodium phosphate buffer (100 mM, pH 6.0) to remove any unadsorbed enzyme.

[0175] Reaction (i) of Example 2 was repeated under the following flow conditions: A liquid feed containing 10 mM 4-nitrophenol in sodium phosphate buffer (100 mM, pH 6.0) (Knauer H-Cube piston pump) was generated by the built-in water tank and electrolytic cell with H 2 used (gas supply pressure was set to 2 bar of H 2 ), and pumped through the H-Cube. The liquid feed flow rate was set to 0.1 ml per minute so as to give a residence time (tRes) of 1.6 seconds through the biocatalyst cartridge. The reaction was carried out at 28 °C. Aliquots were taken periodically for analysis by UV-vis spectroscopy and HPLC.

[0176] Successful complete reduction of the corresponding nitro group to the amine group shown in Scheme 1 was observed under industrially relevant conditions. The reaction proceeded for about 92 hours with high (about 80%) conversion. This is consistent with Hyd1 TTN > 1.4 million.

[0177] Example 4 Example 1 was repeated using 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 of the target compound (1) in buffer and co-solvent at room temperature with H 2It was to mix on a shaker plate under a constant flow rate. The conversion was determined using HPLC after subjecting the reaction mixture to Boc derivatization conditions (see caption, Figure 12). As shown in Table 2, a conversion close to 100% was observed after 21 hours.

[0178] Additional control experiments were carried out to confirm the roles of the redox enzyme and the support material in the supported biocatalyst. The reduction of the azide group was not observed in (i) a control experiment omitting the carbon support material using an equal amount of E. coli Hyd1 in solution; (ii) a control experiment omitting the redox enzyme using an equal amount of the carbon support material; or (iii) a control experiment omitting both the carbon support material and the hydrogenase.

[0179]

Chemical formula

[0180]

Table 2

[0181] Example 5 The nitrogen-containing functional groups of further target compounds were reduced using the system of Example 1. This example demonstrates that (i) aryl azides can be reduced with high efficiency and selectivity using the disclosed method; (ii) further substitution on the aryl group is well tolerated; and (iii) unwanted side reactions such as dehalogenation of the para-chloro substituent can be avoided. Such unwanted side reactions are a common problem when using noble metal catalysts such as palladium on carbon (Pd / C) to catalyze the reduction of aryl nitrogen-containing functional groups such as azides. 2,3

[0182] The results are shown in Table 3. Reaction conditions: 0.25 mL reaction volume, 5 mM azide (target compound), 19 μg of Hyd1 enzyme immobilized on 25 μg of carbon black nanoparticle support material, 1 bar of H 2 mixing under an atmosphere of.

[0183]

Table 3

[0184] The conversion was determined using GC-FID. The conversion was calculated by comparing the peak areas of the azide and the corresponding aniline.

[0185] GC-FID method: Column: CP-Chirasil-Dex CB (Agilent), 25 m in length, 0.25 mm in diameter, 0.25 μm (film thickness), equipped with a 10 m non-deactivated fused silica guard of the same diameter Carrier: He (CP grade), 170 kPa (constant pressure) Inlet temperature: 200 °C Injection conditions: Splitless at a split flow of 60 mL / min, splitless time 0.8 min, purge 5 mL / min. Injection volume = 0.5 μL. Detection: FID (H 2 = 35 mL / min, air = 350 mL / min, makeup N 2 = 40 mL / min, temperature = 200 °C)

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[0187] JPEG2025517686000012.jpg85158

[0188] Example 6 The selectivity of the disclosed method in reducing nitrogen-containing functional groups as compared to other reducible functional groups such as unsaturated C=C double bonds was demonstrated by repeating Example 1 using cinnamyl azide (3). The product distribution was 1 determined using 1H NMR spectroscopy (see Figure 13).

[0189]

Chemical formula

[0190] JPEG2025517686000014.jpg40158

[0191] The control experiment was carried out using the conventional noble metal catalyst (Pd / C) under the same reaction conditions. Both the alkene group and the azide group of compound 3 were reduced to form 5.

[0192] The absence of peaks in the alkene region (i.e., 5 - 7 ppm) of the spectrum showing the product of the reaction with Pd / C as the catalyst confirms that Pd / C reduced the alkene. In contrast, the spectrum for the Hyd1 / C catalyst reaction contains an alkene peak shifted downward relative to the starting azide, demonstrating the formation of the corresponding cinnamylamine.

[0193] This example thus demonstrates the improved chemoselectivity of the disclosed systems and methods compared to conventional catalysts in selectively reducing nitrogen - containing functional groups.

[0194] Example 7 Experiments were conducted to investigate the reaction mechanisms and product distributions described in Examples 1 to 6.

[0195] Figure 1 shows the electrochemistry of 1 mM nitrobenzene in a pH 8 buffer at a pyrolytic graphite edge (PGE) electrode. The reduction wave at a more negative potential indicates the irreversible reduction of the nitro group to an amino group. Figure 1 shows that the onset potential for the reduction of nitrobenzene to aniline is significantly more positive than the hydrogen couple (assuming H at pH 8 and 1 bar). This confirms that electrons provided by hydrogenase, which oxidizes dihydrogen, can be used by the PGE to reduce nitrobenzene to aniline. 2 This confirms that electrons provided by hydrogenase, which oxidizes dihydrogen, can be used by the PGE to reduce nitrobenzene to aniline.

[0196] Figure 2 confirms that carbon black nanopowder (BP2000), like the PGE, can reduce nitrobenzene at a potential more positive than the hydrogen couple under the stated conditions.

[0197] Figure 3 shows the IR difference spectrum of 2 mM nitrobenzene in 1 volume % DMSO in 50 mM Tris-HCl, showing two N-O stretching absorption bands (dark gray dashed lines) of the nitro group and three bands (light gray dotted lines) derived from DMSO. Figure 4 shows that the N-O stretching band (black dashed line) disappears when a reduction potential equal to the potential of the hydrogen couple is applied to the BP working electrode. This indicates that a reduction wave starting at around -0.4 V vs. SHE corresponds to the reduction of the nitro group of nitrobenzene.

[0198] The electrochemical behavior of nitrophenol in an aqueous electrolyte (100 mM sodium phosphate, pH 6) on the BP electrode was also evaluated (Figure 5A). An irreversible reduction wave starting at around -0.3 V vs. SHE is clearly observed. When the potential is scanned at a lower rate (i.e., closer to steady-state conditions, Figure 5B), the reduction wave starts at a potential significantly more positive than the hydrogen couple under these conditions (pH 6, hydrogen at 1 bar, E 0 (H + / H 2 ) = -0.355 V vs. SHE). This again indicates that nitrophenol on the BP surface can be reduced by electrons supplied by an H 2 -oxidizing hydrogenase such as Hyd1.

[0199] The electrochemistry of 5 mM 2-azido-1-phenylethanone on the BP electrode was further investigated in a spectroelectrochemical cell. A reduction wave at a potential below -0.4 V vs. SHE can be observed in the cyclic voltammogram (Figure 6). Similar to the cases of nitrobenzene and nitrophenol, this is significantly more positive than the hydrogen couple under these conditions (pH 8, hydrogen at 1 bar, E 0 (H + / H 2 ) = -0.473 V vs. SHE), and this also indicates again that 2-azido-1-phenylethanone on the BP surface can be reduced by electrons supplied by an H 2 -oxidizing hydrogenase such as Hyd1.

[0200] Figure 7 shows the IR difference spectrum of 5 mM 2 - azido - 1 - phenylethanone in 1% DMSO in 50 mM Tris - HCl, showing the N=N=N stretching absorption band of the azide group (red line) and the C=O stretching of the carbonyl group of 2 - azido - 1 - phenylethanone (blue line). Figure 8 shows the IR difference spectra of 5 mM 2 - azido - 1 - phenylethanone in 50 mM Tris - HCl pH 8 with 1% DMSO at different applied potentials (vs SHE) on a BP - modified activated carbon paper working electrode. Figure 8 shows that when a reduction potential approximately equal to the potential of the hydrogen couple is applied to the BP working electrode, the N=N=N stretching band (at about 2100 cm -1 ) disappears. This indicates that the reduction wave starting at around - 0.4 V vs SHE is actually the reduction of the azide group of 2 - azido - 1 - phenylethanone, and that BP can use electrons supplied by hydrogenase to reduce the azide group. 2 Confirm that BP can reduce the azide group using electrons supplied by hydrogenase.

[0201] Figure 9 shows an enlarged region of the cyclic voltammogram of a PGE electrode modified with BP200 in 100 mM sodium phosphate pH 6 with 1% DMSO and 1 mM tetracyano - benzene at a scan rate of 10 mV s -1 at a rotation speed of 0 rpm obtained at a scan rate of 10 mV s. This indicates that tetracyano - benzene can be reduced at the carbon surface, and the reduction wave starts at a potential slightly positive of the hydrogen couple at pH 6 (assuming a hydrogen pressure of 4 bar). This shows that the supported biocatalysts (e.g., hydrogenase on carbon) described herein can reduce nitrile substrates. 2

[0202] Example 8 Additional experiments were conducted to demonstrate the broad applicability of the disclosed method. As shown in Table 4, a wide range of target compounds were successfully addressed by controlling the reaction conditions. The selection of appropriate reaction conditions is well within the capabilities of those skilled in the art, and the selection of reaction conditions to achieve the desired results is an operating parameter of the method that can be controlled by those skilled in the art.

[0203] Reaction conditions: 10 mM substrate and co-solvent in 100 mM sodium phosphate buffer, pH 6 (unless otherwise indicated) at room temperature, 2 bar of H in a pressure vessel 2 under a constant pressure of, mixed on a shaker plate for 24 hours.

[0204]

Table 4

[0205] HPLC method: Column: Shim-pack GIS 5μm HILIC reverse phase column (Shimadzu), 150 mm in length, 4.6 mm in diameter, with a 5μm Shim-pack GIS (G) guard column attached with a length of 4 mm and a diameter of 10 mm Mobile phase: 80% 20 mM ammonium acetate buffer pH 7.7, 20% acetonitrile at a flow rate of 1 ml / min Oven temperature: 40 °C Injection conditions: Injection volume = 10 μL. Detection: UV at 260 and 310 nm

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[0207] Example 9 This example describes the reduction of various nitro groups using the catalyst "CaHydA1 / C" formed by immobilizing C. acetobutylicum [FeFe]-hydrogenase A1 (CaHydA1) on carbon black nano powder (BP2000). CaHydA1 is a good H that operates without overvoltage 2It is an oxidizing agent and can provide a large driving force as such.

[0208] Table 5 shows the successful conversion of various substituents using CaHydA1 / C. No by-products were identified.

[0209] Reaction conditions: 10 mM substrate in 100 mM sodium phosphate buffer, pH 6, and the indicated co-solvent were mixed on a shaker plate at room temperature for 24 h under a constant pressure of 2 bar of H 2 in a pressure vessel.

[0210]

Table 5

[0211] Example 10 Some compounds with reducible nitrogen-containing functional groups have reduction potentials that are difficult to access. By using redox enzymes that operate at low overpotentials, such compounds can be readily reduced according to the methods disclosed herein.

[0212] Examples of such compounds include 1-nitrohexane and 1-nitropentane. As shown in Figure 14, these compounds have a reduction potential that is only slightly positive relative to the hydrogen couple (pH 6, 1 bar of hydrogen has E 0 (H + / H 2 ) = -0.355 V vs SHE).

[0213] By using hydrogenases with low overpotentials, such as the [FeFe] hydrogenase CaHydA1, the compounds 1-nitrohexane and 1-nitropentane could be readily reduced according to the disclosed methods (see Example 9). Control experiments using redox enzymes with significant overpotentials (e.g., E. coli Hyd1 has an overpotential of approximately 80 mV for H 2Having an overpotential for oxidation) did not result in efficient reduction. It is straightforward to determine the overpotential at which the hydrogenase operates. 1

[0214] Example 11 The reduction of additional substrates was demonstrated using E. coli Hyd-1 modified carbon particles. The reactions were carried out in sodium phosphate buffer pH 6 at room temperature under a hydrogen gas flow at atmospheric pressure. The reaction products were analyzed using NMR. Substrates 8 - 13, 15 - 24, and 26 - 29 were carried out with 10% volume / volume MeCN as a co-solvent.

[0215] The reactions are shown in the following table. All reactions reached complete conversion (i.e., 100% conversion) to the corresponding amine during the indicated time.

[0216] This example demonstrates the broad applicability of the methods disclosed herein. A wide range of substituents on the aromatic group having a reducible nitrogen-containing group (nitro group) are tolerated, and complete reduction of the nitro group was readily achieved under the experimental conditions.

[0217] [Table 6 - 1]

[0218] [Table 6 - 2]

[0219] Example 12 The reduction of additional substrates was demonstrated using E. coli Hyd-2 modified carbon particles. The reactions were carried out in sodium phosphate buffer pH 6 at room temperature under 2 bar of hydrogen gas in a pressure vessel. The reaction products were analyzed using NMR.

[0220] The substrates are shown in the following table. Both substrates were efficiently reduced, and only the amine was detected as the reaction product, indicating complete conversion.

[0221] This example further demonstrates the broad applicability of the methods disclosed herein not only to aromatic compounds but also to aliphatic compounds.

[0222] [Table 7]

[0223] References 1. M. J. Lukey, A. Parkin, M. M. Roessler, B. J. Murphy, J. Harmer, T. Palmer, F. Sargent and F. A. Armstrong, J. Biol. Chem., 2010, 285, 3928 - 38. 2. X. Zhao, S. E. Cleary, C. Zor, N. Grobert, H. A. Reeve and K. A. Vincent, Chem. Sci.,, DOI:10.1039 / D1SC00295C. 3. C. A. Marques, M. Selva and P. Tundo, J. Org. Chem., 1993, 58, 5256 - 5260. 4. US 2020 / 0262784 A1, 2020. 5. M. Rueping, C. Vila and U. Uria, Org. Lett., 2012, 14, 768 - 771.

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Claims

1. A method for reducing a nitrogen-containing functional group in a target compound, wherein the target compound is subjected to oxidation in the presence of a biocatalyst comprising an oxidoreductase or a functional fragment thereof supported on a support material, and a molecular reducing agent for oxidation by the oxidoreductase or its functional fragment. - The molecular reducing agent is oxidized by the oxidoreductase or its functional fragment; and - The nitrogen-containing functional group is reduced in the supporting material: This includes contact under such conditions, Here, the oxidoreductase or its functional fragment transfers electrons to the supporting material, and the reduction of the nitrogen-containing functional group includes direct electron transfer from the supporting material to the target compound; and A method comprising adding the target compound to the reaction medium first or having it present in the reaction medium.

2. The method according to claim 1, comprising contacting a target compound with a support material.

3. The method according to claim 1, comprising reducing a nitrogen-containing functional group to form an amine group or a quaternary ammonium group.

4. The method according to claim 1, wherein an oxidoreductase or a functional fragment thereof is in electronic contact with a support material.

5. The method according to claim 1, wherein an oxidoreductase or a functional fragment thereof transfers electrons to a supporting material via an intramolecular electron conduction pathway.

6. The method according to claim 1, wherein an oxidoreductase or a functional fragment thereof transfers electrons to a supporting material via a series of [FeS] clusters.

7. The method according to claim 1, wherein the molecular reducing agent is selected from hydrogen, carbon monoxide, formates, their isotopes, and mixtures thereof.

8. The method according to claim 1, wherein the molecular reducing agent contains or consists of hydrogen or its isotope.

9. i) The supported biocatalyst comprises a hydrogenase enzyme or a functional fragment thereof; or ii) The supported biocatalyst comprises a carbon monoxide dehydrogenase enzyme or a functional fragment thereof; or iii) The method according to claim 1, wherein the supported biocatalyst comprises a formate dehydrogenase enzyme or a functional fragment thereof.

10. The method according to claim 1, wherein the supported biocatalyst comprises a hydrogenase enzyme or a functional fragment thereof.

11. The supported biocatalyst comprises a hydrogenase enzyme or a functional fragment thereof, The hydrogenase, i) The amino acid sequence of E. 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 E. coli hydrogenase 2 (SEQ ID NO: 3 and / or 4) or an amino acid sequence having at least 60% homology thereto; iii) The amino acid sequence of the membrane-bound hydrogenase moiety of Ralstonia eutropha (SEQ ID NOs. 5 and / or 6 and / or 7) or an amino acid sequence having at least 60% homology thereto; iv) The amino acid sequence of the Ralstonia europha regulatory hydrogenase moiety (SEQ ID NOs: 8 and / or 9) or an amino acid sequence having at least 60% homology thereto; v) The amino acid sequence of Aquifex aeolicus 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 Hydrogenovibrio marinus hydrogenase (SEQ ID NO: 12 and / or 13) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of Thiocapsa roseopersicina hydrogenase (SEQ ID NOs: 14 and 15) or an amino acid sequence having at least 60% homology thereto; viiii) The amino acid sequence of Alteromonas macleodii 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 Allochromium 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 enterica serotype 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 Miyazaki F-hydrogenase (SEQ ID NOs. 23 and / or 24) or an amino acid sequence having at least 60% homology thereto; xi) 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; xiiii) The amino acid sequence of Clostridium pasteurianum [FeFe]-hydrogenase (hydA) (SEQ ID NO: 29) or an amino acid sequence having at least 60% homology thereto; 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; xv) The amino acid sequence of Chlorella variabilis [FeFe]-hydrogenase (SEQ ID NOs. 31 and / or 32) or an amino acid sequence having at least 60% homology thereto; xvi) The amino acid sequence of the soluble hydrogenase moiety of Ralstonia eutropha (SEQ ID NOs. 33 and / or 34) or an amino acid sequence having at least 60% homology thereto; xvii) The amino acid sequence of the soluble hydrogenase portion of Rhodococcus opacus (SEQ ID NO: 35 and / or 36) or an amino acid sequence having at least 60% homology thereto; xviiii) 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; xix) The amino acid sequence of Clostridium acetobutylicum iron-iron hydrogenase (SEQ ID NO: 39) or an amino acid sequence having at least 60% homology thereto; xx) The amino acid sequence of Desulfomicrobium baculatum nickel-iron selenium hydrogenase (SEQ ID NO: 40 and / or 41) or an amino acid sequence having at least 60% homology thereto; xxi) The amino acid sequence of the soluble hydrogenase portion of Hydrogenophyllus thermoluteolus (SEQ ID NOs. 42 and / or 43) or an amino acid sequence having at least 60% homology thereto; xxii) The amino acid sequence of Periplasmic [NiFe]hydrogenase of Desulfovibrio gigas (SEQ ID NOs. 44 and / or 45) or an amino acid sequence having at least 60% homology thereto; or The method according to claim 1, wherein the amino acid sequence is selected from or comprises 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.

12. The supported biocatalyst comprises carbon monoxide dehydrogenase or a functional fragment thereof. The carbon monoxide dehydrogenase, i) The amino acid sequence of Desulfovibrio vulgaris 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 vulgaris Miyazaki 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 psychrotolerans (COOS) (SEQ ID NO: 49) or an amino acid sequence having at least 60% homology thereto; iv) The amino acid sequence of Desulfoluna spongiiphylla 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 Haladesulfovibrio spirochaetisodalis 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 (Ddes) (SEQ ID NO: 52) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of Desulfurivrio alkaliphilus carbon monoxide dehydrogenase (DaAHT2) (SEQ ID NO: 53) or an amino acid sequence having at least 60% homology thereto; viiii) Pseudodesulfovibrio aespoensis: the amino acid sequence of 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 alaskensis 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 ferrophilus 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 carbon monoxide dehydrogenase 2 (cooS2) (SEQ ID NO: 57) or an amino acid sequence having at least 60% homology thereto; xi) The amino acid sequence of Desulfofundulus salinum carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 58) or an amino acid sequence having at least 60% homology thereto; xiiii) The amino acid sequence of Caldanaerobacter subterraneus carbon monoxide dehydrogenase tengcongensis (TTE1708) (SEQ ID NO: 59) or an amino acid sequence having at least 60% homology thereto; xiv) The amino acid sequence of Thermaenaeromonas toyohensis 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 (SEQ ID NO: 61) of Desulfobulbus sp. carbon monoxide dehydrogenase (gene: JT06_17280) or an amino acid sequence having at least 60% homology thereto; xvi) The amino acid sequence of Desulfotomaculum copahuensis (A6M21_00615) (SEQ ID NO: 62) or an amino acid sequence having at least 60% homology thereto; xvii) The amino acid sequence of Pelotomaculum propionicum carbon monoxide dehydrogenase (cooS2) (SEQ ID NO: 63) or an amino acid sequence having at least 60% homology thereto; xviiii) The amino acid sequence of Methylomusa anaerophylla 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 Sporomusa silvacetica 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 Heliobacillus mobilis carbon monoxide dehydrogenase (cooS) (SEQ ID NO: 66) or an amino acid sequence having at least 60% homology thereto; The method according to claim 1, wherein the amino acid sequence is selected from or comprises the amino acid sequence of Desulfocumis palustris carbon monoxide dehydrogenase (DCCM_2691) (SEQ ID NO: 67) or an amino acid sequence having at least 60% homology thereto.

13. The supported biocatalyst comprises a formate dehydrogenase enzyme or a functional fragment thereof, The formate dehydrogenase, i) The amino acid sequence of the major subunit (fdnG) of Escherichia coli formate dehydrogenase (SEQ ID NO: 68) or an amino acid sequence having at least 60% homology thereto; ii) Shigella flexneri formate dehydrogenase-N, nitrate-inducible, amino acid sequence of the alpha subunit (fdnG) (SEQ ID NO: 69) or an amino acid sequence having at least 60% homology thereto; iii) The amino acid sequence of Enterobacteriaceae bacterium formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 70) or an amino acid sequence having at least 60% homology thereto; iv) The amino acid sequence of Salmonella typhurium molybdopterin oxidoreductase (fdnG) (SEQ ID NO: 71) or an amino acid sequence having at least 60% homology thereto; v) Citrobacter rodentium formate dehydrogenase, nitrate-inducible, amino acid sequence of the major subunit (fdnG) (SEQ ID NO: 72) or an amino acid sequence having at least 60% homology thereto; vi) The amino acid sequence of Escherichia alba formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 73) or an amino acid sequence having at least 60% homology thereto; vii) The amino acid sequence of the Enterobacteriaceae bacterium formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 74) or an amino acid sequence having at least 60% homology thereto; viiii) The amino acid sequence of Enterobacteriaceae bacterium 4M9 formate dehydrogenase-N subunit alpha (fdnG) (SEQ ID NO: 75) or an amino acid sequence having at least 60% homology thereto; ix) The amino acid sequence of Erwinia sp. formate dehydrogenase-N subunit alpha (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-N subunit alpha (fdnG) (SEQ ID NO: 77) or an amino acid sequence having at least 60% homology thereto; xi) The method according to claim 1, wherein the amino acid sequence (SEQ ID NO: 78) of the Moellererella wisconsensis selenocysteine-containing formate dehydrogenase N alpha subunit (M992_0960) is selected from or comprises an amino acid sequence having at least 60% homology thereto.

14. The method according to claim 1, wherein an oxidoreductase or a functional fragment thereof is immobilized on a support material.

15. The method according to claim 1, wherein the support material is electronically conductive or semiconducting.

16. The method according to claim 1, wherein the supporting material comprises carbon, a metal or metal alloy, a metal oxide or mixed metal oxide, a metal hydroxide, a metal chalcogenide, a semiconductor material, or a conductive polymer, or a mixture thereof.

17. The method according to claim 1, wherein the support material includes 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 pyrolysis graphite, pyrolysis graphite, doped or surface-modified carbon, or doped diamond.

19. Carbon materials: - 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, lanthanide elements 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, lanthanide elements and transition metals. Including; The method according to claim 17, further and / or the carbon material is comprising or modified to comprise a carboxylic acid surface group.

20. The method according to claim 1, wherein the nitrogen-containing functional group is a nitro, azide, hydroxylamine, nitroso, nitrile, diazo, diazonium, isocyanide, isothiocyanate, isocyanate, hydrazone, hydrazine, amidine, azo, or guanidine group.

21. The method according to claim 1, wherein the target compound is a nitroaromatic compound.

22. The method according to claim 1, wherein the target compound is a nitroaromatic compound comprising a hydrocarbyl aromatic group or heteroaromatic group substituted with a nitro group.

23. The method according to claim 1, wherein the hydrocarbyl aromatic group or heteroaromatic group is further substituted.

24. The method according to claim 1, wherein the biocatalyst does not include an oxidoreductase or a functional fragment thereof that includes an active site capable of catalyzing the enzymatic reduction of a nitrogen-containing functional group.

25. The method according to claim 1, wherein the biocatalyst does not contain a nitroreductase enzyme.

26. The method according to claim 1, wherein the method does not involve the transfer of electrons to the target compound via one or more cofactors.

27. It is a system, i) Biocatalysts comprising an oxidoreductase or a functional fragment thereof supported on a support material; ii) Molecular reducing agents; and iii) Target compounds containing reducible nitrogen-containing functional groups Including; Here, the system is configured such that, during use, (a) the molecular reducing agent is oxidized by the oxidoreductase or its functional fragment; and (b) the nitrogen-containing functional group is reduced by the supporting material; Here, the oxidoreductase or its functional fragment transfers electrons to the supporting material, and the reduction of the nitrogen-containing functional group includes direct electron transfer from the supporting material to the target compound, Here, the target compound is either initially added to the reaction medium or present in the reaction medium, in a system.

28. - An oxidoreductase or a functional fragment thereof as defined in any one of claims 4 to 6 or 9 to 14; and / or - The support material is as defined in any one of claims 15 to 19; and / or - The molecular reducing agent is as defined in any one of claims 7 to 8; and / or - The target compound and / or nitrogen-containing functional group is as defined in any one of claims 20 to 23; and / or - The biocatalyst is as defined in any one of claims 24 to 25: The system according to claim 27.

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