Patent Publication Number: US-2023151401-A1

Title: Method of reducing and recycling oxidized flavin cofactors

Description:
The project leading to this application has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation programme (grant agreement No 819580). 
     FIELD OF THE INVENTION 
     The present invention relates to methods for producing a reaction product from a reactant. The invention also relates to methods of reducing oxidised flavin cofactors and to methods of recycling such flavin cofactors. The invention further relates to systems and apparatuses for the production of such reaction products and the recycling of such cofactors. 
     BACKGROUND 
     Chemical manufacturing processes are typically associated with many environmental concerns. The reagents such as catalysts used are often non-renewable and/or toxic. Extreme operating conditions are typically required, such as elevated temperatures and pressures, with the provision of such conditions being energy inefficient. Toxic solvents are often needed in order to achieve satisfactory yields. Furthermore, the reagents used are often non-selective requiring complex synthetic strategies in order to selectively process only desired functional groups within molecules. 
     Biological catalysis is an approach that has been suggested to address these and related issues. This approach exploits the exquisite chemical control offered by biological systems such as enzymes to process their chemical substrates. Enzymatic processing of chemical reagents offers advantages compared to traditional chemical processing methods. Enzymes are renewable and biodegradable, and thus overcome environmental issues regarding the production and disposal of chemical catalysts. Enzymes are typically nonhazardous and nontoxic, thus addressing safety concerns associated with chemical catalysts. Enzymes typically operate under moderate temperatures and at atmospheric pressure, thus reducing the energy demands associated with conventional chemical processing. Enzymes are also typically highly selective as regards their chemical substrate, and approaches such as rational enzyme engineering and directed mutagenesis continue to expand the range of reactions that can be undertaken. Enzymatic catalysis thus provides many advantages compared to conventional chemical approaches. 
     Whilst enzymatic catalysis has great potential, its use in industry has been limited. One key difficulty has been in the provision of robust systems for recycling enzyme cofactors. 
     Cofactors are non-protein chemical compounds that play an essential role in many enzyme catalysed biochemical reactions, and which typically act to transfer chemical groups between enzymes. Cofactors are also sometimes known as “co-substrates” reflecting their processing by an enzyme in the course of its catalysing of its primary reaction. By way of illustration, a redox enzyme which catalyses an oxidation reaction of a reagent to produce a product may couple that oxidation to the reduction of a cofactor as an electron sink. In this case, the overall reaction catalysed by the enzyme may be represented as: 
       reduced reagent+oxidised cofactor→oxidised product+reduced cofactor
 
     Similarly, enzymes which catalyse the reduction of a reagent to produce a product typically couple that reduction with the oxidation of a cofactor as a source of electrons or reducing equivalents such as hydride ions: 
       oxidised reagent+reduced cofactor→reduced product+oxidised cofactor
 
     Biological use of cofactors is not limited to simple redox reactions as represented above but is also involved in more complex reactions such as atom insertion reactions, rearrangement reactions, etc. 
     There are many cofactors known, including those that occur in nature and synthetic compounds which are designed to have specific properties such as precisely tuned redox potentials, improved stability, etc. One key natural cofactor is nicotinamide adenine dinucleotide (NAD). In vivo, reduction of the oxidised cofactor (NAD + ) by hydride transfer from a reductant yields the reduced cofactor (NADH). The reduced cofactor can be coupled to enzymatic reduction of an oxidised centre (typically an oxidised carbon centre) to yield a reduced centre, in accordance with the general schemes shown above. 
     A difficulty which has limited the industrial exploitation of enzymes which rely on cofactors to catalyse relevant reactions is providing sufficient cofactor for the enzyme to use. One option is to provide the cofactor in superstoichiometric quantities relative to the reagent at issue. However, the high cost and typically low stability of reduced cofactor molecules means that this is not a viable approach. It is thus necessary that systems for regenerating cofactor molecules in their desired form (i.e., recycling the cofactor molecule) are used. 
     Current industrial practices for enzymatic NAD(P)H recycling rely on a superstoichiometric quantity of a carbon-based sacrificial reductant. For example, NAD(P)H is a cofactor for many enzymes used in reduction reactions. The reduction of the reagent to produce the desired product is linked to the enzymatic oxidation of NAD(P)H to NAD(P)+. To regenerate NAD(P)H for subsequent enzyme cycles, glucose or isopropanol is typically used as a sacrificial reductant. However, this leads to additional cost, generates waste products, and requires additional downstream processing steps of the desired product. It is also atom inefficient. In view of these difficulties, there have been extensive efforts in recent years to provide improved methods for recycling NAD(P)H, and some recent developments are promising. 
     A second important class of cofactor in vivo are the flavins. A variety of flavin cofactors exist, including flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin. Many enzymes which couple industrially useful reactions to flavin processing exist. However, unlike the situation for NAD(P)+/NAD(P)H dependent enzymes, widespread industrial exploitation of such flavin-utilising enzymes has been prevented by a lack of suitable means for recycling the flavin cofactor. 
     Some attempts to provide flavin recycling systems have proposed electrochemical reduction of oxidised flavin cofactors. However, in practice such systems are associated with many technical drawbacks. Electrochemical systems are typically difficult to incorporate into industrially relevant contexts. The electrodes used typically require costly materials such as precious metals and highly-processed carbon materials, the production of which is associated with environmental issues and is energy inefficient. Electrochemical side-reaction of the reagents or products may limit the overall efficiency of the reaction process. Furthermore, electrodes are typically subject to fouling by reaction by-products, the reagents or products themselves, or other impurities that may be present. 
     Accordingly, there is a pressing need for improved methods of recycling flavin cofactors. In particular, there is a need for methods for reducing an oxidised flavin cofactor such that the reduced cofactor thus obtained can be used in downstream enzyme-catalysed reactions. There is specifically a need for methods that avoid the requirement for expensive or dangerously reactive chemical reagents; that are atom efficient; that avoid difficulties associated with electrochemical processing of reagents; that do not rely on the use of expensive sacrificial reductants; and/or that avoid the generation of by-products. The present invention aims to address some or all of these problems. 
     SUMMARY OF THE INVENTION 
     The inventors have surprisingly found that it is possible to use hydrogen as a reductant in order to reduce an oxidised flavin cofactor. The hydrogen is processed by a hydrogen-cycling enzyme such as a hydrogenase. Surprisingly, the inventors have found that hydrogenases which do not interact with flavin cofactors in vivo can still enzymatically reduce such cofactors using the electrons generated by hydrogen oxidation. The process is environmentally clean as the hydrogenase enzymes used are renewable and biodegradable. Unlike conventional hydrogen oxidation catalysts such as precious metals, the reactions catalysed by hydrogenases are highly specific and do not lead to unwanted side-reaction. Hydrogenases operate under readily accessible conditions and are amenable to exploitation in industrial contexts such as known reactors (including, but not limited to, hydrogenation reactors). They are not susceptible to fouling by reagent, product or cofactor molecules. By utilising hydrogen as the reductant, the reaction is atom efficient. 
     Accordingly, the invention provides a method of producing a reaction product, comprising:
     i) contacting an oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the oxidised flavin cofactor is reduced to form a reduced flavin cofactor; and   ii) contacting the reduced flavin cofactor and a reactant with a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof under conditions such that (a) the oxidised flavin cofactor is regenerated; and (b) the second polypeptide catalyses the formation of the reaction product from the reactant.   

     Preferably, the method comprises:
     i) contacting an oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the first polypeptide oxidises the hydrogen to produce protons and electrons, and transfers the electrons to the oxidised flavin cofactor, thereby reducing the oxidised flavin cofactor to form a reduced flavin cofactor; and   ii) contacting the reduced flavin cofactor and a reactant with a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof under conditions such that (a) electrons are transferred from the reduced flavin cofactor to an electron acceptor and/or hydride ions are transferred from the reduced flavin cofactor to a hydride ion acceptor; (b) the oxidised flavin cofactor is regenerated; and (c) the second polypeptide catalyses the formation of the reaction product from the reactant.   

     Preferably, the provided method is repeated multiple times thereby recycling the cofactor. 
     This method is illustrated schematically in  FIG.  3   . 
     Typically, the oxidised cofactor is selected from flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), riboflavin, or a derivative thereof. Preferably, the oxidised cofactor is flavin mononucleotide (FMN) or a derivative thereof or flavin adenine dinucleotide (FAD) or a derivative thereof. 
     Usually, the first polypeptide transfers the electrons to the oxidised flavin cofactor via an intramolecular electronically-conducting pathway. The intramolecular electronically-conducting pathway often comprises a series of [FeS] clusters. Preferably, reduction of the oxidised flavin cofactor takes place at an [FeS] cluster within the first polypeptide. 
     Preferably, the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction. 
     Preferably, the first polypeptide is an uptake hydrogenase or a hydrogen-sensing hydrogenase. Preferably, the first polypeptide is a hydrogenase of class 1 or 2b. References to hydrogenase classes such as class 1 and class 2b refer to the established Vignais classification scheme described by Vignais and Billoud, Chem. Rev. 2007, 107, 4206-4272, which is known to those skilled in the art. 
     Preferably, the first polypeptide is selected from or comprises:
         i) the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of  Ralstonia eutropha  membrane-bound hydrogenase moiety (SEQ ID NOs: 5 and/or 6 and/or 7) or an amino acid sequence having at least 60% homology therewith;   iv) the amino acid sequence of  Ralstonia eutropha  regulatory hydrogenase moiety (SEQ ID NOs: 8 and/or 9) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   vi) the amino acid sequence of  Hydrogenovibrio marinus  hydrogenase (SEQ ID NOs: 12 and/or 13) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   viii) the amino acid sequence of  Alteromonas macleodii  hydrogenase (SEQ ID NOs: 16 and/or 17) or an amino acid sequence having at least 60% homology therewith;   ix) the amino acid sequence of  Allochromatium vinosum  membrane bound hydrogenase (SEQ ID NOs: 18 and/or 19) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   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 therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Preferably, in one embodiment, the second polypeptide comprises the electron acceptor and/or hydride ion acceptor. Typically, the second polypeptide comprises a prosthetic group for oxidising the reduced flavin cofactor. This method is illustrated schematically in  FIG.  3   . 
     Preferably, in another embodiment, the electron acceptor and/or hydride ion acceptor comprises a molecular substrate. Typically, the molecular substrate comprises O 2 . This method is illustrated schematically in  FIG.  4   . 
     Preferably, the second polypeptide is a flavin-accepting oxidoreductase, or a functional fragment, derivative or variant thereof. Preferably, the second polypeptide is a flavin-dependent oxidoreductase, or a functional fragment, derivative or variant thereof. Typically, the second polypeptide is a monooxygenase, halogenase, nitro reductase, ene-reductase, peroxidase, or haloperoxidase, or a functional fragment, derivative or variant thereof. Often, the second enzyme is selected from Enzyme Commission (EC) classes 1.1.98.; 1.3.1.; 1.5.1.; 1.6.99.; 1.7.1.; 1.7.99.; 1.11.1.; 1.11.2.; 1.14.14.; and 1.14.99.; or a functional fragment, derivative or variant thereof. 
     In one embodiment, first polypeptide and/or the second polypeptide are preferably in solution. In another embodiment, the first polypeptide and/or the second polypeptide is immobilised on a solid support. The first polypeptide and the second polypeptide may be attached together, as illustrated schematically in  FIG.  5   . The first polypeptide and/or the second polypeptide may be comprised in a biological cell. 
     Preferably, the method is carried out under aerobic conditions. Typically, the method is carried out at a temperature of from about 20° C. to about 80° C. 
     Also provided is a method of reducing an oxidised flavin cofactor, comprising:
         contacting the oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the oxidised flavin cofactor is reduced to form a reduced flavin cofactor;   wherein the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction.       

     This method is illustrated schematically in  FIG.  1   . 
     Preferably, said method further comprises the re-oxidation of the reduced flavin cofactor to regenerate the oxidised flavin cofactor. Typically, the reduction and reoxidation steps are repeated multiple times thereby recycling the cofactor. 
     This method is illustrated schematically in  FIG.  2   . 
     Preferably, in such methods, the oxidised flavin is as defined herein; the first polypeptide is as defined herein; the method is conducted under conditions as described herein; and/or the first polypeptide is immobilised on a solid support or is comprised in a biological cell. 
     The invention also provides a system for reducing an oxidised flavin cofactor, comprising:
         a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof,   the oxidised flavin cofactor; and   molecular hydrogen ( 1 H 2 ) or an isotope thereof;
 
wherein the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction.
       

     Also provided is a system for producing a reaction product, comprising:
         a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof;   a flavin cofactor;   a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof,   molecular hydrogen ( 1 H 2 ) or an isotope thereof, and   a reactant for conversion to said reaction product.       

     Preferably, in the systems provided herein, the flavin cofactor is as defined herein; the first polypeptide is as defined herein; and/or the second polypeptide if present is as defined herein. 
     Without being bound by theory, the inventors believe that, in the methods of the invention, electrons are typically abstracted from hydrogen by the first polypeptide and used to reduce the oxidised flavin cofactor. The reduction takes places at the first polypeptide. The product of the reduction is thus a reduced flavin cofactor. In embodiments of the invention in which the reduced flavin cofactor is exploited in the production of a reaction product from a reactant, the reduced flavin cofactor is typically oxidised at a second polypeptide. For example, the reduced flavin may be oxidised by the second polypeptide, e.g. at an active site or prosthetic group of the second polypeptide. The reduced flavin may be oxidised by an electron or hydride ion acceptor such as O 2 . In these embodiments, the second polypeptide catalyses the formation of the product by reaction of the reagent with the oxidised flavin cofactor. 
     The second polypeptide thus catalyses the conversion of the reactant to the product. The reaction catalysed by the second polypeptide may for example be a reduction reaction, e.g. the reduction of a C═C double bond to a C—C single bond. Such reactions are catalysed by enzymes such as ene reductases. The reaction catalysed by the second polypeptide may be an atom insertion reaction such as the insertion of an oxygen atom into a chemical bond. Such reactions are catalysed by enzymes such as monooxygenases and peroxidases. The reaction may be a halogenation reaction. Such reactions are catalysed by enzymes such as halogenases and haloperoxidases. The reaction may be the reduction of a nitro group e.g. a nitroaromatic group, or a quinone; such reactions are catalysed by enzymes such as nitroreductases. 
     DESCRIPTION OF THE SEQUENCE LISTING 
     
         
         SEQ ID NO: 1—the amino acid sequence of the  Escherichia coli  hydrogenase 1 (large subunit). 
         SEQ ID NO: 2—the amino acid sequence of the  Escherichia coli  hydrogenase 1 (small subunit). 
         SEQ ID NO: 3—the amino acid sequence of the  Escherichia coli  hydrogenase 2 (large subunit). 
         SEQ ID NO: 4—the amino acid sequence of the  Escherichia coli  hydrogenase 2 (small subunit). 
         SEQ ID NO: 5—the amino acid sequence of the  Ralstonia eutropha  membrane-bound hydrogenase moiety (HoxG). 
         SEQ ID NO: 6—the amino acid sequence of the  Ralstonia eutropha  membrane-bound hydrogenase moiety (HoxK). 
         SEQ ID NO: 7—the amino acid sequence of the  Ralstonia eutropha  membrane-bound hydrogenase moiety (HoxZ). 
         SEQ ID NO: 8—the amino acid sequence of the  Ralstonia eutropha  regulatory hydrogenase moiety (HoxB). 
         SEQ ID NO: 9—the amino acid sequence of the  Ralstonia eutropha  regulatory hydrogenase moiety (HoxC). 
         SEQ ID NO: 10—the amino acid sequence of the  Aquifex  aeolicus hydrogenase 1 (large subunit). 
         SEQ ID NO: 11—the amino acid sequence of the  Aquifex  aeolicus hydrogenase 1 (small subunit). 
         SEQ ID NO: 12—the amino acid sequence of the  Hydrogenovibrio marinus  hydrogenase (large subunit). 
         SEQ ID NO: 13—the amino acid sequence of the  Hydrogenovibrio marinus  hydrogenase (small subunit). 
         SEQ ID NO: 14—the amino acid sequence of the  Thiocapsa roseopersicina  hydrogenase HupL. 
         SEQ ID NO: 15—the amino acid sequence of the  Thiocapsa roseopersicina  hydrogenase HupS. 
         SEQ ID NO: 16—the amino acid sequence of the  Alteromonas macleodii  hydrogenase small subunit. 
         SEQ ID NO: 17—the amino acid sequence of the  Alteromonas macleodii  hydrogenase large subunit. 
         SEQ ID NO: 18—the amino acid sequence of the  Allochromatium vinosum  Membrane Bound Hydrogenase large subunit. 
         SEQ ID NO: 19—the amino acid sequence of the  Allochromatium vinosum  Membrane Bound Hydrogenase small subunit. 
         SEQ ID NO: 20—the amino acid sequence of the  Salmonella enterica  serovar  Typhimurium  LT2 nickel-iron hydrogenase 5 Large subunit. 
         SEQ ID NO: 21—the amino acid sequence of the  Salmonella enterica  serovar  Typhimurium  LT2 nickel-iron hydrogenase 5 Small subunit. 
         SEQ ID NO: 22—the amino acid sequence of the  Escherichia coli  cytochrome HyaC. 
         SEQ ID NO: 23—the amino acid sequence of the  Desulfovibrio vulgaris  Miyazaki F hydrogenase (large subunit). 
         SEQ ID NO: 24—the amino acid sequence of the  Desulfovibrio vulgaris  Miyazaki F hydrogenase (small subunit). 
         SEQ ID NO: 31—the amino acid sequence of Chromate Reductase, ‘TsOYE’ from  Thermus scotoductus.    
         SEQ ID NO: 32—the amino acid sequence of NADPH Dehydrogenase 1, ‘OYE-1 ’, Saccharomyces pastorianus.    
         SEQ ID NO: 33: the amino acid sequence of NADPH Dehydrogenase 2, ‘OYE-2’,  Saccharomyces cerevisiae  strain ATCC 204508 S288c. 
         SEQ ID NO: 34: the amino acid sequence of NADPH Dehydrogenase, ‘YqjM’,  Bacillus subtilis.    
         SEQ ID NO: 35: the amino acid sequence of Xenobiotic Reductase A, ‘XenA’,  Pseudomonas putida.    
         SEQ ID NO: 36: the amino acid sequence of NADPH dehydrogenase, ‘FOYE-1’, ‘Ferrovum’ strain JA12. 
         SEQ ID NO: 37: the amino acid sequence of Oxidored_FMN domain-containing protein, ‘MgER’, Meyerozyma  guilliermondii.    
         SEQ ID NO: 38: the amino acid sequence of Oxidored_FMN domain-containing protein, ‘CER’,  Clavispora  ( Candida )  lusitaniae.    
         SEQ ID NO: 39: the amino acid sequence of Tryptophan 2-Halogenase, ‘CmdE’, Chondromyces crocatus. 
         SEQ ID NO: 40: the amino acid sequence of Tryptophan 5-Halogenase, ‘PyrH’,  Streptomyces  rugosporus. 
         SEQ ID NO: 41: the amino acid sequence of Flavin-Dependent Tryptophan Halogenase, ‘RebH’,  Lentzea aerocolonigenes  ( Lechevalieria aerocolonigenes ) ( Saccharothrix aerocolonigenes ). 
         SEQ ID NO: 42: the amino acid sequence of Flavin-Dependent Tryptophan Halogenase, ‘PrnA’,  Pseudomonas fluorescens.    
         SEQ ID NO: 43: the amino acid sequence of Thermophilic Tryptophan Halogenase, ‘Th-Hal’,  Streptomyces  violaceusnige. 
         SEQ ID NO: 44: the amino acid sequence of Tryptophan 6-Halogenase, ‘SttH’,  Streptomyces toxytricini.    
         SEQ ID NO: 45: the amino acid sequence of KtzQ, ‘KtzQ’, Kutzneria sp. 744. 
         SEQ ID NO: 46: the amino acid sequence of Monodechloroaminopyrrolnitrin halogenase, ‘PrnC’,  Pseudomonas fluorescens.    
         SEQ ID NO: 47: the amino acid sequence of FADH2-dependent halogenase, ‘PitA’,  Pseudomonas protegens  Pf-5. 
         SEQ ID NO: 48: the amino acid sequence of Halogenase, ‘PltM’,  Pseudomonas fluorescens  (strain ATCC BAA-477 NRRL B-23932 Pf-5). 
         SEQ ID NO: 49: the amino acid sequence of Flavin-Dependent Halogenase, ‘Clz5’,  Streptomyces  sp. CNH-287. 
         SEQ ID NO: 50: the amino acid sequence of Pyrrole Halogenase, ‘Bmp2’,  Pseudoalteromonas piscicida.    
         SEQ ID NO: 51: the amino acid sequence of Non-Heme Halogenase, ‘Rdc2’, Metacordyceps chlamydosporia (Pochonia chlamydosporia). 
         SEQ ID NO: 52: the amino acid sequence of Tryptophan 6-Halogenase, ‘BorH’, uncultured bacteria. 
         SEQ ID NO: 53: the amino acid sequence of Styrene Monooxygenase, ‘StyA’,  Pseudomonas  sp. 
         SEQ ID NO: 54: the amino acid sequence of 4-Nitrophenol 2-Monooxygenase Oxygenase Component, ‘PheA1 ’, Rhodococcus erythropolis  ( Arthrobacter picolinophilus ). 
         SEQ ID NO: 55: the amino acid sequence of 4-Hydroxyphenylacetate 3-Monooxygenase Oxygenase Component, ‘HpaB’,  Klebsiella oxytoca.    
         SEQ ID NO: 56: the amino acid sequence of Chlorophenol Monooxygenase, ‘HadA’,  Ralstonia pickettii  ( Burkholderia pickettii ). 
         SEQ ID NO: 57: the amino acid sequence of Tetrachlorobenzoquinone Reductase, ‘PcpD’,  Sphingobium chlorophenolicum.    
         SEQ ID NO: 58: the amino acid sequence of 2-Methyl-6-ethyl-4-monooxygenase Oxygenase Component, ‘MeaX’,  Sphingobium baderi.    
         SEQ ID NO: 59: the amino acid sequence of Alkanesulfonate Monooxygenase, ‘SsuD’,  Escherichia coli  (strain K12). 
         SEQ ID NO: 60: the amino acid sequence of p-Hydroxyphenylacetate 3-Hydroxylase, Oxygenase Component, ‘C2-HpaH’,  Acinetobacter baumannii  (SEQ ID NO:60). 
         SEQ ID NO: 61: the amino acid sequence of FADH(2)-Dependent Monooxygenase, ‘TftD’,  Burkholderia cepacia  ( Pseudomonas cepacia ). 
         SEQ ID NO: 62: the amino acid sequence of 4-Nitrophenol 2-Monooxygenase, Oxygenase Component, ‘NphA1’,  Rhodococcus  sp. 
         SEQ ID NO: 63: the amino acid sequence of Putative dehydrogenase/oxygenase subunit, ‘VpStyA1 ’, Variovorax paradoxus  (strain EPS). 
         SEQ ID NO: 64: the amino acid sequence of Oxygenase, ‘RoIndA1’ {from styA1 gene},  Rhodococcus opacus  ( Nocardia opaca ). 
         SEQ ID NO: 65: the amino acid sequence of Smoa_sbd domain-containing protein, ‘AbIndA’,  Acinetobacter baylyi  (strain ATCC 33305 BD413 ADPI). 
         SEQ ID NO: 66: the amino acid sequence of 2,5-Diketocamphane 1,2-Monooxygenase 1, ‘CamP’,  Pseudomonas putida  ( Arthrobacter siderocapsulatus ). 
         SEQ ID NO: 67: the amino acid sequence of 3,6-Diketocamphane 1,6-Monooxygenase, ‘CamE36’,  Pseudomonas putida  ( Arthrobacter siderocapsulatus ). 
         SEQ ID NOs: 68 and 69: the amino acid sequence of Alkanal monooxygenase, alpha and beta chain, ‘LuxAB’,  Vibrio harveyi  ( Beneckea harveyi ); SEQ ID NOs: 70 and 71: the amino acid sequence of Alkanal monooxygenase, alpha and beta chain, ‘LuxAB’,  Photorhabdus luminescens  (Xenorhabdus  luminescens ). 
         SEQ ID NO: 72: the amino acid sequence of Alkane Monooxygenase, ‘LadA’,  Geobacillus thermodenitrificans.    
         SEQ ID NO: 73: the amino acid sequence of EDTA Monooxygenase, ‘EmoA’,  Chelativorans multitrophicus.    
         SEQ ID NO: 74: the amino acid sequence of Isobutylamine N-hydroxylase, ‘IBAH’,  Streptomyces viridifaciens.    
         SEQ ID NO: 75: the amino acid sequence of ActVA 6 Protein, ‘ActVA-Orf6’,  Streptomyces coelicolor.    
         SEQ ID NO: 76: the amino acid sequence of Pyrimidine Monooxygenase, ‘ RutA’, Escherichia coli  (strain K12). 
         SEQ ID NO: 77: the amino acid sequence of p-Hydroxyphenylacetate 2-Hydroxylase Reductase Component, ‘C1-HpaH’,  Acinetobacter baumannii.    
         SEQ ID NO: 78: the amino acid sequence of FMN_red Domain-Containing Protein, ‘YdhA’,  Bacillus subtilis  subsp. natto (strain BEST195). 
         SEQ ID NO: 79: the amino acid sequence of NAD(P)H-Flavin Reductase, ‘Fre’,  Escherichia coli  (strain K12). 
         SEQ ID NO: 80: the amino acid sequence of 4-hydroxyphenylacetate 3-monooxygenase reductase component, ‘HpaC’,  Escherichia coli.    
         SEQ ID NO: 81: the amino acid sequence of nitroreductase ‘NfsB’,  Escherichia coli  (strain K12). 
         SEQ ID NO: 82: the amino acid sequence of vanadium chloroperoxidase ‘CPO’ or ‘CiVHPO’, Curvularia  inaequalis.    
         SEQ ID NO: 83: the amino acid sequence of aromatic unspecified peroxygenase ‘APO1’ or ‘AaeUPO’,  Agrocybe aegerita  (Blackpoplar mushroom) ( Agaricus aegerita ). 
       
    
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1    shows a schematic diagram of the production of a reduced flavin cofactor from an oxidised flavin cofactor in accordance with the methods of the invention. 
         FIG.  2    shows a schematic diagram of the recycling of a flavin cofactor in accordance with the methods of the invention. 
         FIG.  3    shows a schematic diagram of the production of a product from a reactant in accordance with the methods of the invention, wherein electrons and/or hydride ions are transferred from the reduced flavin cofactor to an electron and/or hydride ion acceptor comprised within the second polypeptide. 
         FIG.  4    shows a schematic diagram of the production of a product from a reactant in accordance with the methods of the invention, wherein electrons and/or hydride ions are transferred from the reduced flavin cofactor to a molecular substrate (in this case shown in non-limiting form as O 2 ). 
         FIG.  5    shows a schematic diagram of the production of a product from a reactant in accordance with the methods of the invention wherein the first polypeptide and second polypeptide are attached together, for example in the form of a fusion protein or by being cross-linked together. 
         FIG.  6    shows a schematic of two-electron flavin reduction by Hyd1. H 2  oxidation at the [NiFe] active site provides 2 electrons that are transferred to the surface of the protein via FeS clusters. Figure prepared using PyMOL™ 2.3.4 (PDB: 6FPW). 
         FIG.  7    shows results of an activity assay for H 2 -driven Hyd1 reduction of flavin measured by in situ UV-visible spectroscopy. A) Hyd1 reducing FMN. B) Hyd1 reducing FAD. C) Calculated [FMN] based on λ max =445 nm (ε=12.50 mM −1  cm −1 ). D) Calculated [FAD] based on λ max =450 nm (ε=11.30 mM −1  cm −1 ). Reaction conditions: General Procedure A in Tris-HCl buffer (50 mM, pH 8.0, 25° C.). Results are described in example 1. 
         FIG.  8 A  shows Hyd1-catalysed flavin reduction at different temperatures. Reaction conditions: General Procedure A in phosphate buffer (50 mM, pH 8.0). Conversion was calculated after 30 min using UV-visible spectroscopy. Results are described in example 1.  FIG.  8 B  shows Hyd1-catalysed flavin reduction at different temperatures. 
       
         
           
             
               
                 Conversion 
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                 relative 
                 ⁢ 
                     
                 to 
                 ⁢ 
                     
                 standard 
               
               = 
               
                 
                   
                     Conversion 
                     ⁢ 
                         
                     at 
                     ⁢ 
                         
                     temp 
                   
                   
                     Conversion 
                     ⁢ 
                         
                     at 
                     ⁢ 
                         
                     25 
                     ⁢ 
                     ° 
                     ⁢ 
                         
                     
                       C 
                       . 
                     
                   
                 
                 × 
                 100 
                 ⁢ 
                 
                   % 
                   . 
                 
               
             
           
         
       
       The FMN 25-50° C. bars Conversion at 25° C. represent the average of relative conversions calculated from duplicate experiments, with the range represented as error bars. Reaction conditions: General Procedure A (Supporting Information, Example 1) in phosphate buffer (50 mM, pH 8.0). Conversion was calculated after 30 min using UV-visible spectroscopy. 
         FIG.  9    shows current applications and methods of flavin recycling. 
         FIGS.  10  to  12    show the results of control experiments, discussed in example 1.  FIG.  10    shows background flavin reduction in absence of H 2 .  FIG.  11    shows background flavin reduction in absence of Hyd1.  FIG.  12    shows background flavin reduction in absence of Hyd1. 
         FIG.  13    shows UV-visible spectra of FMN and FMNH 2  produced by Hyd1 under H 2  or sodium dithionite (gray). Results discussed in example 1. 
         FIG.  14    shows exemplary chiral-phase GC-FID traces of enzymatic H 2 -driven reduction of ketoisophorone to (R)-levodione. Results discussed in example 1. 
         FIG.  15    shows exemplary GC-FID traces of enzymatic  E. coli  Hyd1 H 2 -driven reduction of 4-phenyl-3-buten-2-one (5) reduction to 4-phenyl-2-butanone (6). Results discussed in example 2. 
         FIG.  16    shows exemplary GC-FID traces of enzymatic  E. coli  Hyd1 H 2 -driven reduction of dimethyl itaconate (3) to dimethyl (R)-methyl succinate (4). Results discussed in example 2. 
         FIG.  17    shows  1 H NMR spectra of compound standards in H 2 O/D 2 O, run with water suppression.  FIG.  17 A  shows full speactrum; B shows zoom-in of the aromatic proton region. Results described in example 3. 
         FIG.  18    shows activity assay results for  E. coli  Hyd2 catalysed reduction of flavins. Results described in example 4. 
         FIG.  19    shows activity assay results for  Desulfovibrio vulgaris  Miyazaki F catalysed reduction of flavins. Results described in example 4. 
         FIG.  20    shows the specific activity of  E. coli  Hyd1 for FAD reduction measured at different mixtures of water:solvent. A: measurements taken at different mixtures of water:DMSO. B: measurements taken at different mixtures of water:acetonitrile. Results described in example 5. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Methods of the Invention 
     As described above, the invention provides a method of producing a reaction product, comprising:
         i) contacting an oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the oxidised flavin cofactor is reduced to form a reduced flavin cofactor; and   ii) contacting the reduced flavin cofactor and a reactant with a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof under conditions such that the oxidised flavin cofactor is regenerated; and (b) the second polypeptide catalyses the formation of the reaction product from the reactant.       

     In the first step of the method above, an oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof are contacted with a first polypeptide. The first polypeptide typically oxidises the molecular hydrogen to produce protons and electrons. The first polypeptide is described in more detail herein. The electrons generated by the oxidation of the molecular hydrogen preferably reduce the oxidised flavin cofactor to form a reduced flavin cofactor. This is shown schematically in  FIG.  1   . Flavin cofactors suitable for use in the invention are described below. 
     Isotopes of molecular hydrogen suitable for use in the invention include  2 H 2  and  3 H 2 . Mixed isotopes (e.g.  1 H 2 H and  1 H 3 H) are also embraced. Preferably, in the invention, the hydrogen is  1 H 2 . It will be apparent that, as used herein, organic molecules such as glucose, formate, and ethanol, isopropanol, etc, are not sources of molecular hydrogen. 
     The molecular hydrogen is typically provided in the form of a gas. The gas may be mixed with an aqueous solution in which the first polypeptide and other reaction components such as the second polypeptide and reactant are present. At 1 bar H 2  the solubility of H 2  in water is 0.8 mM. In other words, by providing the hydrogen in the form of molecular hydrogen gas, the first polypeptide typically operates under concentrations of 0.8 mM hydrogen. Other pressures may also be used. For example, the gas pressure in the reaction vessel may be from 0.01 to about 100 bar, such as from 0.1 to 10 bar, e.g. from about 0.2 to about 5 bar, e.g. from 0.5 to 2 bar, such as approximately 1 bar. 
     The hydrogen may be provided as a mixture of hydrogen and other gases such as CO, CO 2 , air, O 2 , N 2 , Ar, etc. When provided as a mixture, the mixture may comprise from about 0.1% to about 99% hydrogen, such as from 1% to about 95%, e.g. from about 2% to about 10% H 2 . 
     The hydrogen used in the invention may be of any suitable purity. For example, hydrogen of 99% purity or greater (e.g. 99.9%, 99.99% or 99.999%) may be used when it is important to control impurity levels in the final product mixture. In other aspects, lower purity hydrogen may be used when it is not so important to control impurity levels in the final product mixture. For example, relatively low purity hydrogen may be provided in the form of “syngas”. Syngas produced by coal gasification generally is a mixture of 30 to 60% carbon monoxide, 25 to 30% hydrogen, 5 to 15% carbon dioxide, and 0 to 5% methane, and may optionally comprise lesser amount of other gases also. 
     For avoidance of doubt, the molecular hydrogen may also be provided in the form of a solution (e.g. an aqueous solution, e.g. comprising buffer salts as described in more detail here) in which molecular hydrogen is dissolved. 
     The molecular hydrogen may be provided from any suitable source, such as a gas cylinder. Alternatively, the molecular hydrogen or isotope thereof can be produced in situ e.g. by electrolysis of water. 
     In the second step of the method above, the reduced flavin cofactor generated in the first step and a reactant are contacted with a second polypeptide. The second polypeptide is described in more detail herein. Usually, electrons and/or hydride ions are transferred from the reduced flavin cofactor to an acceptor therefor. The acceptor may be comprised in the second polypeptide, for example the acceptor may comprise an active site of the second polypeptide or may comprise a prosthetic group comprised in the second polypeptide. The acceptor may comprise a molecular substrate. The molecular substrate is typically exogenous, i.e. is not part of the second polypeptide. Preferred substrates for use in this aspect of the invention include O 2 . 
     The transfer of electrons and/or hydride to the acceptor generates oxidised flavin cofactor. Accordingly, the oxidation of the flavin cofactor leads to the regeneration of the oxidised flavin cofactor used in the provided methods. The process is typically as shown schematically in  FIGS.  2  and  3   . The second polypeptide catalyses the formation of the reaction product from the reactant. Suitable reactants and the products thereby produced are described in more detail herein. 
     Accordingly, the method of producing a reaction product preferably comprises:
         i) contacting an oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the first polypeptide oxidises the hydrogen to produce protons and electrons, and transfers the electrons to the oxidised flavin cofactor, thereby reducing the oxidised flavin cofactor to form a reduced flavin cofactor; and   ii) contacting the reduced flavin cofactor and a reactant with a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof under conditions such that (a) electrons are transferred from the reduced flavin cofactor to an electron acceptor and/or hydride ions are transferred from the reduced flavin cofactor to a hydride ion acceptor, (b) the oxidised flavin cofactor is regenerated; and (c) the second polypeptide catalyses the formation of the reaction product from the reactant.       

     In preferred embodiments of the invention, method steps (i) and (ii) are repeated multiple times thereby recycling the cofactor. For avoidance of doubt, by recycling the cofactor, it is meant that a single cofactor molecule can be reduced in a method of the invention from the oxidised form to a reduced form. The reduced cofactor can subsequently transfer electrons and/or a hydride ion to an electron acceptor and/or hydride acceptor as described above, thus oxidising the cofactor, which can be re-reduced as described. The repeated reduction and oxidation of the cofactor corresponds to recycling of the cofactor. The net result is that the cofactor itself is not spent. 
     In methods of the invention which involve recycling the cofactor, each cofactor molecule is typically recycled as defined herein at least 10 times, such as at least 50 times, e.g. at least 100 times, more preferably at least 1000 times e.g. at least 10,000 times or at least 100,000 times, such as at least 1,000,000 times. Accordingly, in methods of the invention, the turnover number (TN) is typically at least 10, such as at least 100, more preferably at least 1000 e.g. at least 10,000 or at least 100,000, such as at least 1,000,000. As those skilled in the art will appreciate, the TN indicates the number of moles of product generated per mole of cofactor, and is thus a measure of the number of times each cofactor molecule is used. 
     Enzyme turnover can be calculated in a number of ways. The Total Turnover Number (TTN, also known as the TON) is a measure of the number of moles of product per mole of enzyme (specifically per mole of the first polypeptide). As those skilled in the art will appreciate, the TTN thus indicates the number of times that the enzyme (i.e. the first polypeptide) has turned over. Preferably, the TTN is at least 10, such as at least 100, more preferably at least 1000 e.g. at least 10,000 or at least 100,000, such as at least 1,000,000, preferably at least 10 7  such as at least 10 8 , e.g. at least 10 9 . 
     The Turnover Frequency (TOF) is a measure of the number of moles of product generated per second per mole of enzyme (first polypeptide) present. Accordingly, in methods of the invention for the production of a reduced cofactor, the TOF indicates the number of moles of reduced cofactor generated per second per mole of first polypeptide. Accordingly, the TOF is identified with the number of catalytic cycles undertaken by each enzyme molecule per second. Preferably, in the methods of the invention, the first polypeptide has a TOF of 0.1 to 1000 s −1 , more preferably 1 to 100 s −1  such as from about 10 to about 50 s −1 . 
     Flavin Cofactor 
     The methods of the invention involve the reduction and optional re-oxidation of an oxidised cofactor. 
     Preferably, the oxidised cofactor is a flavin cofactor. Flavin cofactors exist in the oxidised form (FI) and the reduced form (FIH2). The oxidized form acts as an electron acceptor, by being reduced. The reduced form, in turn, can act as a reducing agent, by being oxidized. 
     Preferably, the flavin cofactor is based on isoalloxazine. Typically, the flavin cofactor is a compound of Formula (I): 
     
       
         
         
             
             
         
       
     
     Typically, the compound of Formula (I) is a compound of Formula (Ia) or Formula (Ib), preferably (Ia): 
     
       
         
         
             
             
         
       
     
     In formula (I), (Ia) and (Ib):
         X is CH or N;   R 1  and R 2  are each independently selected from hydrogen, C 1-4  alkyl, halogen, —OH, —SH, nitro, —NR 10 R 11  and —N + R 10 R 11 R 12 ; and when R 1  and/or R 2  is an alkyl group the alkyl group is independently unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, —OH, —SH, and nitro, —NR 10 R 11  and —N + R 10 R 11 R 12 ;   R 4  is selected from hydrogen and C 1-4  alkyl; and when R 4  is an alkyl group the alkyl group is unsubstituted or is substituted by 1, 2 or 3 substituents independently selected from halogen, —OH, and nitro, —NR 10 R 11  and —N + R 10 R 11 R 12 ;   R 10 , R 11  and R 12  are each independently H or methyl;   R 5  is H or is an alkylene group and is attached to X to form an optionally substituted cyclic group;   R is an optionally substituted alkyl group;   and   R 3  is absent or R 3  is an alkylene group and is attached to R to form a cyclic group.       

     A C 1-4  alkyl group is a linear or branched alkyl group containing from 1 to 4 carbon atoms. 
     A C 1-4  alkyl group is often a C 1-3  alkyl group or a C 1-2  alkyl group. Examples of C 1  to C 4  alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl, often methyl is preferred. Where two alkyl groups are present, the alkyl groups may be the same or different. An alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from an alkane. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. Typically an alkylene group is a C 1  to C 4  alkylene group such as methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene and tert-butylene. Where two alkylene groups are present, the alkylene groups may be the same or different. An alkyl or alkylene group may be unsubstituted or substituted, e.g. by one or more, e.g. 1, 2, 3 or 4 substituents selected from halogen, —OH, —SH, and nitro, —NR 10 R 11  and -N+R 10 R 11 R 12 , where R 10-12  are as defied herein. The substituents on a substituted alkyl or alkylene group are typically themselves unsubstituted. Where more than one substituent is present, these may be the same or different. 
     A cyclic group is typically a 4- to 10-membered carbocyclic group or a 4-10 membered heterocyclic group. A carbocyclic group is a cyclic hydrocarbon. A carbocyclic group may be saturated or partially unsaturated, but is typically saturated. A 4- to 10-membered partially unsaturated carbocyclic group is a cyclic hydrocarbon containing from 4 to 10 carbon atoms and containing 1 or 2, e.g. 1 double bond. Often, a 4- to 10-membered carbocyclic group is a 4- to 6-membered (e.g. 5- to 6-membered) carbocyclic group. Examples of 4- to 6-membered saturated carbocyclic groups include cyclobutyl, cyclopentyl and cyclohexyl groups. A 4- to 10-membered heterocyclic group is a cyclic group containing from 4 to 10 atoms selected from C, O, N and S in the ring, including at least one heteroatom, and typically one or two heteroatoms. The heteroatom or heteroatoms are typically selected from O, N, and S, most typically N. A heterocyclic group may be saturated or partially unsaturated. A 4- to 10-membered partially unsaturated heterocyclic group is a cyclic group containing from 4 to 10 atoms selected from C, O, N and S in the ring and containing 1 or 2, e.g. 1 double bond. Often, a 4- to 10-membered heterocyclic group is a monocyclic 4- to 6-membered heterocyclic group or a monocyclic 5- or 6-membered heterocyclic group, such as piperazine, piperidine, morpholine, 1,3-oxazinane, pyrrolidine, imidazolidine, and oxazolidine. 
     Preferably, X is N. Preferably, R 1  and R 2  are each independently selected from hydrogen and unsubstituted C 1-2  alkyl. Most preferably, R 1  and R 2  are each methyl. 
     Preferably, R 3  is absent. When R 3  is other than absent, the nitrogen atom to which R 3  is attached is typically positively charged. 
     Preferably, R 4  is hydrogen or unsubstituted C 1-2  alkyl. Most preferably, R 4  is hydrogen. 
     Preferably, R 5  is hydrogen or is attached to X to form a 6-membered heterocyclic group which is optionally substituted by 1 or 2 methyl groups. Most preferably, R 5  is hydrogen. 
     Preferably, R is alkyl (preferably C 1-6 alkyl) optionally substituted by one or more groups independently selected from —OH, —OC(O)—C 1-4 alkyl (e.g. —OC(O)—CH 3 ), phenyl, and phosphate, wherein each phosphate group is optionally substituted. Preferred R moieties include —CH 2 (CHOH) 3 CH 2 OH;
     —CH 2 (CHOH) 3 CH 2 OPO 3 ; —CH 2 (CHOH) 3 CH 2 (OPO 3 ) 2 -Adenine; —CH 2 (CHOC(O)CH 3 ) 3 CH 2  OC(O)CH 3 ;   —CH 2 CH(CH 2 CH 3 ) 2 ; and —CH 2 CH(CH 2 C6H 5 ) 2 .   

     Preferably, therefore, X is N; R 1  and R 2  are each methyl; R 4  is hydrogen; R 3  is absent and R is alkyl substituted by one or more groups selected from —OH, —OC(O)—CH 3 , phenyl and phosphate. 
     Preferably, the flavin cofactor is selected from flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), riboflavin, or a derivative thereof. The structures of riboflavin, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) in their respective oxidised forms are shown below. 
     
       
         
         
             
             
         
       
     
     Derivatives of flavin cofactors may also be modified at the position corresponding to group R in formula (I). For example, in any of FMN, FAD and riboflavin the alkyl group attached to the isoalloxazine moiety may be modified, e.g. by modification of one or more of the —OH groups. Alternatively, in flavin cofactors which comprise a substituted phosphate group (as in FAD, for example), the phosphate group may be modified to alter the substituents thereon. Derivatives of flavin cofactors may also be modified at the positions corresponding to R 1 , R 2 , and R 4  of Formula (I). Typically, such derivatives are modified in accordance with the definitions for Formula (I). 
     The reduction of the flavin cofactor occurs as shown in the reaction scheme below, in which X is depicted as N and R 3  is depicted as being absent. 
     
       
         
         
             
             
         
       
     
     Preferably, in the invention, the flavin cofactor is selected from riboflavin, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). More preferably, the cofactor is flavin adenine dinucleotide (FAD) or a derivative thereof or flavin mononucleotide (FMN) or a derivative thereof. Most preferably the cofactor is flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). 
     First Polypeptide 
     In the invention, the first polypeptide is a hydrogenase enzyme or a functional fragment or derivative thereof. Any suitable hydrogenase can be used. The hydrogenase may comprise an active site comprising iron atoms (as in the [FeFe]-hydrogenases) or both nickel and iron atoms (as in the [NiFe]- and [NiFeSe]-hydrogenases). Preferably, the hydrogenase comprises an active site comprising both nickel and iron atoms. Suitable proteins are described below. 
     The first polypeptide is preferably selected or modified to catalyze H 2  oxidation close to the thermodynamic potential E° of the 2H + /H 2  couple (“E°(2H + /H 2 )”) under the experimental conditions. (Those skilled in the art will appreciate that E°(2H + /H 2 )=−0.413 V at 25° C., pH 7.0 and 1 bar H 2 , and varies according to the Nernst equation). Preferably, the first polypeptide is selected or modified to catalyze H 2  or  x H 2  oxidation at applied potentials of less than 100 mV more positive than E°(2H + /H 2 ); more preferably at applied potentials of less than 50 mV more positive than E°(2H + /H 2 ). Methods of determining the ability of a polypeptide to catalyze H 2  oxidation close to E°(2H + /H 2 ) under the experimental conditions at issue are routine for those skilled in the art and are, for example, described in Vincent et al,  J. Am. Chem. Soc . (2005) 127, 18179-18189. 
     In the invention, the first polypeptide typically transfers the electrons to the oxidised flavin cofactor via an intramolecular electronically-conducting pathway. In other words, the electron transfer from the hydrogen electron source to the flavin cofactor is a direct electron transfer. While the invention does embrace the use of electron mediators (e.g. redox active dyes such as methyl or benzyl viologen) to mediate electron transfer from the hydrogen electron source (e.g. from the first polypeptide) to the flavin cofactor, the electron transfer is typically not mediated by electron transfer agents such as mediators, e.g. is typically not mediated by a redox active dye such as methyl or benzyl viologen. Typically, the intramolecular electronically-conducting pathway comprises a series of [FeS] clusters. As those skilled in the art will appreciate, [FeS]-clusters include [3Fe4S] and [4Fe4S] clusters. 
     Without being bound by theory, the inventors consider that the reduction of the oxidised flavin cofactor preferably takes place at an [FeS] cluster within the first polypeptide, preferably at the distal [FeS] cluster. The notation “distal” in this context is routine in the art. For a protein which contains an active site and a chain or series of [FeS] clusters, the proximal cluster is the [FeS] cluster at closest proximity to the active site. The distal cluster is the [FeS] cluster closest to a solvent-accessible surface of the protein, and thus furthest away from the active site. [FeS] clusters between the proximal and distal clusters are referred to as medial clusters. The distal cluster is often solvent accessible. Sometimes, the distal cluster can be accessed by the oxidised flavin cofactor. 
     Preferably, in the invention, the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction. Some known hydrogenases do comprise such an active site. Hydrogenase enzymes which do comprise a native flavin active site for NAD(P)+ reduction include the soluble hydrogenase (SH) enzymes from  R. eutropha, Rhodococcus opacus, Hydrogenophilus thermoluteolus  and  Pyrococcus furiosus . Accordingly, the first polypeptide is typically not selected from the soluble hydrogenase (SH) enzymes from  R. eutropha, Rhodococcus opacus, Hydrogenophilus thermoluteolus  and  Pyrococcus furiosus . Native flavin sites are also sometimes referred to as flavin prosthetic groups. Accordingly, the first polypeptide preferably does not comprise a flavin prosthetic group. Without being bound by theory, it is believed that hydrogenases lacking such groups typically have increased stability compared to hydrogenases comprising such prosthetic groups. Examples of hydrogenases lacking a flavin prosthetic group include  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1-2),  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3-4),  Ralstonia eutropha  membrane-bound hydrogenase (SEQ ID NOs: 5-7),  Ralstonia eutropha  regulatory hydrogenase (SEQ ID NOs:8-9),  Aquifex  aeolicus hydrogenase 1 (SEQ ID NOs:10-11), and  Hydrogenovibrio marinus  membrane-bound hydrogenase (SEQ ID NOs: 12-13). 
     It is a surprising finding of the present invention that reduction of oxidised flavin cofactors can be catalysed by hydrogenase enzymes which do not comprise a native flavin prosthetic group. Without being bound by theory, it is believed that such enzymes typically operate in vivo by shuttling electrons into the quinone pool of the parent organism and as such there is no requirement in vivo for a redox cofactor such as flavin to be accessible to the electron transfer pathway in the protein. It was previously unknown that electrons could be passed between the active site of such enzymes and an exogenous cofactor such as a flavin. 
     Preferably, in the invention, the first polypeptide is an uptake hydrogenase or a hydrogen-sensing hydrogenase. Uptake hydrogenases are used by organisms in vivo to generate energy by oxidation of molecular hydrogen in their environment. In vivo, they link oxidation of H 2  to reduction of anaerobic acceptors such as nitrate and sulfate, or O 2 . Typically, uptake hydrogenases comprise a signal peptide (often of length from about 30 to about 60 amino acid residues) at the N terminus of the small subunit. Typically, the signal peptide comprises a [DENST]RRxFxK motif Hydrogen sensing hydrogenases (also known as regulatory hydrogenases) are used by organisms in vivo to sense hydrogen levels in order to control biosynthesis of uptake hydrogenases in response to H 2 . Regulatory hydrogenases typically do not comprise the signal peptide characteristic of uptake hydrogenases. Regulatory hydrogenases are often insensitive to O 2 . 
     Preferably, the hydrogenase is selected or modified to be oxygen tolerant. Oxygen tolerant hydrogenases are capable of oxidising H 2  or H 2  in the presence of oxygen, such as in the presence of at least 0.01% O 2 , preferably at least 0.1% O 2 , more preferably at least 1% O 2 , such as at least 5% O 2 , e.g. at least 10% O 2  such as at least 20% O 2  or more whilst retaining at least 1%, preferably at least 5%, such as at least 10%, preferably at least 20%, more preferably at least 50% such as at least 80% e.g. at least 90% preferably at least 95% e.g. at least 99% of their H 2 -oxidation activity under anaerobic conditions. Various oxygen-tolerant hydrogenases are known to those skilled in the art. 
     Preferably, in the invention, the first polypeptide is a hydrogenase of class 1 or 2b. References to hydrogenase classes such as class 1 and class 2b refer to the established Vignais classification scheme described by Vignais and Billoud,  Chem. Rev.  2007, 107, 4206-4272, which is known to those skilled in the art. The hydrogenase may be any of the hydrogenases of class 1 or class 2b listed in Vignais and Billoud,  Chem. Rev.  2007, 107, 4206-4272, the contents of which are incorporated by reference. 
     Preferably, the first polypeptide is selected from or comprises:
         i) the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of  Ralstonia eutropha  membrane-bound hydrogenase moiety (SEQ ID NOs: 5 and/or 6 and/or 7) or an amino acid sequence having at least 60% homology therewith;   iv) the amino acid sequence of  Ralstonia eutropha  regulatory hydrogenase moiety (SEQ ID NOs: 8 and/or 9) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   vi) the amino acid sequence of  Hydrogenovibrio marinus  hydrogenase (SEQ ID NOs: 12 and/or 13) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   viii) the amino acid sequence of  Alteromonas macleodii  hydrogenase (SEQ ID NOs: 16 and/or 17) or an amino acid sequence having at least 60% homology therewith;   ix) the amino acid sequence of  Allochromatium vinosum  membrane bound hydrogenase (SEQ ID NOs: 18 and/or 19) or an amino acid sequence having at least 60% homology therewith;   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 therewith; or   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 therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Preferably, the first polypeptide is selected from or comprises:
         i) the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith; ii) the amino acid sequence of  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of  Ralstonia eutropha  membrane-bound hydrogenase moiety (SEQ ID NOs: 5 and/or 6 and/or 7) or an amino acid sequence having at least 60% homology therewith;   iv) the amino acid sequence of  Ralstonia eutropha  regulatory hydrogenase moiety (SEQ ID NOs: 8 and/or 9) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   vi) the amino acid sequence of  Hydrogenovibrio marinus  hydrogenase (SEQ ID NOs: 12 and/or 13) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   viii) the amino acid sequence of  Alteromonas macleodii  hydrogenase (SEQ ID NOs: 16 and/or 17) or an amino acid sequence having at least 60% homology therewith;   ix) the amino acid sequence of  Allochromatium vinosum  membrane bound hydrogenase (SEQ ID NOs: 18 and/or 19) or an amino acid sequence having at least 60% homology therewith; or   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 therewith;
 
or a functional fragment, derivative or variant thereof.
       

     More preferably, the first polypeptide is selected from or comprises:
         i) the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of  Ralstonia eutropha  membrane-bound hydrogenase moiety (SEQ ID NOs: 5 and/or 6 and/or 7) or an amino acid sequence having at least 60% homology therewith;   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 therewith;   v) the amino acid sequence of  Hydrogenovibrio marinus  hydrogenase (SEQ ID NOs: 12 and/or 13) or an amino acid sequence having at least 60% homology therewith;   vi) the amino acid sequence of  Alteromonas macleodii  hydrogenase (SEQ ID NOs: 16 and/or 17) or an amino acid sequence having at least 60% homology therewith; 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 therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Sometimes, the first polypeptide is selected from or comprises:
         i) the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4) or an amino acid sequence having at least 60% homology therewith; or   iii) 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 therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Most preferably, the first polypeptide comprises the amino acid sequence of  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2) or an amino acid sequence having at least 60% homology therewith; or a functional fragment, derivative or variant thereof. 
     Preferably, when the first polypeptide comprises or consists of one or more amino acid sequences having at least 60% homology with a specified sequence, each amino acid sequence independently has at least 70%, such as at least 80%, more preferably at least 90%, e.g. at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% homology with the specified sequence. More preferably, each amino acid sequence independently has at least 70%, such as at least 80%, more preferably at least 90%, e.g. at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% identity with the specified sequence. For avoidance of doubt, if the first polypeptide comprises two or more amino acid sequences, the percentage homology of each of the two or more sequences with respect to their respective specified sequences can be the same or different, preferably the same. Percentage homology and/or percentage identity are each preferably determined across the length of the specified reference sequence as described herein. 
     It will be apparent to the skilled person that the first polypeptide may either be a single polypeptide or may comprise multiple polypeptides. The first polypeptide may also be a portion such as one or more domains of a multidomain polypeptide. Those skilled in the art will appreciate that hydrogenase enzymes typically comprise two or more subunits. As used herein, the term “first polypeptide” relates to one or more of the subunits of the relevant protein. For example, when the first polypeptide is  Escherichia coli  hydrogenase 1 (SEQ ID NOs:1 and/or 2), the first polypeptide may comprise (i) SEQ ID NO: 1 but not SEQ ID NO: 2; (ii) SEQ ID NO: 2 but not SEQ ID NO: 1; or (iii) both SEQ ID NO: 1 and SEQ ID NO: 2. When the first polypeptide is  Escherichia coli  hydrogenase 2 (SEQ ID NOs:3 and/or 4), the first polypeptide may comprise (i) SEQ ID NO: 3 but not SEQ ID NO: 4; (ii) SEQ ID NO: 4 but not SEQ ID NO: 3; or (iii) both SEQ ID NO: 3 and SEQ ID NO: 4. When the first polypeptide is  Desulfovibrio vulgaris  Miyazaki F hydrogenase (SEQ ID NOs: 23 and/or 24), the first polypeptide may comprise (i) SEQ ID NO: 23 but not SEQ ID NO: 24; (ii) SEQ ID NO: 24 but not SEQ ID NO: 23; or (iii) both SEQ ID NO: 23 and SEQ ID NO: 24. Typically, when the first polypeptide is a hydrogenase enzyme having two or more subunits, the first polypeptide comprises said two or more subunits. 
     Furthermore, the first polypeptide may be used in the invention in the form of a monomer or a multimer. For example, when the first polypeptide comprises a hydrogenase which can exist in a monomeric or dimeric form, the first polypeptide used in the invention can be provided in the form of the monomer or the dimer. For example,  Escherichia coli  hydrogenase 1 may be purified either as a dimer or a monomer or a mixture thereof. When the first polypeptide comprises  Escherichia coli  hydrogenase 1 (i.e. SEQ ID NOs: 1 and/or 2) the first polypeptide may be provided as a monomer (1×SEQ ID NO: 1 and/or 1×SEQ ID NO 2) or as a dimer (2×SEQ ID NO: 1 and/or 2×SEQ ID NO: 2), or as a mixture thereof. When the first polypeptide is provided as a mixture of a monomer and dimer, the mixture typically contains from about 1% to about 99% of the monomer and from about 99% to about 1% of the dimer. Sometimes, the amount of monomer and dimer may be approximately similar, and the first polypeptide may thus comprise from about 30% to about 70% monomer and from about 70% to about 30% dimer, such as from about 40% to about 60% monomer and from about 60% to about 40% dimer. Sometimes, the first polypeptide comprises from about 1 to about 10% monomer/about 90% to about 99% dimer, e.g. from about 1% to about 5% monomer/about 95% to about 99% dimer. Sometimes, the first polypeptide comprises from about 1 to about 10% dimer/about 90% to about 99% monomer, e.g. from about 1% to about 5% dimer/about 95% to about 99% monomer. 
     It will also be apparent to those skilled in the art that the first polypeptide may comprise associated proteins which may for example be co-purified with the first polypeptide. For example, when the first polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and/or 2 (or a functional fragment, derivative or variant thereof), the first polypeptide may further comprise a native cytochrome electron transfer partner such as the cytochrome of SEQ ID NO: 22 (or a functional fragment, derivative or variant thereof). Thus, in embodiments of the invention in which the first polypeptide comprises SEQ ID NO: 1 and/or 2 (or a functional fragment, derivative or variant thereof), the first polypeptide may also comprise SEQ ID NO: 22 (or a functional fragment, derivative or variant thereof). 
     Second Polypeptide 
     In inventive methods which comprise the use of a second polypeptide to catalyse the conversion or a reagent to a product, any suitable second polypeptide may be used. Typically, electrons are transferred from the reduced flavin cofactor to an electron acceptor and/or hydride ions are transferred from the reduced flavin cofactor to a hydride ion acceptor, thereby regenerating the oxidised flavin cofactor. Usually, in the invention, electrons are transferred from the reduced flavin cofactor to an electron acceptor. 
     In one embodiment, the second polypeptide comprises an electron acceptor and/or hydride ion acceptor. The acceptor group may consist of or comprise a prosthetic group or active site within the second polypeptide. Thus, in this embodiment, the second polypeptide typically comprises a prosthetic group for oxidising the reduced flavin cofactor. The second polypeptide typically comprises a flavin prosthetic group. Preferably, the flavin group is an FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide) group. This embodiment is shown schematically in  FIG.  3   . 
     In another embodiment, electrons and/or hydride are transferred at the second polypeptide from the reduced cofactor to a molecular substrate. The molecular substrate is preferably an exogenous substrate; i.e. it is not part of the second polypeptide. Examples of molecular substrates for use in the invention include O 2 . For example, when O 2  is used as the molecular substrate, the reduced cofactor may form an oxidised cofactor comprising a product of the O 2  reduction such as a peroxo group. For example, an oxidised cofactor comprising a peroxo group obtained from O 2  reduction may be of the form: 
     
       
         
         
             
             
         
       
     
     (with reference to Formula (I) above, in which X is depicted as N and R 3  is depicted as being absent). 
     The second polypeptide may catalyse the reaction of the product of the molecular substrate reduction (e.g. the peroxo group) with the reagent in order to form the product. When O 2  is used as the molecular substrate, water is typically produced as a by-product. This embodiment is shown schematically as  FIG.  4   . 
     In another embodiment, electrons and/or hydride are transferred from the reduced cofactor to a molecular substrate. The molecular substrate is preferably an exogenous substrate; i.e. it is not part of the second polypeptide. Examples of molecular substrates for use in the invention include O 2 . For example, when O 2  is used as the molecular substrate, the reduced cofactor may form an oxidised cofactor comprising a product of the O 2  reduction such as a peroxo group, e.g. as discussed above. The peroxy oxidised cofactor may release H 2 O 2  to regenerate the oxidised cofactor. The H 2 O 2  thus released may be used to convert a reactant to a product in accordance with the invention. Preferably, the second polypeptide catalyses the conversion of the reactant to the product. 
     Typically, the second polypeptide is a flavin-accepting oxidoreductase, or a functional fragment, derivative or variant thereof. In one embodiment, a flavin-accepting oxidoreductase is an enzyme which is facultatively capable of abstracting electrons and/or hydride from a reduced flavin cofactor. Those skilled in the art will appreciate that in consuming the reduced flavin, oxidised flavin is obtained. Thus, the flavin cofactor is not destroyed by the second polypeptide. In another embodiment, a flavin-accepting oxidoreductase is an enzyme which is facultatively capable of catalysing the reaction of an oxidised form of a flavin cofactor (e.g. that obtained by oxidation of a reduced cofactor with a molecular substrate such as O 2 ) with a reagent, thus forming a product and regenerating the oxidised flavin. Thus, the flavin cofactor is not destroyed by the second polypeptide. 
     More typically, the second polypeptide is a flavin-dependent oxidoreductase, or a functional fragment, derivative or variant thereof. A flavin-dependent oxidoreductase as used herein requires flavin cofactors such as FMN and FAD. Thus, a flavin-accepting oxidoreductase is capable of utilising various reduced cofactors including flavins as an electron/hydride source. A flavin-dependent oxidoreductase is only capable of using flavins as an electron/hydride source. 
     It will be apparent to the skilled person that the second polypeptide may either be a single polypeptide or may comprise multiple polypeptides, e.g. additional peptides in addition to the flavin-accepting or flavin-dependent oxidoreductase. The second polypeptide may also be a portion such as one or more domains of a multidomain polypeptide. 
     Preferably, the second polypeptide is a monooxygenase, halogenase or ene-reductase, or a functional fragment, derivative or variant thereof. More preferably, the second polypeptide is a flavin monooxygenase, a flavin halogenase, a flavin ene-reductase, a nitro reductase, a peroxidase or a haloperoxidase. Still more preferably, the second polypeptide is a flavin monooxygenase, a flavin halogenase, or a flavin ene-reductase. Preferably, the second polypeptide is of the “Old Yellow Enzyme” (OYE) type. OYEs are flavin-dependent redox enzymes and include e.g. OYE ene reductases. 
     Such enzymes catalyse commercially useful reactions. For example, halogenases catalyse chlorination, bromination, and iodination reactions. Ene-reductase catalyse reactions such as alkene reduction and nitro reduction. Monooxygenase enzymes catalyse reactions such as epoxidations, hydroxylations, and Baeyer-Villiger oxidations. Nitro reductases catalyse reactions such as the reduction of aromatic nitro groups and quinones. Peroxidases catalyse reaction such as O atom insertion reactions. Haloperoxidases catalyse reactions such as the conversion of C—H groups to C—Y groups (wherein Y is a halogen). Such reactions are wide in utility, including in natural product synthesis, biodegradation of environmental pollutants, and non-native light-driven reactions. 
     Often, the second enzyme is selected from Enzyme Commission (EC) classes 1.1.98.; 1.3.1.; 1.5.1.; 1.6.99.; 1.7.1.; 1.7.99; 1.11.1.; 1.11.2.; 1.14.14.; and 1.14.99.; or a functional fragment, derivative or variant thereof. 
     Preferably, the second polypeptide is selected from Enzyme Commission (EC) classes 1.1.98.; 1.5.1.; 1.6.99.; 1.7.99.; 1.14.14.; and 1.14.99.; 1.3.1; 1.11.1.; 1.11.2.; or a functional fragment, derivative or variant thereof; more preferably the second polypeptide is selected from Enzyme Commission (EC) classes 1.1.98.; 1.5.1.; 1.6.99.; 1.7.99.; 1.14.14.; and 1.14.99.; or a functional fragment, derivative or variant thereof. 
     More preferably, the second polypeptide is selected from or comprises
         i) the amino acid sequence of Chromate Reductase, ‘TsOYE’ from Thermus scotuductus (SEQ ID NO:31) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of the NADPH Dehydrogenase 1, ‘OYE-1 ’, Saccharomyces pastorianus  (SEQ ID NO:32) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of NADPH Dehydrogenase 2, ‘OYE-2’,  Saccharomyces cerevisiae  strain ATCC 204508 S288c (SEQ ID NO:33) or an amino acid sequence having at least 60% homology therewith;   iv) the amino acid sequence of NADPH Dehydrogenase, ‘YqjM’,  Bacillus subtilis  (SEQ ID NO:34) or an amino acid sequence having at least 60% homology therewith;   v) the amino acid sequence of Xenobiotic Reductase A, ‘XenA’,  Pseudomonas putida  (SEQ ID NO:35) or an amino acid sequence having at least 60% homology therewith;   vi) the amino acid sequence of NADPH dehydrogenase, ‘FOYE-1’, ‘Ferrovum’ strain JA12 (SEQ ID NO:36) or an amino acid sequence having at least 60% homology therewith;   vii) the amino acid sequence of Oxidored_FMN domain-containing protein, ‘MgER’, Meyerozyma  guilliermondii  (SEQ ID NO:37) or an amino acid sequence having at least 60% homology therewith;   viii) the amino acid sequence of Oxidored_FMN domain-containing protein, ‘CER’,  Clavispora  ( Candida )  lusitaniae  (SEQ ID NO: 38) or an amino acid sequence having at least 60% homology therewith;   ix) the amino acid sequence of Tryptophan 2-Halogenase, ‘CmdE’, Chondromyces crocatus, (SEQ ID NO:39) or an amino acid sequence having at least 60% homology therewith;   x) the amino acid sequence of Tryptophan 5-Halogenase, ‘PyrH’,  Streptomyces  rugosporus (SEQ ID NO:40) or an amino acid sequence having at least 60% homology therewith;   xi) the amino acid sequence of Flavin-Dependent Tryptophan Halogenase, ‘RebH’,  Lentzea aerocolonigenes  ( Lechevalieria aerocolonigenes ) ( Saccharothrix aerocolonigenes ), (SEQ ID NO:41) or an amino acid sequence having at least 60% homology therewith;   xii) the amino acid sequence of Flavin-Dependent Tryptophan Halogenase, ‘PrnA’,  Pseudomonas fluorescens  (SEQ ID NO:42) or an amino acid sequence having at least 60% homology therewith;   xiii) the amino acid sequence of Thermophilic Tryptophan Halogenase, ‘Th-Hal’, Streptomyces violaceusnige (SEQ ID NO:43) or an amino acid sequence having at least 60% homology therewith;   xiv) the amino acid sequence of Tryptophan 6-Halogenase, ‘SttH’,  Streptomyces toxytricini  (SEQ ID NO: 44) or an amino acid sequence having at least 60% homology therewith;   xv) the amino acid sequence of KtzQ, ‘KtzQ’, Kutzneria sp. 744 (SEQ ID NO:45) or an amino acid sequence having at least 60% homology therewith;   xvi) the amino acid sequence of Monodechloroaminopyrrolnitrin halogenase, ‘PrnC’,  Pseudomonas fluorescens  (SEQ ID NO:46) or an amino acid sequence having at least 60% homology therewith;   xvii) the amino acid sequence of FADH 2 -dependent halogenase, ‘PltA’, Pseudomonasprotegens Pf-5 (SEQ ID NO:47) or an amino acid sequence having at least 60% homology therewith;   xviii) the amino acid sequence of Halogenase, ‘PltM’,  Pseudomonas fluorescens  (strain ATCC BAA-477 NRRL B-23932 Pf-5) (SEQ ID NO:48) or an amino acid sequence having at least 60% homology therewith;   xix) the amino acid sequence of Flavin-Dependent Halogenase, ‘Clz5’,  Streptomyces  sp. CNH-287 (SEQ ID NO:49) or an amino acid sequence having at least 60% homology therewith;   xx) the amino acid sequence of Pyrrole Halogenase, ‘Bmp2’,  Pseudoalteromonas piscicida  (SEQ ID NO:50) or an amino acid sequence having at least 60% homology therewith;   xxi) the amino acid sequence of Non-Heme Halogenase, ‘Rdc2’, Metacordyceps chlamydosporia (Pochonia chlamydosporia) (SEQ ID NO:51) or an amino acid sequence having at least 60% homology therewith;   xxii) the amino acid sequence of Tryptophan 6-Halogenase, ‘BorH’, uncultured bacteria (SEQ ID NO:52) or an amino acid sequence having at least 60% homology therewith;   xxiii) the amino acid sequence of Styrene Monooxygenase, ‘StyA’,  Pseudomonas  sp. (SEQ ID NO:53) or an amino acid sequence having at least 60% homology therewith;   xxiv) the amino acid sequence of 4-Nitrophenol 2-Monooxygenase Oxygenase Component, ‘PheA1’,  Rhodococcus erythropolis  ( Arthrobacter picolinophilus ) (SEQ ID NO:54) or an amino acid sequence having at least 60% homology therewith;   xxv) the amino acid sequence of 4-Hydroxyphenylacetate 3-Monooxygenase Oxygenase Component, ‘HpaB’,  Klebsiella oxytoca  (SEQ ID NO:55) or an amino acid sequence having at least 60% homology therewith;   xxvi) the amino acid sequence of Chlorophenol Monooxygenase, ‘HadA’,  Ralstonia pickettii  ( Burkholderia pickettii ) (SEQ ID NO:56) or an amino acid sequence having at least 60% homology therewith;   xxvii) the amino acid sequence of Tetrachlorobenzoquinone Reductase, ‘PcpD’,  Sphingobium chlorophenolicum  (SEQ ID NO:57) or an amino acid sequence having at least 60% homology therewith;   xxviii) the amino acid sequence of 2-Methyl-6-ethyl-4-monooxygenase Oxygenase Component, ‘MeaX’,  Sphingobium baderi  (SEQ ID NO:58) or an amino acid sequence having at least 60% homology therewith;   xxix) the amino acid sequence of Alkanesulfonate Monooxygenase, ‘SsuD’,  Escherichia coli  (strain K12) (SEQ ID NO:59) or an amino acid sequence having at least 60% homology therewith;   xxx) the amino acid sequence of p-Hydroxyphenylacetate 3-Hydroxylase, Oxygenase Component, ‘C2-HpaH’,  Acinetobacter baumannii  (SEQ ID NO:60) or an amino acid sequence having at least 60% homology therewith; xxxi) the amino acid sequence of FADH(2)-Dependent Monooxygenase, ‘TftD’,  Burkholderia cepacia  ( Pseudomonas cepacia ) (SEQ ID NO:61) or an amino acid sequence having at least 60% homology therewith;   xxxii) the amino acid sequence of 4-Nitrophenol 2-Monooxygenase, Oxygenase Component, ‘NphA1 ’, Rhodococcus  sp. (SEQ ID NO:62) or an amino acid sequence having at least 60% homology therewith;   xxxiii) the amino acid sequence of Putative dehydrogenase/oxygenase subunit, ‘VpStyA1 ’, Variovorax paradoxus  (strain EPS) (SEQ ID NO:63) or an amino acid sequence having at least 60% homology therewith;   xxxiv) the amino acid sequence of Oxygenase, ‘RoIndA1’ {from styA1 gene},  Rhodococcus opacus  ( Nocardia opaca ) (SEQ ID NO:64) or an amino acid sequence having at least 60% homology therewith;   xxxv) the amino acid sequence of Smoa_sbd domain-containing protein, ‘AbIndA’,  Acinetobacter baylyi  (strain ATCC 33305 BD413 ADP1) (SEQ ID NO:65) or an amino acid sequence having at least 60% homology therewith;   xxxvi) the amino acid sequence of 2,5-Diketocamphane 1,2-Monooxygenase 1, ‘CamP’,  Pseudomonas putida  ( Arthrobacter siderocapsulatus ) (SEQ ID NO:66) or an amino acid sequence having at least 60% homology therewith; xxxvii) the amino acid sequence of 3,6-Diketocamphane 1,6-Monooxygenase, ‘CamE36’,  Pseudomonas putida  ( Arthrobacter siderocapsulatus ), (SEQ ID NO:67) or an amino acid sequence having at least 60% homology therewith; xxxviii) the amino acid sequence of Alkanal monooxygenase, alpha and beta chain, ‘LuxAB’,  Vibrio harveyi  ( Beneckea harveyi ) (SEQ ID NOs:68 and 69) or an amino acid sequence having at least 60% homology therewith;   xxxix) the amino acid sequence of Alkanal monooxygenase, alpha and beta chain, ‘LuxAB’,  Photorhabdus luminescens  (Xenorhabdus  luminescens ) (SEQ ID NOs:70 and 71) or an amino acid sequence having at least 60% homology therewith;   xl) the amino acid sequence of Alkane Monooxygenase, ‘LadA’,  Geobacillus thermodenitrificans  (SEQ ID NO:72) or an amino acid sequence having at least 60% homology therewith;   xli) the amino acid sequence of EDTA Monooxygenase, ‘EmoA’,  Chelativorans multitrophicus  (SEQ ID NO:73) or an amino acid sequence having at least 60% homology therewith;   xlii) the amino acid sequence of Isobutylamine N-hydroxylase, ‘IBAH’,  Streptomyces viridifaciens  (SEQ ID NO:74) or an amino acid sequence having at least 60% homology therewith;   xliii) the amino acid sequence of ActVA 6 Protein, ‘ActVA-Orf6’,  Streptomyces coelicolor , (SEQ ID NO:75) or an amino acid sequence having at least 60% homology therewith;   xliv) the amino acid sequence of Pyrimidine Monooxygenase, ‘ RutA’, Escherichia coli  (strain K12) (SEQ ID NO:76) or an amino acid sequence having at least 60% homology therewith;   xlv) the amino acid sequence of p-Hydroxyphenylacetate 2-Hydroxylase Reductase Component, ‘C1-HpaH’,  Acinetobacter baumannii  (SEQ ID NO:77) or an amino acid sequence having at least 60% homology therewith;   xlvi) the amino acid sequence of FMN_red Domain-Containing Protein, ‘YdhA’,  Bacillus subtilis  subsp. natto (strain BEST195) (SEQ ID NO:78) or an amino acid sequence having at least 60% homology therewith;   xlvii) the amino acid sequence of NAD(P)H-Flavin Reductase, ‘Fre’,  Escherichia coli  (strain K12) (SEQ ID NO:79) or an amino acid sequence having at least 60% homology therewith;   xlviii) the amino acid sequence of 4-hydroxyphenylacetate 3-monooxygenase reductase component, ‘HpaC’,  Escherichia coli  (SEQ ID NO:80) or an amino acid sequence having at least 60% homology therewith;   xlix) the amino acid sequence of nitroreductase ‘NfsB’,  Escherichia coli  (strain K12) (SEQ ID NO:81) or an amino acid sequence having at least 60% homology therewith;   1) the amino acid sequence of vanadium chloroperoxidase ‘CPO’ or ‘CiVHPO’, Curvularia  inaequalis  (SEQ ID NO:82) or an amino acid sequence having at least 60% homology therewith;   li) the amino acid sequence of aromatic unspecified peroxygenase ‘APO1’ or ‘AaeUPO’,  Agrocybe aegerita  (Black poplar mushroom) ( Agaricus aegerita ) (SEQ ID NO:83) or an amino acid sequence having at least 60% homology therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Still more preferably, the second polypeptide is selected from or comprises
         i) the amino acid sequence of Chromate Reductase, ‘TsOYE’ from  Thermus scotoductus  (SEQ ID NO:31) or an amino acid sequence having at least 60% homology therewith;   ii) the amino acid sequence of NADPH Dehydrogenase 2, ‘OYE-2’,  Saccharomyces cerevisiae  strain ATCC 204508 S288c (SEQ ID NO:33) or an amino acid sequence having at least 60% homology therewith;   iii) the amino acid sequence of Xenobiotic Reductase A, ‘XenA’,  Pseudomonas putida  (SEQ ID NO:35) or an amino acid sequence having at least 60% homology therewith;   iv) the amino acid sequence of Tryptophan 5-Halogenase, ‘PyrH’,  Streptomyces  rugosporus (SEQ ID NO:40) or an amino acid sequence having at least 60% homology therewith;   v) the amino acid sequence of Thermophilic Tryptophan Halogenase, ‘Th-Hal’,  Streptomyces  violaceusnige (SEQ ID NO:43) or an amino acid sequence having at least 60% homology therewith;   vi) the amino acid sequence of Halogenase, ‘PltM’,  Pseudomonas fluorescens  (strain ATCC BAA-477 NRRL B-23932 Pf-5) (SEQ ID NO:48) or an amino acid sequence having at least 60% homology therewith;   vii) the amino acid sequence of Styrene Monooxygenase, ‘StyA’,  Pseudomonas  sp. (SEQ ID NO:53) or an amino acid sequence having at least 60% homology therewith;   viii) the amino acid sequence of Chlorophenol Monooxygenase, ‘HadA’,  Ralstonia pickettii  ( Burkholderia pickettii ) (SEQ ID NO:56) or an amino acid sequence having at least 60% homology therewith;   ix) the amino acid sequence of 2,5-Diketocamphane 1,2-Monooxygenase 1, ‘CamP’,  Pseudomonas putida  ( Arthrobacter siderocapsulatus ) (SEQ ID NO:66) or an amino acid sequence having at least 60% homology therewith;   x) the amino acid sequence of Alkanal monooxygenase, alpha and beta chain, ‘LuxAB’,  Vibrio harveyi  ( Beneckea harveyi ) (SEQ ID NOs:68 and 69) or an amino acid sequence having at least 60% homology therewith;   xi) the amino acid sequence of Isobutylamine N-hydroxylase, ‘IBAH’,  Streptomyces viridifaciens  (SEQ ID NO:74) or an amino acid sequence having at least 60% homology therewith;   xii) the amino acid sequence of NAD(P)H-Flavin Reductase, ‘Fre’,  Escherichia coli  (strain K12) (SEQ ID NO:79) or an amino acid sequence having at least 60% homology therewith;
 
or a functional fragment, derivative or variant thereof.
       

     Preferably, when the second polypeptide comprises or consists of one or more amino acid sequences having at least 60% homology with a specified sequence, each amino acid sequence independently has at least 70%, such as at least 80%, more preferably at least 90%, e.g. at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% homology with the specified sequence. More preferably, each amino acid sequence independently has at least 70%, such as at least 80%, more preferably at least 90%, e.g. at least 95%, preferably at least 97%, such as at least 98%, preferably at least 99% identity with the specified sequence. For avoidance of doubt, if the second polypeptide comprises two or more amino acid sequences, the percentage homology of each of the two or more sequences with respect to their respective specified sequences can be the same or different, preferably the same. Percentage homology and/or percentage identity are each preferably determined across the length of the specified sequence. 
     As explained above, the choice of the second polypeptide is an operational parameter which can be controlled in order to obtain the desired reaction product. The methods of the invention can thus be used to produce a variety of functional groups within molecules. For example, Olefins (alkenes) can be reduced to alkane groups, e.g. using ene reductases. Halogenated products (e.g. brominated, chlorinated or fluorinated products) can be obtained using halogenases. Nitro groups such as aromatic nitro groups can be reduced (e.g. to their corresponding amine or hydroxylamine groups) using nitroreductases. Oxygen insertion reactions can be used to produce e.g. alcohols and ethers, etc, using monooxygenases. The methods of the invention find utility particularly in the production of complex products such as in synthesis or derivatisation of natural products, and in pharmaceutical production. The stereochemistry of the reaction can typically be controlled by appropriate selection of the second polypeptide. The choice of appropriate second polypeptide and the characterisation of the products obtained from the methods of the invention is well within the capacity of those skilled in the art. For example, products can be characterised by chemical analytical techniques such as IR spectroscopy, NMR, GC (including chiral-phase GC), polarimetry etc. 
     Polypeptides Used in the Invention 
     Methods for expression of proteins in cellular (e.g. microbial) expression systems are well known and routine to those skilled in the art. For example, the first polypeptide and the second polypeptide (if present) can be independently isolated from their host organisms using routine purification methods. For example, host cells can be grown in a suitable medium. Lysing of cells allows internal components of the cells to be accessed. Membrane proteins can be solubilised 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 chromatographic techniques such as size exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography. Alternatively, the first polypeptide and the second polypeptide (if present) can be independently encoded in one or more nucleotide vector and subsequently expressed in an appropriate host cell (e.g. a microbial cell, such as  E. coli ). Purification tags such as a HIS (hexa-histidine) tag can be encoded (typically at the C- or N-terminal of the relevant polypeptide) and can be used to isolate the tagged protein using affinity chromatography for example using nickel- or cobalt-NTA chromatography. If desired, protease recognition sequences can be incorporated between the first and/or second polypeptide and the affinity purification tag to allow the tag to be removed post expression. Such techniques are routine to those skilled in the art and are described in, for example, Sambrook et al, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press. 
     As described herein the first polypeptide and/or the second polypeptide (if present) may be a functional fragment, derivative or variant of an enzyme or amino acid sequence. As those skilled in the art will appreciate, fragments of amino acid sequences include deletion variants of such sequences wherein one or more, such as at least 1, 2, 5, 10, 20, 50 or 100 amino acids are deleted. Deletion may occur at the C-terminus or N-terminus of the native sequence or within the native sequence. Typically, deletion of one or more amino acids does not influence the residues immediately surrounding the active site of an enzyme. Derivatives of amino acid sequences include post-translationally modified sequences including sequences which are modified in vivo or ex vivo. Many different protein modifications are known to those skilled in the art and include modifications to introduce new functionalities to amino acid residues, modifications to protect reactive amino acid residues or modifications to couple amino acid residues to chemical moieties such as reactive functional groups on linkers or substrates (surfaces) for attachment to such amino acid residues. 
     Derivatives of amino acid sequences include addition variants of such sequences wherein one or more, such as at least 1, 2, 5, 10, 20, 50 or 100 amino acids are added or introduced into the native sequence. Addition may occur at the C-terminus or N-terminus of the native sequence or within the native sequence. Typically, addition of one or more amino acids does not influence the residues immediately surrounding the active site of an enzyme. 
     Variants of amino acid sequences include sequences wherein one or more amino acid such as at least 1, 2, 5, 10, 20, 50 or 100 amino acid residues in the native sequence are exchanged for one or more non-native residues. Such variants can thus comprise point mutations or can be more profound e.g. native chemical ligation can be used to splice non-native amino acid sequences into partial native sequences to produce variants of native enzymes. Variants of amino acid sequences include sequences carrying naturally occurring amino acids and/or unnatural amino acids. Variants, derivatives and functional fragments of the aforementioned amino acid sequences retain at least some of the activity/functionality of the native/wild-type sequence. Preferably, variants, derivatives and functional fragments of the aforementioned sequences have increased/improved activity/functionality when compared to the native/wild-type sequence. 
     Variants of an enzyme, such as the first polypeptide or second polypeptide as described herein, may preferably be modified to have an increased catalytic activity for their respective substrates. Preferably, the catalytic activity is increased at least 2 times, such as at least 5 times, e.g. at least 10 times, such as at least 100 times, preferably at least 1000 times. Catalytic activity can be determined in any suitable method. For example, the catalytic activity can be associated with the Michaelis constant K M  (with increased activity being typically associated with decreased K M  values) or with the catalytic rate constant, k cat  (with increased activity being typically associated with increased k cat  values). 
     Measuring K M  and k cat  is routine to those skilled in the art. For example, the K M  of a polypeptide for a substrate can be determined spectrophotometrically, e.g. by measuring absorption at 578 nm under anaerobic conditions at 30° C. in 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM substrate, 5 mM benzyl viologen (oxidized; F=8.9 mM −1  cm −1 ), 90 μM dithionite, and 10 to 30 pmol of enzyme. Examples of solution assays in which the absorbance of oxidised and reduced flavins are determined are described in the examples. 
     Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984)  Nucleic Acids Research  12, p 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described 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 analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). 
     Similarity can be measured using pairwise identity or by applying a scoring matrix such as BLOSUM62 and converting to an equivalent identity. Since they represent functional rather than evolved changes, deliberately mutated positions would be masked when determining homology. Similarity may be determined more sensitively by the application of position-specific scoring matrices using, for example, PSIBLAST on a comprehensive database of protein sequences. A different scoring matrix could be used that reflect amino acid chemico-physical properties rather than frequency of substitution over evolutionary time scales (e.g. charge). Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table A below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table B. 
     
       
         
           
               
             
               
                 TABLE A 
               
               
                   
               
               
                 Chemical properties of amino acids 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Ala 
                 aliphatic, hydrophobic, 
                 Met 
                 hydrophobic, neutral 
               
               
                   
                 neutral 
               
               
                 Cys 
                 polar, hydrophobic, 
                 Asn 
                 polar, hydrophilic, 
               
               
                   
                 neutral 
                   
                 neutral 
               
               
                 Asp 
                 polar, hydrophilic, 
                 Pro 
                 hydrophobic, neutral 
               
               
                   
                 charged (−) 
               
               
                 Glu 
                 polar, hydrophilic, 
                 Gln 
                 polar, hydrophilic, 
               
               
                   
                 charged (−) 
                   
                 neutral 
               
               
                 Phe 
                 aromatic, hydrophobic, 
                 Arg 
                 polar, hydrophilic, 
               
               
                   
                 neutral 
                   
                 charged (+) 
               
               
                 Gly 
                 aliphatic, neutral 
                 Ser 
                 polar, hydrophilic, 
               
               
                   
                   
                   
                 neutral 
               
               
                 His 
                 aromatic, polar, 
                 Thr 
                 polar, hydrophilic, 
               
               
                   
                 hydrophilic, charged (+) 
                   
                 neutral 
               
               
                 Ile 
                 aliphatic, hydrophobic, 
                 Val 
                 aliphatic, hydrophobic, 
               
               
                   
                 neutral 
                   
                 neutral 
               
               
                 Lys 
                 polar, hydrophilic, 
                 Trp 
                 aromatic, hydrophobic, 
               
               
                   
                 charged(+) 
                   
                 neutral 
               
               
                 Leu 
                 aliphatic, hydrophobic, 
                 Tyr 
                 aromatic, polar, 
               
               
                   
                 neutral 
                   
                 hydrophobic 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 Hydropathy scale 
               
            
           
           
               
               
               
            
               
                   
                 Side Chain 
                 Hydropathy 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 Ile 
                 4.5 
               
               
                   
                 Val 
                 4.2 
               
               
                   
                 Leu 
                 3.8 
               
               
                   
                 Phe 
                 2.8 
               
               
                   
                 Cys 
                 2.5 
               
               
                   
                 Met 
                 1.9 
               
               
                   
                 Ala 
                 1.8 
               
               
                   
                 Gly 
                 −0.4 
               
               
                   
                 Thr 
                 −0.7 
               
               
                   
                 Ser 
                 −0.8 
               
               
                   
                 Trp 
                 −0.9 
               
               
                   
                 Tyr 
                 −1.3 
               
               
                   
                 Pro 
                 −1.6 
               
               
                   
                 His 
                 −3.2 
               
               
                   
                 Glu 
                 −3.5 
               
               
                   
                 Gln 
                 −3.5 
               
               
                   
                 Asp 
                 −3.5 
               
               
                   
                 Asn 
                 −3.5 
               
               
                   
                 Lys 
                 −3.9 
               
               
                   
                 Arg 
                 −4.5 
               
               
                   
                   
               
            
           
         
       
     
     Preferably, sequence homology can be assessed in terms of sequence identity. Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Preferred methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680 (1994)) and iterative refinement (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)). Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes). 
     Alignment Scores for Determining Sequence Identity 
       
                                                                                                                 A   R   N   D   C   Q   E   G    H   I   L   K   M   F   P   S   T   W   Y   V                                                                                                                    A   4                                                                                   R   −1    5                                                                               N   −2    0    6                                                                           D   −2    −2   1    6                                                                       C   0    −3    −3   −3   9                                                                   Q   −1    1   0   0   −3   5                                                               E   −1    0    0   2   −4   2   5                                                           G   0    −2    0   −1   −3    −2    −2   6                                                       H   −2    0    1    −1   −3    0    0   −2    8                                                   I   −1    −3    −3    −3   −1    −3    −3   −4    −3    4                                               L   −1    −2    −3    −4    −1    −2    −3   −4   −3    2    4                                           K   −1    2   0   −1    −3    1   1    −2   −1   −3   −2   5                                       M   −1    −1   −2   −3   −1    0   −2   −3    −2   1    2    −1   5                                   F   −2    −3    −3    −3    −2    −3   −3    −3   −1   0    0    −3   0   6                               P   −1    −2    −2    −1   −3   −1   −1   −2   −2   −3    −3    −1   −2   −4   7                           S   1    −1    1   0   −1   0   0   0    −1   −2    −2   0   −1   −2   −1   4                       T   0    −1   0    −1   −1   −1   −1   −2   −2   −1   −1   −1   −1   −2   −1   1   5                   W   −3    −3    −4    −4    −2   −2    −3    −2   −2   −3   −2   −3   −1   1   −4   −3   −2   11               Y   −2    −2   −2    −3   −2   −1   −2   −3   2   −1   −1   −2   −1   3   −3   −2   −2   2   7           V   0    −3   −3   −3   −1   −2   −2   −3   −3   3   1   −2   1   −1   −2   −2   0   −3   −1   4                    
Percent identity is then calculated as:
 
       100×( T/L )
 
     where
 
T=Total number of identical matches
 
L=Length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences
 
     The first polypeptide and the second polypeptide when present may be distinct. However, in other embodiments, the first polypeptide is attached to the second polypeptide. This is shown schematically in  FIG.  5   . Any suitable attachment means may be used. 
     For example, the first polypeptide and second polypeptide may be attached together by chemical means. For example, cross-linking reagents can be used to attach the first polypeptide to the second polypeptide. Any suitable cross-linking reagent can be used. Suitable cross-linking reagents are known in the art. Functional groups that can be targeted with cross-linking agents include primary amines, carboxyls, sulfhydryls, carbohydrates and carboxylic acids. The cross-linking agent may be homobifunctional or heterobifunctional. Cross-linking reagents include bis(2-[succinimidooxycarbonyloxy]ethyl) sulfone, 1,4-di-(3′-[2′pyridyldithio]-propionamido) butane, disuccinimidyl suberate, disuccinimidyl tartrate, sulfodisuccinimidyl tartrate, dithiobis(succinimidyl propionate) (Lomant&#39;s reagent), 3,3′-dithiobis(sulfosuccinimidyl propionate), ethylene glycol bis(succinimidyl succinate), m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-γ-maleimidobutyryloxysuccinimide ester, N-γ-maleimidobutyryloxysulfosuccinimide ester, N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidocaproyloxy) succinimide ester, N-(ε-maleimidocaproyloxy) sulfo succinimide ester, N-(p-maleimidophenyl) isocyanate, N-succinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(N-maleimidomethyl) cyclohexane1-carboxylate, succinimidyl 4-(p-maleimidophenyl) butyrate, N-sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfo succinimidyl 4-(p-maleimidophenyl) butyrate, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, maleimide PEG N-hydroxysuccinimide ester, ρ-azidobenzoyl hydrazide, N-5-azido-2-nitrobenzyloxysuccinimide, ρ-azidophenyl glyoxal monohydrate, N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio) propionamide, bis(p-[4-azidosalicylamido]-ethyl) disulphide, N-hydroxysuccinimideyl-4-azidosalicyclic acid, N-hydroxysulfosuccinimidyl-4-azidobenzoate, sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate, sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-propionate, sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate, sulfosuccinimidyl (4-azidophenyl dithio)propionate, sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3-dithiopropionate, and the like. Glutaraldehyde may be used. The first and second polypeptide can be attached together by ECD/NHS coupling. 
     Alternatively, the first and second polypeptide may be genetically fused together. The first polypeptide and second polypeptide may for example by attached by a genetically encoded linker e.g. comprising (SG)n units (wherein n is typically from about 4 to about 50). A construct between the first and second polypeptide may be produced by native chemical ligation. Methods of cloning and expressing fusion polypeptides are well known to those skilled in the art and are described in, for example, Sambrook et al, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press. 
     A construct comprising the first and second polypeptide attached together, for example, as described above, is provided as a further aspect of the invention. 
     Reaction Conditions 
     In the invention, the first polypeptide and optionally the second polypeptide if present may be in solution. The concentration of the first and/or second polypeptide in solution is an operational parameter that can be controlled by the user to obtain required outputs. Typical concentrations are in the range of from about 1 to about 1000 μg of protein per mL of solution, such as from about 10 to about 100 μg/mL, e.g. from about 25 to about 75 μg/mL. The first polypeptide may be provided in a first solution and the second polypeptide provided in a second solution, and the first and second solutions may be mixed. Alternatively the first and second polypeptide may be co-formulated in a single solution. 
     Alternatively, the first polypeptide and optionally the second polypeptide if present may be immobilised on a support e.g. a solid support. The first polypeptide may be immobilised on a different solid support to the second polypeptide if present. The first polypeptide and second polypeptide if present may be immobilised on separate supports wherein the supports are the same type of support or are different types of support. The first polypeptide and second polypeptide may be immobilised on the same support. The first polypeptide and the second polypeptide may be co-immobilised on the support. The first polypeptide and the second polypeptide may be mixed and the mixture of the first and second polypeptides may be immobilised on the support. Alternatively the first polypeptide may be immobilised on the support and then the second polypeptide may be immobilised on the support. Still alternatively, the second polypeptide may be immobilised on the support and then the first polypeptide may be immobilised on the support. The first polypeptide may be provided in solution and the second polypeptide may be immobilised on a support. Alternatively the first polypeptide may be immobilised on a support and the second polypeptide may be provided in solution. 
     As used herein, the term “immobilized” embraces adsorption, entrapment and/or cross-linkage between the support and the polypeptide. Adsorption embraces non-covalent interactions including electrostatic interactions, hydrophobic interactions, and the like. A charged adsorption enhancer such as polymyxin B sulphate can be used to enhance adsorption. Entrapment embraces containment of the polypeptide onto the surface of the support, e.g. within a polymeric film or in a hydrogel. Cross-linkage embraces covalent attachment, either directly between the polypeptide (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 cross-linkers such as thiol-terminated linkers or crosslinking reagents. Immobilization means comprising or consisting of adsorption are preferred. Combination of some or all of the above mentioned immobilization means may be used. 
     Typically, the or each support independently comprises a material comprising carbon, silica, a metal or metal alloy, a metal oxide (include mixed metal oxides, e.g. titanium, aluminium and zirconium oxides), a metal hydroxide (including layered double hydroxides), a metal chalcogenide, or a polymer (e.g. polyaniline, polyamide, polystyrene, etc); or mixtures thereof. As those skilled in the art will appreciated, suitable support materials can include mixtures of materials described herein. Any suitable support material can be used. Resins and glasses can be used. 
     Sometimes, the or each support material comprises a carbon material. Suitable carbon materials include graphite, carbon nanotube(s), carbon black, activated carbon, carbon nanopowder, vitreous carbon, carbon fibre(s), carbon cloth, carbon felt, carbon paper, graphene, and the like. Sometimes the or each support material comprises a mineral such as bentonite, halloysite, kaolinite, montmorillonite, sepiolitem and hydroxyapatite. 
     Sometimes the or each support material comprises a biological material such as collagen, cellulose, keratin, carrageenan, chitin, chitosan, alginate and agarose. 
     Suitable materials may be in the form of a particle. Typical particle sizes are from about 1 nm to about 100 μm, such as from about 10 nm to about 10 μm e.g. from about 100 nm to about 1 μm. Methods of determining particle size are routine in the art and include, for example, dynamic light scattering. Support materials of appropriate size are readily available from commercial suppliers. For example, carbon black particles such as “Black Pearls 2000” particles are available from Cabot corp (Boston, Mass., USA). 
     A benefit which arises from the support of the first and/or second polypeptide is that the polypeptides can be easily removed from the reaction mixture. For example, the support(s) can be removed by sedimentation, filtration, centrifugation, or the like. Many such methods are known to those skilled in the art, e.g. filtration can be achieved using a simple filter paper to remove solid components from a liquid composition; or a mixed solid/liquid composition can be allowed to settle and the liquid then decanted from the settled solids. 
     The first polypeptide and second polypeptide if present may be present in a biological cell. This is an example of whole cell catalysis. The first polypeptide may be present in a different biological cell to the second polypeptide if present. The first polypeptide and second polypeptide if present may be present in biological cells wherein the cells are the same type of support or are different types of cell. The first polypeptide and second polypeptide may be present in the same biological cell. 
     The first polypeptide may be an exogenous polypeptide which is expressed non-natively by the cell. Alternatively the first polypeptide may be a native polypeptide which is natively expressed by the cell. The first polypeptide may be a native polypeptide in the cell and expressed under native conditions. The first polypeptide may be a native polypeptide in the cell, but expressed under non-native conditions, e.g. the first polypeptide may be overexpressed in the cell. The second polypeptide if present may be an exogenous polypeptide which is expressed non-natively by the cell. Alternatively the second polypeptide may be a native polypeptide which is natively expressed by the cell. The second polypeptide may be a native polypeptide in the cell and expressed under native conditions. The second polypeptide may be a native polypeptide in the cell, but expressed under non-native conditions, e.g. the second polypeptide may be overexpressed in the cell. Methods of cloning and expressing polypeptides in cells are well known to those skilled in the art and are described in, for example, Sambrook et al, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press. When the first and/or second polypeptide is present in a cell, the cell may be supported on a support as described herein. 
     When the first and/or second polypeptide is present in a cell, any suitable cell may be used. Typically, the cell is a bacterial or archaeal cell. Usually, the cell is a bacterial cell. Suitable bacterial cells include  Escherichia  cells,  Ralstonia  (also referred to as  Cupriavidus ) cells and  Pseudomonas  cells. For example, the bacterial cell may be an  E. coli  cell, a  Pseudomonas aeruginosa  cell or a  Ralstonia eutropha  (also known as  Cupriavidus necator ) cell. 
     In the methods of the invention, the cofactor is preferably initially added to or present in an aqueous solution at a concentration of 1 μM to 1 M, such as from 5 μM to 800 mM, e.g. from 10 μM to 600 mM such as from 25 μM to 400 mM e.g. from 50 μM to 200 mM such as from 100 μM to about 100 mM e.g. from about 250 μM to about 10 mM such as from about 500 μM to about 1 mM. 
     As explained above, the methods of the invention are typically conducted under a gas atmosphere; i.e. in the presence of gas (for example in the headspace of a reactor). Preferably, the gas atmosphere comprises hydrogen or an isotope thereof and optionally an inert gas. O 2  or an isotope thereof may be present. Preferred inert gases include nitrogen, argon, helium, neon, krypton, xenon, radon and sulfur hexafluoride (SF 6 ) and mixtures thereof, more preferably nitrogen and/or argon, most preferably nitrogen. 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%, with the remaining gas comprising an inert gas as defined herein and/or O 2 . Preferred gas atmospheres include from 80-100% H 2  with the remaining gas comprising one or more inert gases; and from 0-20% H 2  with the remaining gas comprising one or more inert gases and/or O 2  (such as from 14% H 2  in air). The gas atmosphere may optionally also include non-inert gases such as ammonia, carbon dioxide and hydrogen sulphide. Preferably, however, the gas atmosphere is free of ammonia, carbon dioxide and hydrogen sulphide. The methods of the invention may be conducted at any suitable pressure: selecting an appropriate pressure is an operational parameter of the methods of the invention which can be controlled by the operator. Sometimes, the methods of the invention are conducted at ambient pressure (e.g. about 1 bar). Sometimes, the methods of the invention are conducted at reduced pressure (e.g. less than 1 bar) or at elevated pressure (e.g. greater than 1 bar). For example, increasing the operating pressure can increase hydrogen solubility in the reaction medium. Preferably, the methods of the invention are carried out at a pressure of from about 0.1 bar to about 20 bar, such as from about 1 bar to about 10 bar, e.g. from about 2 bar to about 8 bar such as from about 4 bar to about 6 bar, e.g. about 5 bar. 
     Typically, the methods of the invention are carried out under aerobic conditions. As used herein, “aerobic conditions” refers to the gas atmosphere not being strictly anaerobic, e.g. comprising at least trace O 2 . Suitable O 2  levels are typically greater than 100 ppm, e.g. greater than 1000 ppm (0.1%), such as greater than 1% O 2 , for example greater than 2% 02. Usually, 02 levels do not exceed the O 2  levels in atmospheric air, i.e. 21% O 2 , however greater O 2  levels are not excluded. 
     The methods of the invention are typically conducted in an aqueous composition which may optionally comprise e.g. buffer salts. For some applications buffers are not required and the methods of the invention can be conducted without any buffering agents. Preferred buffer salts which can be used in the methods of the invention include Tris; phosphate; citric acid/Na 2 HPO 4 ; citric acid/sodium citrate; sodium acetate/acetic acid; Na 2 HPO 4 /NaH 2 PO 4 ; imidazole (glyoxaline)/HCl; sodium carbonate/sodium bicarbonate; ammonium carbonate/ammonium bicarbonate; MES; Bis-Tris; ADA; aces; PIPES; MOPSO; Bis-Tris Propane; BES; MOPS; TES; HEPES; DIPSO; MOBS; TAPSO; Trizma; HEPPSO; POPSO; TEA; EPPS; Tricine; Gly-Gly; Bicine; HEPBS; TAPS; AMPD; TABS; AMPSO; CHES; CAPSO; AMP; CAPS and CABS. Selection of appropriate buffers for a desired pH is routine to those skilled in the art, and guidance is available at e.g. http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html. Buffer salts are preferably used at concentrations of from 1 mM to 1 M, preferably from 10 mM to 100 mM such as about 50 mM in solution. Most preferred buffers for use in methods of the invention include 50 mM phosphate, pH 8.0. 
     The methods of the invention are typically conducted in an aqueous composition. However, non-aqueous components can optionally be used instead or as well as water in the compositions used in the methods of the invention. For example, one or more organic solvents (e.g. alcohols, DMSO, acetonitrile, etc) or one or more ionic liquids may be used or included in the compositions. 
     The methods of the invention are typically carried out at a temperature of from about 20° C. to about 80°, such as from about 25° C. to about 60° C., e.g. from about 30° C. to about 50° C. 
     The methods of the invention may be performed in an apparatus as provided herein. The apparatus typically comprises a reaction vessel. The reaction vessel typically comprises one or more inlets for molecular hydrogen gas or hydrogen-containing liquids (e.g. hydrogen saturated liquids such as buffer solutions as described herein); and/or one or more inlet for reagents; and/or one or more outlets for product. Further equipment such a pressure controls, temperature controls, mixing apparatus, flow controls, etc may be incorporated. The apparatus may be comprised as a part of an apparatus for converting initial reagents into final products and thus be configured to perform an intermediate reaction step. The apparatus may be controlled by equipment such as a computer controller. The apparatus may comprise means for detecting cofactor turnover, reagent utilisation and/or product production, e.g. spectrophotometric means. The apparatus may be configured to be operated in flow mode (i.e. continuous mode) or in batch mode. 
     Accordingly, the methods provided herein may be performed in a flow setup, e.g. in a flow reaction cell. The methods provided herein may alternatively be performed in a batch setup e.g. in a batch reaction cell. 
     Further Methods 
     The invention also provides a method of reducing an oxidised flavin cofactor, comprising:
         contacting the oxidised flavin cofactor and molecular hydrogen ( 1 H 2 ) or an isotope thereof with a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof under conditions such that the oxidised flavin cofactor is reduced to form a reduced flavin cofactor;   wherein the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction.       

     Preferably, such methods further comprise the re-oxidation of the reduced flavin cofactor to regenerate the oxidised flavin cofactor. Preferably, such method steps are repeated multiple times thereby recycling the cofactor. 
     In such methods, the oxidised flavin is typically as defined herein. The first polypeptide is typically as defined herein. In some embodiments the first polypeptide is in solution. In other embodiments the first polypeptide is immobilised on a solid support. Sometimes, the first polypeptide is comprised in a biological cell as defined here. Often, the reaction conditions are as set out in more detail herein. Other features of this aspect of the invention are typically as set out herein. 
     System 
     The invention also provides a system for performing a method of the invention. 
     The invention thus provides a system for reducing an oxidised flavin cofactor, comprising:
         a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof,   the oxidised flavin cofactor; and   molecular hydrogen ( 1 H 2 ) or an isotope thereof;
 
wherein the first polypeptide does not comprise a native flavin active site for NAD(P) +  reduction.
       

     The invention also provides a system for producing a reaction product, comprising:
         a first polypeptide which is a hydrogenase enzyme or a functional fragment or derivative thereof,   a flavin cofactor;   a second polypeptide which is an oxidoreductase or a functional fragment or derivative thereof,   molecular hydrogen ( 1 H 2 ) or an isotope thereof, and   a reactant for conversion to said reaction product.       

     In the systems of the invention, the flavin cofactor is typically as defined herein. The first polypeptide and second polypeptide if present are typically each as defined herein. The first polypeptide and second polypeptide is present are typically independently in solution, or are immobilised on a solid support. The first polypeptide and second polypeptide if present may be comprised in a biological cell. The first polypeptide and second polypeptide if present may be attached to each other. The system may be configured to be operated as described for the methods provided herein. 
     Typically, in the systems of the invention, one or both of the enzymes is in solution or is supported on a support as described herein. Typically, the system may further comprise means for controlling the gas atmosphere in the system, such as a gas flow system. The system is often configured as a flow cell containing reagents as described herein. The system (e.g. a flow cell) may comprise features of the provided apparatus described herein, such as one or more inlets for molecular hydrogen gas or hydrogen-containing liquids and/or one or more inlet for reagents; and/or one or more outlets for product; and/or one or more pressure controls, temperature controls, mixing apparatus, flow controls, etc 
     The following Examples illustrate the invention. They do not, however, limit the invention in any way. In this regard, it is important to understand that the particular assays used in the Examples section are designed only to provide an indication of the efficacy of the method of the invention. There are many assays available to determine reaction efficiency, and a negative result in any one particular assay is therefore not determinative. 
     EXAMPLES 
     Example 1 
     This example demonstrates a new activity for the [NiFe] uptake hydrogenase 1 of  Escherichia coli  (Hyd1) in accordance with the invention. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H 2  as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70° C.) and extended reaction times. The utility of this system as a simple, easy to implement FMNH 2  regenerating system is then demonstrated by supplying reduced flavin to an Old Yellow Enzyme for asymmetric alkene reductions with up to 100% conversion. High Hyd1 turnover frequencies (up to 20.4 min −1 ) and total turnover numbers (&gt;20,000) during flavin recycling demonstrate the efficacy of this biocatalytic system. 
     As the need to make chemical manufacturing more sustainable becomes urgent, academic and industrial fields increasingly turn to biotechnology. [1]  Enzymes provide many advantages over other catalysts: they are renewable, biodegradable, nonhazardous, and provide high selectivity. The once-limited scope of known enzyme reactions has rapidly expanded, aided by enzyme engineering and ongoing discovery and characterisation of new enzymatic functions. [2,3]   
     One class of synthetically useful enzymes are flavoenzymes, which contain or rely upon biological flavin cofactors (e.g. FMN, FAD; see below). For biotechnology, important flavoenzymes are halogenases (chlorination, bromination, iodination), [4]  ene-reductases (activated alkene reduction), [5]  and flavoprotein monooxygenases (epoxidations, hydroxylations, Baeyer-Villiger oxidation). [6]  Potential applications of these enzymes include natural product and pharmaceutical synthesis, [7]  biodegradation of environmental pollutants, [8]  and non-native light-driven reactions ( FIG.  9   ). [9]  These reactions require one equivalent of the reduced cofactors FMNH 2  or FADH 2 . To lower cost and waste, a catalytic quantity of more stable oxidised FMN/FAD is supplied, and is reduced in situ by means of photochemistry, electrochemistry, metal-catalysis or biocatalysis ( FIG.  9   ). [4,5,10]   
     In general, biocatalyzed cofactor recycling is the most straightforward option for coupling with flavoenzyme reactions because the alternative catalysts can face biocompatibility challenges (e.g. mutual inactivation, mismatched ideal solvent, pH or temperature). [4,11]  A common, yet cumbersome, strategy is to recycle the reduced flavin using an NAD(P)H-dependent reductase which produces FMNH 2  or FADH 2  at the expense of NAD(P)H [12]  or oxidised nicotinamide cofactor analogues. [13]  A catalytic quantity of the reduced nicotinamide cofactors must in turn be regenerated due to their high cost. This is typically achieved via glucose dehydrogenase-catalysed oxidation of glucose, which elevates cost, waste and downstream processing. [14]  The complexity of the currently-available recycling systems for reduced flavins may explain the under-utilisation of flavoenzymes in biotechnology, despite the important reactions they catalyse. [15]   
     
       
         
         
             
             
         
       
     
     Alternatively, H 2  has previously been demonstrated for cleaner enzymatic NADH cofactor recycling. [16,17]  The soluble hydrogenase from  Cupriavidus necator  (formerly  Alcaligenes eutrophus  or  Ralstonia eutropha ) couples the reduction of NADY to NADH using H 2 . This enzyme contains a prosthetic flavin cofactor at the NAD +  binding site where electrons from H 2  oxidation accumulate to reduce NAD + . [17]  Reduction of various substrates by this enzyme under H 2  has also been investigated. [18]  However, this enzyme is produced in the native host and requires extended incubation (7-10 days) in energy-depleted growth conditions for significant expression levels. [19]  Additionally, it lacks stability at elevated temperatures, [20]  preventing broad applicability. 
     This inspired us to test whether a simple hydrogenase system ( FIG.  6   ) could be suitable for H 2 -driven flavin reduction. The thermodynamic potential for the H + /H 2  redox couple (−0.472 V, pH 8) is negative of the potential for flavin reduction (−0.231 V, pH 8) [21] , making the reduction of flavin by H 2  thermodynamically favourable. We selected  E. coli  [NiFe]-hydrogenase (Hyd1), which is a good H 2  oxidiser [22,23]  and well-characterised in terms of X-ray crystal structures [24,25]  and spectroscopy. [23,26]  Hyd1 is natively expressed in  E. coli  and, unlike many hydrogenases, [27]  it is O 2 -tolerant [23]  and active over a wide pH range. [28]  Like other uptake hydrogenases, the basic unit of Hyd1 is a 100 kDa heterodimer of the large subunit (L) housing the NiFe active site, and the small subunit (S) housing the iron-sulfur cluster electron transfer relay. Natively, Hyd1 is coupled to a cytochrome electron acceptor, and exists as a homodimer of SL units. The isolated enzyme we utilize here comprises predominantly dimeric SL units. 
     The H 2  oxidation activity of Hyd1 is typically measured using the artificial electron acceptor benzyl viologen in colourimetric assays. [28]  Electrons from H 2  oxidation at the [NiFe] active site ( FIG.  6   ) are relayed through FeS clusters where, evidence suggests, benzyl viologen reduction occurs rather than directly at the NiFe active site. [29]  Herein, we demonstrate that both FMN and FAD can accept electrons from H 2  oxidation by Hyd1 to generate FMNH 2  and FADH 2 , and we show that Hyd1 can be used as an effective FMNH 2  regeneration system to support asymmetric alkene reduction by an Old Yellow Enzyme (OYE)-type ene-reductase. 
       FIG.  7    shows the results of in situ UV-visible spectrophotometric assays to explore the possibility of FMN and FAD reduction by Hyd1 (38 mg, produced and isolated as described below, see Supporting Information) under H 2  (General Procedure A, Supporting Information). The flavin moiety of FMN gives λ max  at 445 nm and FAD at 450 nm, both of which disappear upon two-electron reduction ( FIG.  7 A-B ; see  FIG.  13    for spectra of fully reduced FMN) [30,31] . The decrease in [oxidised flavin] over time was used to calculate initial enzyme activity ( FIG.  7 C-D ). Control experiments indicated that omission of Hyd1 or H 2  led to negligible flavin reduction ( FIGS.  10 - 11   ). 
     Upon addition of Hyd1, a lag phase was observed during FMN and FAD reduction, which is attributed to the well-characterised H 2 -dependent activation phase for aerobically purified Hyd1. [23]  Later experiments (when indicated) used Hyd1 that was first activated under a H 2  atmosphere. [32]  The lag phase was followed by a decrease in absorbance consistent with FMNIIH 2 /FADH 2  formation, and clear isosbestic points at 330 nm corroborated a lack of side products. Specific initial activities for FMN and FAD reduction (76 and 32 nmol min −1  mg −1  Hyd1, respectively) were determined during the linear reaction phase. The higher activity for FMN reduction compared with FAD cannot be attributed to thermodynamic driving force since both cofactors have similar reduction potentials, [21]  but could relate to the cofactors&#39; ability to interact at the protein surface. 
     Hyd1 is known to be robust which inspired us to test H 2 -driven flavin reduction activity at different temperatures (25-70° C., General Procedure A). Percentage conversion of FMN and FAD to the reduced forms after 30 min reaction time increased with temperature ( FIG.  8   ), though FMN reduction was not enhanced past 60° C. This temperature and pH tolerance of Hyd1 is likely to open new doors to cofactor recycling for flavoenzymes with optimal activity at higher temperatures. 
     In order to demonstrate the utility of Hyd1 in biotechnologically-relevant flavin recycling, we coupled Hyd1-catalysed flavin reduction with the OYE-type ene-reductase from  Thermus scotoductus , TsOYE, [33,34]  to catalyze enantioselective reduction of ketoisophorone (1) to (R)-levodione (2, see scheme below). Reactions were conducted according to General Procedure B (Supporting Information) and monitored using chiral-phase GC-FID after extraction of the mixture into ethyl acetate (see  FIG.  14   ). Enantiomeric excess (ee) was always &gt;99% at the first time point but decreased to 86-92% from slow racemisation under alkaline conditions. Control experiments confirmed that each component is required for conversion (Table 2). 
     
       
         
         
             
             
         
       
     
     Quantitative conversion and the highest Hyd1 turnover frequency (TOF, 0.34 s −1 ) was achieved with 0.5 mM FMN and 2 mM 1 (entry 1, Table 1). This TOF compares with or improves upon two-component NAD(P)H:flavin reductases, which unlike Hyd1 are rate-limited by the active site binding or release of two substrates. [15,35]   
     When 0.1 mM FMN was used with varying [1] (entries 2-5), up to 97 FMN turnovers (TN) were achieved. This is comparable to the FMN TN reported for a formate-driven homogeneous Rh-catalyzed method for FMNH 2  recycling coupled to TsOYE. [33]  That system required careful balance between enzyme and Rh-catalyst loading to prevent non-enantioselective alkene reduction by FMNH 2  or [Cp*Rh(bpy)H] + , which was not an appreciable issue with our biocatalytic system (Table 2). 
     The highest Hyd1 total turnover number (TTN, 10,200) was achieved using 10 mM 1 (entry 4). This TTN is of an appropriate order of magnitude for industrial catalysis, [36]  but there remains room for further optimisation to that end. At 20 mM 1, Hyd1 TTN and conversion were boosted using 4 bar H 2 , which also improved Hyd1 TOF from 0.9 s −1  to 0.11 s −1  (compare entries 5-6). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 H 2 -Driven enzymatic alkene reduction under various conditions 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 [1] 
                 [FMN] 
                 Conv. to 2 
                 Hyd1 
                 FMN 
               
               
                   
                 Entry 
                 (mM) 
                 (mM) 
                 (%) a   
                 TTN b   
                 TN b   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 0.5 
                 100 
                 2,100 
                 4 
               
               
                   
                 2 
                 2 
                 0.1 
                 100 
                 2,100 
                 20 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 3 
                 5 
                 0.1 
                 95 
                 {100} 
                 5,200 
                 50 
               
               
                   
                 4 
                 10 
                 0.1 
                 62 
                 {97} 
                 10,200 
                 97 
               
               
                   
                 5 
                 20 
                 0.1 
                 24 
                 {37} 
                 7,800 
                 74 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                      6 c   
                 20 
                 0.1 
                 {44} 
                 9,300 
                 88 
               
               
                   
                   
               
               
                   
                 Reaction conditions: General procedure B using consistent catalyst loadings. 
               
               
                   
                   a Chiral-phase GC conversion to 2 at 15 h {and 24 h}. 
               
               
                   
                   b Hyd1 total turnover number (mol 2 per mol Hyd1) and FMN turnover number (mol 2 per mol flavin) were determined at the end of the reaction. 
               
               
                   
                   c 4 bar H 2  was used. 
               
            
           
         
       
     
     Like Hyd1, TsOYE has enhanced activity at elevated temperatures, [33]  therefore entry 4 was replicated at 35° C. (data not shown): Hyd1 TOF nearly doubled to 0.16 s −1  and full conversion was achieved after 24 h, however GC-FID showed that some of 1 and 2 likely evaporated. 
     To test stability over time, Hyd1 (57 μg) was activated under H 2  at 22° C. over 58 h, then incubated in 0.08 mM FMN under H 2  (1 bar) in a sealed vessel for 62 h. Upon release of H 2 , FMNH 2  partially oxidised under the N 2  atmosphere to 0.05 mM FMN (determined using UV-visible spectroscopy). The Hyd1 and FMN/FMNH 2  solution was placed back under H 2 , and full reduction to FMNH 2  was noticed after 3.5 h (see Figure S 5 ), which demonstrates appreciable Hyd1 stability over 125 h (&gt;5 days). 
     The simplified, H 2 -driven biocatalysed flavin recycling method coupled with TsOYE led to high Hyd1 TOF and TTN that correspond with commercial grade enzymes. [37]  Further modifications to Hyd1, which is tolerant of mutagenesis, [25,32]  might enhance the non-native activity. Additionally, process development is underway to improve industrially-relevant metrics such as cofactor TN. This proof of concept work shows that the robust Hyd1, tolerant to a range of conditions, is a promising catalyst to bring clean flavin recycling into biotechnology. This work thus demonstrates the efficacy and utility of the invention provided herein. 
     Supporting Information 
     Reagents 
     Buffer salts (Sigma Aldrich), FAD (disodium salt, &gt;98%, Cayman Chemical Company), and FMN (monosodium salt dihydrate, Applichem Panreac) were all used as received. All aqueous solutions were prepared with deoxygenated MilliQ water (Millipore, 18 MΩcm). The hydrogenase ( E. coli  hydrogenase 1, Hyd1, molecular weight 100 kDa) was produced by homologous over-expression of the genes encoding the structural subunits of the enzyme and key maturases. After Hyd1 overexpression under anaerobic bacterial growth, the enzyme was isolated following published protocols [S2]  and stored at −80° C. as a 3.8 mg/mL solution in Tris-HCl buffer (20 mM Tris-HCl pH 7.2, 350 mM NaCl, 0.02% Triton X, 1 mM DTT). The  Thermus scotoductus  ene-reductase of the Old Yellow Enzyme family (TsOYE, molecular weight of homodimer 72.4 kDa) [S3,S4]  was isolated following published protocols. 
     Analytical Tools 
     UV-visible spectra were recorded by a Cary 60 spectrophotometer with a cell holder (Agilent) and a Peltier accessory for temperature control using a quartz cuvette (path length 1 cm, cell volume 1 mL, Hellma). The indicated buffer was used to take a baseline scan. In some of the experiments, there was a uniform shift of the baseline across the entire spectral region (200-800 nm), which was corrected for during data processing. The concentration of FMN was directly calculated based on the absorbance at λ=445 nm (ε=12.50 mM −1  cm −1 ) and FAD based on the absorbance at λ=450 nm (ε=11.30 mM −1  cm −1 ). 
     The decrease in [oxidized flavin] over time was determined in order to calculate specific initial enzyme activity (not counting any lag phase). [S5]   
     Chiral phase GC-FID to monitor alkene reductions was performed as follows: 
     Column: CP-Chirasil-Dex CB (Agilent), 25 m length, 0.25 mm diameter, 0.25 μm (film thickness), fitted with a guard of 10 m undeactivated fused silica of the same diameter 
     Carrier: He (CP grade), 170 kPa (constant pressure) 
     Inlet temperature: 200° C. 
     Injection conditions: Splitless with split flow 60 mL/min, splitless time 0.8 mins, 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, temp=200° C.) 
     Oven Heating Profile: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Temperature 
               
               
                   
                   
               
             
            
               
                   
                 0 → 5 
                 Hold at 70° C. 
               
               
                   
                  5 → 30 
                 Ramp to 120° C. at 2° C./min 
               
               
                   
                 30 → 36 
                 Ramp to 180° C. at 10° C./min 
               
               
                   
                 36 → 45 
                 Hold at 180° C. for 5 minutes 
               
               
                   
                   
               
            
           
         
       
     
     Compound Retention Times (Reduction of 1): 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Compound 
               
               
                   
                   
               
             
            
               
                   
                 12.27 
                 Ketoisophorone (1) 
               
               
                   
                 12.68 
                 (R)-Levodione (2) 
               
               
                   
                 12.80 
                 (S)-Levodione (2) 
               
               
                   
                   
               
            
           
         
       
     
     Experimental Procedures 
     All experiments were carried out in a glovebox (Glove Box Technology Ltd) under a protective N 2  atmosphere (O 2 &lt;0.1 ppm). Stock solutions of FAD and FMN were prepared using deoxygenated buffer. Different concentrations of stock solutions of 1 were prepared in DMSO such that DMSO was 1 vol % in the final reaction mixture. 
     General Procedure A (Flavin Reduction) 
     The indicated volume of Tris-HCl buffer (50 mM, pH 8.0) or phosphate buffer (50 mM, pH 8.0) was added to a UV-visible quartz cuvette, which was placed in the cell holder and allowed to warm to the indicated temperature (pre-set on the Peltier accessory) for 5 min. A baseline was recorded using the UV-visible spectrophotometer. A solution of 0.1 mM flavin (unless otherwise noted) in the designated buffer was next prepared in the cuvette, which was then capped with a rubber septum that was pierced with two needles to provide a gas inlet and outlet. An H 2 -line was then connected and bubbled through the flavin solution via the inlet needle for 10 minutes. The needle was then moved up to the headspace through which a continuous H 2  flow was supplied. About 0.4 mL of the flavin solution was then used to transfer the designated quantity of Hyd1 into the cuvette using a syringe and needle, and the needle and syringe rinsed by drawing solution in and out of the cuvette. The assay was carried out by taking one scan (200-800 nm) every 30 seconds over 30 minutes. 
     General Procedure B (Alkene Reduction) 
     Using a syringe and needle, 600 μL of H 2 -saturated Tris-HCl buffer (50 mM, pH 8.0, 25° C.) was transferred to a centrifuge tube (Eppendorf, 1.5 mL) that contained the required quantities of FMN and 1 in DMSO (1 vol % DMSO in total reaction mixture). A portion of this solution (approx. 0.2 mL) was used to transfer Hyd1 (57 μg, activated under H 2  for 3-15 h) and TsOYE (145 μg) into the reaction tube in sequence via a needle and syringe. The lid of the centrifuge tube was pierced once with a needle, capped, and placed in a Büchi Tinyclave pressure vessel which was then charged to the designated pressure of H 2 . The pressure vessel was then removed from the glovebox and wrapped in aluminum foil to exclude light in order to prevent photodecomposition of the FMN, flavoenzyme, or both. [S6]  The vessel was placed on a Stuart® mini see-saw rocker set to 30 oscillations/min. The extent of conversion and enantiomeric excess (% ee) of (R)-2 was determined by chiral GC-FID (General Procedure C). 
     General Procedure C (Preparing Samples for Chiral GC Analysis) 
     Aliquots (25 μL) of reaction mixture were taken for analysis at 1 h and 15 h (and 24 h when indicated) then extracted into 200 μL EtOAc with 2 mM undecane as an internal standard. The biphasic solution was centrifuged to separate out any solids (12,000×g, 2 min), then 150 μL of the EtOAc layer was removed, dried over Na 2 SO 4  and 75 μL of the solution was taken for GC analysis. 
     Supplementary Data and Results 
       FIG.  9    shows current applications and methods of flavin recycling A. Examples of flavoenzymes applied toward natural products and analogues, [S7-9]  degradation of an environmental pollutant, [S10]  and a non-native light-driven cyclisation. [S11]  B. Current enzymatic flavin regeneration methods rely on NAD(P)H, which itself is continually regenerated using expensive, carbon-based sacrificial reductants. C. Other catalytic methods for flavin recycling tend to rely on cosubstrate additives. D. (This work) A simplified H 2 -driven direct flavin reduction method using Hyd1 enzyme. 
       FIGS.  10  to  12    show control experiments to confirm role of Hyd1 and H 2  in flavin reduction:
           FIG.  10    shows Background flavin reduction in absence of H 2 . Reaction conditions: 800 μL scale, 0.1 mM flavin in Tris-HCl buffer (50 mM, pH 8, 25° C.), g Hyd1, 25° C. controlled by Peltier accessory. The Hyd1 specific activity for FAD and FMN reduction during this control reaction was 0.06 nmol min −1  mg −1  and 2.08 nmol min −1  mg −1  respectively.     FIG.  11    shows background flavin reduction in absence of Hyd1. Reaction conditions: 800 μL scale, 0.1 mM flavin in Tris-HCl buffer (50 mM, pH 8, 25° C.), H 2  flow (cuvette head space), 25° C. controlled by Peltier accessory. The overall decrease in [FAD] and [FMN] amounts to 0.000 mM and 0.005 mM after 30 minutes respectively.     FIG.  12    shows background flavin reduction in the absence of H 2  and Hyd1. Reaction conditions: 800 μL scale, 0.1 mM flavin in Tris-HCl buffer (50 mM, pH 8, 25° C.), 25° C. controlled by Peltier accessory. The overall decrease in [FAD] and [FMN] amounts to 0.000 mM and 0.080 mM after 30 minutes respectively.       

     Complete reduction of FMN to FMNH 2  was also demonstrated.  FIG.  13    shows UV-visible spectra of FMN and FMNH 2  produced by Hyd1 under H 2  or sodium dithionite (gray). 
     Reaction conditions for FMN reduction by Hyd1: 800 μL scale, 0.1 mM FMN in Tris-HCl buffer (50 mM, pH 8, 25° C.), H 2  flow (cuvette head space), 57 μg Hyd1, 25° C. controlled by Peltier accessory. The full reduction of FMN by Hyd1 was completed during the experiment designed to test the stability of Hyd1 over time (&gt;5 days). 
     Reaction conditions for FMN reduction by sodium dithionite (gray): 800 μL scale, 0.1 mM FMN in Tris-HCl buffer (50 mM, pH 8, 25° C.), 0.15 mM sodium dithionite, 25° C. controlled by Peltier accessory. 
     Control experiments for H 2 -driven ketoisophorone reduction were also conducted. Control experiments were performed to see if Hyd1 (entry 1) or TsOYE (entry 2), alone, could lead to 2. In addition, similar experiments were done in the absence of FMN (entry 3) or no enzyme (entry 4). The control experiments demonstrated the need for each reaction component for the reaction to be successful. The results of the experiment are shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Control experiments for H 2 -driven ketoisophorone reduction 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Conversion 
               
               
                 Entry 
                 FMN 
                 TsOYE 
                 Hyd1 
                 to 2 (%) 
               
               
                   
               
               
                 1 
                 ✓ 
                 — 
                 ✓ 
                 0 
               
               
                 2 
                 ✓ 
                 ✓ 
                 — 
                 0 
               
               
                 3 
                 — 
                 ✓ 
                 ✓ 
                 0 
               
               
                 4 
                 ✓ 
                 — 
                 — 
                 0 
               
               
                   
               
               
                 Reaction conditions: 600 μL scale, 0.1 mM FAD, 57 μg Hyd1, TsOYE (145 μg), 10 mM ketoisophorone, Tris-HCl buffer (50 mM, pH 8.0, 25° C.), 1 vol % DMSO at ambient temperature in pressure vessel (1 bar H 2 ), 24 h. 
               
            
           
         
       
     
     Exemplary chiral-phase GC-FID spectra of enzymatic H 2 -driven reduction of ketoisophorone to (R)-levodione was demonstrated. Reduction of 1 (entry 4, Table 1) was carried out and the reaction mixture was analysed by chiral GC-FID according to General Procedure C. Conversion to 1 and the enantiomeric excess (% ee) were calculated based on the peak area of their respective peaks as shown in  FIG.  14   , which shows GC-FID results of ketoisophorone reductions (gray). Ketoisophorone, (rac)-levodione and (R)-levodione standards were diluted using EtOAc with 2 mM undecane as internal standard. 
     Example 2 
     This example demonstrates further examples of flavin-cycling coupled to ene-reductions in accordance with the claimed invention. H 2  is used as a reductant with Hyd1 catalysing flavin reduction to allow catalytic reduction of the alkenes dimethyl itaconate and 4-phenyl-3-buten-2-one. 
     We extended the H 2 -driven system described in Example 1 to a suite of commercially-available ene-reductases (Johnson Matthey). The alkene reductions demonstrated were dimethyl itaconate (3) reduction to dimethyl (R)-methyl succinate (4) and 4-phenyl-3-buten-2-one (5) reduction to 4-phenyl-2-butanone (6) (Table 1), using the same protocols established for TsOYE in Example 1. Control experiments demonstrated conversion in the presence of Hyd1, FMN, and ENE. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 H 2 -driven enzymatic alkene reductions using commercial ene-reductases 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 [FMN] 
                 Ene- 
                 Time 
                 Conv. 
                 ee 
               
               
                 Entry 
                 Substrate 
                 (mM) 
                 reductase 
                 (h) 
                 (%) a   
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                  1 
                 3 
                 0.5 
                 ENE-101 
                 17 
                  75 
                 n.d. b   
               
               
                  2 
                 3 
                 0.1 
                 ENE-103 
                 42 
                  81 
                 &gt;99 
               
               
                  3 
                 3 
                 0.5 
                 ENE-103 
                 42 
                  98 
                 &gt;99 
               
               
                  4 
                 3 
                 0.5 
                 ENE-108 
                 17 
                  99 
                 n.d. b   
               
               
                  5 
                 3 
                 0.5 
                 ENE-109 
                 17 
                 100 
                 n.d. b   
               
               
                  6 
                 5 
                 0.5 
                 ENE-103 
                 17 
                 100 
                 n.a. d   
               
               
                  7 c   
                 5 
                 0.1 
                 ENE-107 
                 24 
                  20 ± 1 
                 n.a. d   
               
               
                  8 c   
                 5 
                 0.5 
                 ENE-107 
                 24 
                  33 ± 3 
                 n.a. d   
               
               
                  9 
                 5 
                 0.1 
                 ENE-107 
                 40 
                  35 
                 n.a. d   
               
               
                 10 
                 5 
                 0.5 
                 ENE-107 
                 40 
                 100 
                 n.a. d   
               
               
                 11 
                 5 
                 0.5 
                 ENE-108 
                 17 
                 100 
                 n.a. d   
               
               
                 12 
                 5 
                 0.5 
                 ENE-109 
                 17 
                 100 
                 n.a. d   
               
               
                   
               
               
                 Reaction conditions: 23.7 μg/mL Hyd1, 0.5 mg/mL ene-reductase and 5 mM substrate in Tris-HCl (50 mM, pH 8), 1 vol % DMSO at room temperature (20-30° C.) in a sealed vessel under an H 2  (1 bar) atmosphere. 
               
               
                   a GC conversions to 4 or 6. 
               
               
                   b Not determined. 
               
               
                   c Entries 3 and 4 were performed in triplicate and are shown ±1 standard deviation, and were separate experiments from entries 5 and 6. 
               
               
                   c Not applicable. 
               
            
           
         
       
     
     High conversions to (R)-4 (measured by GC) were reached with ENE-101 (https://matthey.com/en/products-and-services/pharmaceutical-and-medical/catalysts/ene-101), -103 (https://matthey.com/en/products-and-services/pharmaceutical-and-medical/catalysts/ene-103), -108 (https://matthey.com/en/products-and-services/pharmaceutical-and-medical/catalysts/ene-108), and -109 (https://matthey.com/en/products-and-services/pharmaceutical-and-medical/catalysts/ene-109) (Table 3, entries 1-5). With ENE-103, we confirmed that enantioselective (&gt;99% ee) reduction to (R)-4 was achieved using chiral-phase GC-FID (entries 2-3). Conversion of 5 to 6 was successful using ENE-103, -107 (https://matthey.com/en/products-and-services/pharmaceutical-and-medical/catalysts/ene-107), -108, and -109 (entries 6-12). Entries 7-8 were performed in triplicate in order to demonstrate the high reproducibility of the reaction. 
       FIG.  15    shows characterisation data for the enzymatic reduction of 5 to 6 by GC-FID.  FIG.  15    confirms that the methods of the invention can be used to reduce 5 to 6 with extremely high conversion efficiency. 
     Experimental conditions for the data collected in  FIG.  15    were as follows: 
     Methods: 
     4-phenyl-3-buten-2-one (commercially available, 99%) and 4-phenyl-2-butanone (commercially available, 98%) standards were diluted using EtOAc. 
     GC Method: 
     Instrument: ThermoScientific Trace 1310 GC; Column: CP-Chirasil-Dex CB (Agilent), 25 m length, 0.25 mm diameter, 0.25 m (film thickness), fitted with a guard of 10 m undeactivated fused silica of the same diameter; Carrier: He (CP grade), 170 kPa (constant pressure); Inlet temperature: 200° C.; Injection conditions: Splitless with split flow 60 mL/min, splitless time 0.8 min, purge 5 mL/min. Injection volume=0.1 μL. Detection: FID (H 2 =35 mL/min, air=350 mL/min, makeup N 2 =40 mL/min, temp=200° C.) 
     Oven Heating Profile: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Temperature 
               
               
                   
                   
               
             
            
               
                   
                 0 → 5 
                 Hold at 70° C. 
               
               
                   
                  5 → 30 
                 Ramp to 120° C. at 2° C./min 
               
               
                   
                 30 → 36 
                 Ramp to 180° C. at 10° C./min 
               
               
                   
                 36 → 45 
                 Hold at 180° C. for 5 minutes 
               
               
                   
                   
               
            
           
         
       
     
     Compound Retention Times: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Compound 
               
               
                   
                   
               
             
            
               
                   
                 14.48 
                 4-Phenyl-3-buten-2-one (5) 
               
               
                   
                 12.86 
                 4-Phenyl-2-butanone (6) 
               
               
                   
                   
               
            
           
         
       
     
       FIG.  16    shows characterisation data for the enzymatic reduction of 3 to 4 by GC-FID. 
       FIG.  15    confirms that the methods of the invention can be used to reduce 3 to 4 at with up to 100% conversion. 
     Experimental conditions for the data collected in  FIG.  16    were as follows: 
     Methods: 
     GC-FID results of dimethyl itaconate reductions (top 2 panels). Dimethyl itaconate (commercially available, 98%), (rac)-dimethyl methyl succinate (commercially available, 98%) and dimethyl (R)-methyl succinate (commercially available, 99%) standards were diluted using EtOAc. 
     GC Method: 
     Instrument: ThermoFinnigan Trace GC; Column: Cyclosil-B (Agilent), 30 m length, 0.25 mm diameter, 0.25 m (film thickness); Carrier: He (CP grade), 100 kPa (constant pressure); Inlet temperature: 220° C.; Injection volume: 2 μL; Detection: FID (H 2=35  mL/min, air=350 mL/min, N2=30 mL/min, temp=250° C. 
     Oven Heating Profile: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Temperature 
               
               
                   
                   
               
             
            
               
                   
                  0 → 160 
                 Hold at 70° C. 
               
               
                   
                 160 → 170 
                 Ramp to 180° C. at 20° C./min 
               
               
                   
                   
               
            
           
         
       
     
     Compound Retention Times: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Time (minutes) 
                 Compound 
               
               
                   
                   
               
             
            
               
                   
                 83.67 
                 Dimethyl itaconate (3) 
               
               
                   
                 58.50 
                 (R)-Dimethyl methyl succinate (4) 
               
               
                   
                 60.76 
                 (S)-Dimethyl methyl succinate (4) 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 thus confirms the broad applicability of the methods provided herein. 
     Example 3 
     This example demonstrates further examples of flavin-cycling, coupled to other enzymatic reductions. In this example, H 2 -driven flavin reduction was used to allow nitroreductases to reduce a nitro group (here 2-methyl-5-nitropyridine) to the corresponding amine, in accordance with the methods provided herein. 
     Hyd1-catalysed H 2 -driven FMN and FAD recycling was coupled with nitroreductase (NR) enzymes (engineered, prepared and provided by Johnson Matthey; E. C. 1.7.1.16). These nitroreductase enzymes contain an FMN prosthetic group, and have been reported for use with GDH (glucose dehydrogenase)/glucose to continually supply a catalytic quantity of NADPH to the nitroreductase, which in turn will reduce aromatic nitro groups with the assistance of V 2 O 5  as a co-catalyst (Scheme 1a). 38  However, such methods provide a mixture of the corresponding amine, hydroxylamine, and other undesired side products. 
     a. NR and Vanadium-Catalysed Nitro Reduction 
     
       
         
         
             
             
         
       
     
     b. NR-Catalysed Nitro Reduction by H 2 -Driven FMN Recycling (this Work) 
     
       
         
         
             
             
         
       
     
     Scheme 1. Contrast between previously known methods of nitroreductase-catalysed nitro reductions and methods provided herein. 
     By contrast, the method provided herein for supplying an externally reduced FMN to OYE-type ene-reductases (see Example 1) can be extended for use other flavin reductases without generating unwanted side products. Here, we used Johnson Matthey nitroreductases (Scheme 1b). Thus, using the methods provided herein efficient conversions to the desired 5-amino-2-methylpyridine were confirmed using  1 H NMR spectroscopy, with comparison against commercially available authentic starting material and product standards. Over the time course of the reaction conducted, conversion of up to 70% was observed; longer reaction times would be expected to lead to more complete conversion. The hydroxylamine product was prepared following a known procedure, 39  which resulted in a mixture of amine and hydroxylamine products as confirmed by  1 H NMR spectroscopy (see  FIG.  17   ). 
     Reactions conditions were as follows: 2-methyl-5-nitropyridine (5 mM), 1 mM FMN, Hyd1 (32 μg/mL) and NR (80 μg/mL) were dissolved in H 2 -saturated Tris-HCl (50 mM, pH 8.0) with 2 vol % DMSO at room temperature, then left to react under an atmosphere (1 bar) of H 2  in a 40° C. water bath for 3 h. 
       1 H NMR spectra were obtained at room temperature on a Bruker Advance III HD nanobay (400 MHz) using water suppression. Samples was prepared with a total volume of 500 μL, containing 200 μL reaction solution, 250 μL deionised water, and 50 μL D 2 O. Compound standards were prepared for NMR spectroscopic analysis using: 
     2-Methyl-5-aminopyridine:  1 H NMR (400 MHz, H 2 O+D 2 O, pH 8.0) δ 7.93 (d, J=2.8 Hz, 1H), 7.21 (dd, J=8.4, 2.8 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 2.38 (s, 3H). 
     2-Methyl-5-hydroxylaminopyridine 39 :  1 H NMR (400 MHz, H 2 O+D 2 O) δ 8.13 (d, J=2.7 Hz, 1H), 7.42 (dd, J=8.4, 2.7 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 2.44 (s, 3H). 
     2-Methyl-5-nitropyridine:  1 H NMR (400 MHz, H 2 O+D 2 O, pH 8.0) δ 9.24 (d, J=2.7 Hz, 1H), 8.51 (dd, J=8.7, 2.7 Hz, 1H), 7.54 (d, J=8.7 Hz, 1H), 2.65 (s, 3H). 
     Example 4 
     This example demonstrates flavin reduction by the methods of the invention using a range of other hydrogenase enzymes. 
     The reduction of flavin cofactors, FAD and FMN by hydrogenases different to Hyd1 was shown using Hydrogenase-2 (Hyd2) (PDB code: 6EHQ, EC 1.12.99.6—SEQ ID NOs: 3/4) from  E. coli  and the NiFe hydrogenase from  Desulfovibrio vulgaris  Miyazaki F (DvMF) (PDB code: 1WUJ, EC 1.12.2.1; SEQ ID NOs: 23/24) 
       E. coli  Hyd2 is relatively more oxygen sensitive than  E. coli  Hyd1 and works reversibly catalysing both H 2  oxidation and evolution efficiently. On supply of H 2 , flavin reduction activity by Hyd2 was observed and the resulting UV-vis spectra showing flavin reduction are shown in  FIG.  18   . Activities were measured during the linear phase of the reaction.  FIG.  18    shows activity assay results for Hyd2 catalysed reduction of A) FMN and B) FAD measured by in situ UV-visible Spectroscopy, C) calculated [FMN] based on λ max =445 nm (ε=12.50 mM −1  cm −1 ), D) calculated [FAD] based on λ max =450 nm (ε=11.30 mM −1  cm −1 ). Reaction conditions: 800 μL scale, 0.1 mM flavin, 0.048 mg/mL Hyd2 (activated under 1 bar H 2  for 20 h), TrisHCl buffer (pH 8), temp: 25° C. Hydrogenase specific activity was calculated as: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Hydrogenase 
               
               
                   
                   
                 specific activity 
               
               
                   
                 Cofactor 
                 (nmol min −1  mg −1 ) 
               
               
                   
                   
               
             
            
               
                   
                 FMN 
                 31 
               
               
                   
                 FAD 
                 17 
               
               
                   
                   
               
            
           
         
       
     
     Similar reduction of flavins by  Desulfovibrio vulgaris  Miyazaki F (DvMF) NiFe hydrogenase was also observed and their activities calculated.  FIG.  19    shows activity assay for DvMF catalysed reduction of A) FMN and B) FAD measured by in situ UV-visible Spectroscopy, C) calculated [FMN] based on λ max =445 nm (ε=12.50 mM −1  cm −1 ), D) calculated [FAD] based on λ max =450 nm (ε=11.30 mM −1  cm −1 ). Reaction conditions: 800 ul scale, 0.1 mM flavin, 0.045 mg/ml DvMF (activated under a flow of H 2  for 45 min), Tris HCl buffer (pH 8), temp: 25° C. Hydrogenase specific activity was calculated as: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Specific activity 
               
               
                   
                 Cofactor 
                 (nmol min −1  mg −1 ) 
               
               
                   
                   
               
             
            
               
                   
                 FMN 
                 70 
               
               
                   
                 FAD 
                 51 
               
               
                   
                   
               
            
           
         
       
     
     For FMN, only a 20 minute activity assay was performed, however, almost full conversion of FMN to FMNH 2  was observed during this reaction time. Similar to Hyd1, the flavin reduction activity was higher for FMN when compared to that of FAD during reduction reactions by both the enzymes. 
     Those skilled in the art will appreciate that a direct comparison of the reduction activities by the two enzymes (Hyd2 and DvMF) cannot be made, due to differences in techniques used for enzyme activation under hydrogen, and uncertainty with regard to the activation states and purities of the enzymes. However, the data presented in this example confirms that the methods of the invention are widely applicable to and are not restricted to specific hydrogenases. 
     Example 5 
     This example demonstrates the solvent tolerance of the methods provided herein. 
     The specific activity of  E. coli  Hyd1 for FAD reduction was measured by UV/vis assay (recording A 450 nm  over time,    FAD λ=450 nm) =0.0113 mol −1  dm 3  cm −1 ) at different mixtures of water:DMSO and water:acetonitrile. 
       FIG.  20 A  shows the specific activity of  E. coli  Hyd1 for FAD reduction (pmol min −1  mg −1 ) measured at different mixtures of water: DMSO. The reaction mixture included 0.055 mg mL −1  Hyd1, and 0.1 mM FAD under 1 bar H 2  at 25° C. Specific activity was measured from the initial linear phase of activity over approx. 20 minutes, following a brief lag phase corresponding to hydrogenase activation. 
       FIG.  20 B  shows the specific activity of  E. coli  Hyd1 for FAD reduction (μmol min −1  mg −1 ) measured at different mixtures of water:acetonitrile (CH 3 CN). The reaction mixture included 0.055 mg mL −1  Hyd1, and 0.1 mM FAD under 1 bar H 2  at 25° C. Specific activity was measured from the initial linear phase of activity over approx. 20 minutes, following a brief lag phase corresponding to hydrogenase activation. 
     The results in this example confirm that the methods of the invention are applicable to a range of reaction conditions including different ratios of solvent and water. Enzymatic reduction can be carried out even at elevated solvent levels. 
     REFERENCES 
     
         
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                 SEQUENCE LISTING 
                   
               
               
                 SEQ ID NO: 1 -  Escherichia coli  hydrogenase 1 (large subunit). 
               
               
                 MSTQYETQGYTINNAGRRLVVDPITRIEGHMRCEVNINDQNVITNAVSCGTMFRGLEIIL 
               
               
                   
               
               
                 QGRDPRDAWAFVERICGVCTGVHALASVYAIEDAIGIKVPDNANIIRNIMLATLWCHDHL 
               
               
                   
               
               
                 VHFYQLAGMDWIDVLDALKADPRKTSELAQSLSSWPKSSPGYFFDVQNRLKKFVEGGQLG 
               
               
                   
               
               
                 IFRNGYWGHPQYKLPPEANLMGFAHYLEALDFQREIVKIHAVFGGKNPHPNWIVGGMPCA 
               
               
                   
               
               
                 INIDESGAVGAVNMERLNLVQSIITRTADFINNVMIPDALAIGQFNKPWSEIGTGLSDKC 
               
               
                   
               
               
                 VLSYGAFPDIANDFGEKSLLMPGGAVINGDFNNVLPVDLVDPQQVQEFVDHAWYRYPNDQ 
               
               
                   
               
               
                 VGRHPFDGITDPWYNPGDVKGSDTNIQQLNEQERYSWIKAPRWRGNAMEVGPLARTLIAY 
               
               
                   
               
               
                 HKGDAATVESVDRMMSALNLPLSGIQSTLGRILCRAHEAQWAAGKLQYFFNKLMTNLKNG 
               
               
                   
               
               
                 NLATASTEKWEPTTWPTECRGVGFTEAPRGALGHWAAIRDGKIDLYQCVVPTTWNASPRD 
               
               
                   
               
               
                 PKGQIGAYEAALMNTKMAIPEQPLEILRTLHSFDPCLACSTHVLGDDGSELISVQVR 
               
               
                   
               
               
                 SEQ ID NO: 2 -  Escherichia coli  hydrogenase 1 (small subunit). 
               
               
                 MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGAGMAPKIAWALENKPRIPVVWIHGL 
               
               
                   
               
               
                 ECTCCTESFIRSAHPLAKDVILSLISLDYDDTLMAAAGTQAEEVFEDIITQYNGKYILAV 
               
               
                   
               
               
                 EGNPPLGEQGMFCISSGRPFIEKLKRAAAGASAIIAWGTCASWGCVQAARPNPTQATSID 
               
               
                   
               
               
                 KVITDKPIIKVPGCPPIPDVMSAIITYMVTFDRLPDVDRMGRPLMFYGQRIHDKCYRRAH 
               
               
                   
               
               
                 FDAGEFVQSWDDDAARKGYCLYKMGCKGPTTYNACSSTRWNDGVSFPIQSGHGCLGCAEN 
               
               
                   
               
               
                 GFWDRGSFYSRVVDIPQMGTHSTADTVGLTALGVVAAAVGVHAVASAVDQRRRHNQQPTE 
               
               
                   
               
               
                 TEHQPGNEDKQA 
               
               
                   
               
               
                 SEQ ID NO: 3 -  Escherichia coli  hydrogenase 2 (large subunit). 
               
               
                 MSQRITIDPVTRIEGHLRIDCEIENGVVSKAWASGTMWRGMEEIVKNRDPRDAWMIVQRICGVCTT 
               
               
                   
               
               
                 THALSSVRAAESALNIDVPVNAQYIRNIILAAHTTHDHIVHFYQLSALDWVDITSALQADPTKASE 
               
               
                   
               
               
                 MLKGVSTWHLNSPEEFTKVQNKIKDLVASGQLGIFANGYWGHPAMKLPPEVNLIAVAHYLQALECQ 
               
               
                   
               
               
                 RDANRVVALLGGKTPHIQNLAVGGVANPINLDGLGVLNLERLMYIKSFIDKLSDFVEQVYKVDTAV 
               
               
                   
               
               
                 IAAFYPEWLTRGKGAVNYLSVPEFPTDSKNGSFLFPGGYIENADLSSYRPITSHSDEYLIKGIQES 
               
               
                   
               
               
                 AKHSWYKDEAPQAPWEGTTIPAYDGWSDDGKYSWVKSPTFYGKTVEVGPLANMLVKLAAGRESTQN 
               
               
                   
               
               
                 KLNEIVAIYQKLTGNTLEVAQLHSTLGRIIGRTVHCCELQDILQNQYSALITNIGKGDHTTFVKPN 
               
               
                   
               
               
                 IPATGEFKGVGFLEAPRGMLSHWMVIKDGIISNYQAVVPSTWNSGPRNFNDDVGPYEQSLVGTPVA 
               
               
                   
               
               
                 DPNKPLEVVRTIHSFDPCMACAVH 
               
               
                   
               
               
                 SEQ ID NO: 4 -  Escherichia coli  hydrogenase 2 (small subunit). 
               
               
                 MEMAESVTNPQRPPVIWIGAQECTGCTESLLRATHPTVENLVLETISLEYHEVLSAAFGHQVEENK 
               
               
                   
               
               
                 HNALEKYKGQYVLVVDGSIPLKDNGIYCMVAGEPIVDHIRKAAEGAAAIIAIGSCSAWGGVAAAGV 
               
               
                   
               
               
                 NPTGAVSLQEVLPGKTVINIPGCPPNPHNFLATVAHIITYGKPPKLDDKNRPTFAYGRLIHEHCER 
               
               
                   
               
               
                 RPHFDAGRFAKEFGDEGHREGWCLYHLGCKGPETYGNCSTLQFCDVGGVWPVAIGHPCYGCNEEGI 
               
               
                   
               
               
                 GFHKGIHQLANVENQTPRSQKPDVNAKEGGHHHHHH 
               
               
                   
               
               
                 SEQ ID NO: 5 -  Ralstonia eutropha  membrane-bound hydrogenase 
               
               
                 moiety (HoxG). 
               
               
                 MSAYATQGFNLDDRGRRIVVDPVTRIEGHMRCEVNVDANNVIRNAVSTGTMWRGLEVILK 
               
               
                   
               
               
                 GRDPRDAWAFVERICGVCTGCHALASVRAVENALDIRIPKNAHLIREIMAKTLQVHDHAV 
               
               
                   
               
               
                 HFYHLHALDWVDVMSALKADPKRTSELQQLVSPAHPLSSAGYFRDIQNRLKRFVESGQLG 
               
               
                   
               
               
                 PFMNGYWGSKAYVLPPEANLMAVTHYLEALDLQKEWVKIHTIFGGKNPHPNYLVGGVPCA 
               
               
                   
               
               
                 INLDGIGAASAPVNMERLSFVKARIDEIIEFNKNVYVPDVLAIGTLYKQAGWLYGGGLAA 
               
               
                   
               
               
                 TNVLDYGEYPNVAYNKSTDQLPGGAILNGNWDEVFPVDPRDSQQVQEFVSHSWYKYADES 
               
               
                   
               
               
                 VGLHPWDGVTEPNYVLGANTKGTRTRIEQIDESAKYSWIKSPRWRGHAMEVGPLSRYILA 
               
               
                   
               
               
                 YAHARSGNKYAERPKEQLEYSAQMINSAIPKALGLPETQYTLKQLLPSTIGRTLARALES 
               
               
                   
               
               
                 QYCGEMMHSDWHDLVANIRAGDTATANVDKWDPATWPLQAKGVGTVAAPRGALGHWIRIK 
               
               
                   
               
               
                 DGRIENYQCVVPTTWNGSPRDYKGQIGAFEASLMNTPMVNPEQPVEILRTLHSFDPCLAC 
               
               
                   
               
               
                 STHVMSAEGQELTTVKVR 
               
               
                   
               
               
                 SEQ ID NO: 6 -  Ralstonia eutropha  membrane-bound hydrogenase 
               
               
                 moiety (HoxK). 
               
               
                 MVETFYEVMRRQGISRRSFLKYCSLTATSLGLGPSFLPQIAHAMETKPRTPVLWLHGLEC 
               
               
                   
               
               
                 TCCSESFIRSAHPLAKDVVLSMISLDYDDTLMAAAGHQAEAILEEIMTKYKGNYILAVEG 
               
               
                   
               
               
                 NPPLNQDGMSCIIGGRPFIEQLKYVAKDAKAIISWGSCASWGCVQAAKPNPTQATPVHKV 
               
               
                   
               
               
                 ITDKPIIKVPGCPPIAEVMTGVITYMLTFDRIPELDRQGRPKMFYSQRIHDKCYRRPHFD 
               
               
                   
               
               
                 AGQFVEEWDDESARKGFCLYKMGCKGPTTYNACSTTRWNEGTSFPIQSGHGCIGCSEDGF 
               
               
                   
               
               
                 WDKGSFYDRLTGISQFGVEANADKIGGTASVVVGAAVTAHAAASAIKRASKKNETSGSEH 
               
               
                   
               
               
                 SEQ ID NO: 7 -  Ralstonia eutropha  membrane-bound hydrogenase 
               
               
                 moiety (HoxZ). 
               
               
                 MSTKMQADRIADATGTDEGAVASGKSIKATYVYEAPVRLWHWVNALAIVVLAVTGFFIGS 
               
               
                   
               
               
                 PPATRPGEASANFLMGYIRFAHFVAAYIFAIGMLGRIYWATAGNHHSRELFSVPVFTRAY 
               
               
                   
               
               
                 WQEVISMLRWYAFLSARPSRYVGHNPLARFAMFFIFFLSSVFMILTGFAMYGEGAQMGSW 
               
               
                   
               
               
                 QERMFGWVIPLLGQSQDVHTWHHLGMWFIVVFVIVHVYAAIREDIMGRQSVVSTMVSGYR 
               
               
                   
               
               
                 TFKD 
               
               
                   
               
               
                 SEQ ID NO: 8 -  Ralstonia eutropha  regulatory hydrogenase moiety 
               
               
                 (HoxB). 
               
               
                 MNAPVCTGLASAKPGVLNVLWIQSGGCGGCSMSLLCADTTDFTGMLKSAGIHMLWHPSLS 
               
               
                   
               
               
                 LESGVEQLQILEDCLQGRVALHALCVEGAMLRGPHGTGRFHLLAGTGVPMIEWVSRLAAV 
               
               
                   
               
               
                 ADYTLAVGTCAAYGGITAGGGNPTDACGLQYEGDQPGGLLGLNYRSRAGLPVINVAGCPT 
               
               
                   
               
               
                 HPGWVTDALALLSARLLTASDLDTLGRPRFYADQLVHHGCTRNEYYEFKASAEKPSDLGC 
               
               
                   
               
               
                 MMENMGCKGTQAHADCNTRLWNGEGSCTRGGYACISCTEPGFEEPGHPFHQTPKVAGIPI 
               
               
                   
               
               
                 GLPTDMPKAWFVALASLSKSATPKRVKLNATADHPLIAPAIRKTRLK 
               
               
                   
               
               
                 SEQ ID NO: 9 -  Ralstonia eutropha  regulatory hydrogenase moiety 
               
               
                 (HoxC). 
               
               
                 MERLVVGPFNRVEGDLEVNLEVASGRVCSARVNATMYRGLEQILLHRHPLDALVYAPRVC 
               
               
                   
               
               
                 GICSVSQSVAASRALADLAGVTVPANGMLAMNLMLATENLADHLTHFYLFFMPDFTREIY 
               
               
                   
               
               
                 AGRPWHTDATARFSPTHGKHHRLAIAARQRWFTLMGTLGGKWPHTESVQPGGSSRAIDAA 
               
               
                   
               
               
                 ERVRLLGRVREFRCFLEQTLYAAPLEDVVALDSEVALWRWHAQAPQAGDLRCFLTIAQDA 
               
               
                   
               
               
                 ALDQMGPGPGTYLSYGAYPQPEGGFCFAQGVWRSAQGRLDALDLAAISEDATSAWLVDQG 
               
               
                   
               
               
                 GARHPANGLTAPAPDKVGAYTWNKAPRLAGAVLETGAIARQLAGAQPLVRDAVARCGATV 
               
               
                   
               
               
                 YTRVLARLVELARVVPLMEDWLQSLEIGAPYWASAHLPDQGAGVGLTEAARGSLGHWVSV 
               
               
                   
               
               
                 RDGRIDNYQIVAPTSWNFSPRDIAGQPGAVEKALEGAPVLQGETTPVAVQHIVRSFDPCM 
               
               
                   
               
               
                 VCTVH 
               
               
                   
               
               
                 SEQ ID NO: 10 -  Aquifex aeolicus  hydrogenase 1 (large subunit). 
               
               
                 MKRVVVDPVTRIEGHLRIEIMVDEETGQVKDALSAGTMWRGIELIVRNRDPRDVWAFTQR 
               
               
                   
               
               
                 ICGVCTSIHALASLRAVEDALEITIPKNANYIRNIMYGSLQVHDHVVHFYHLHALDWVSP 
               
               
                   
               
               
                 VEALKADPVATAALANKILEKYGVLNEFMPDFLGHRAYPKKFPKATPGYFREFQKKIKKL 
               
               
                   
               
               
                 VESGQLGIFAAHWWDHPDYQMLPPEVHLIGIAHYLNMLDVQRELFIPQVVFGGKNPHPHY 
               
               
                   
               
               
                 IVGGVNCSISMDDMNAPVNAERLAVVEDAIYTQVESTDFFYIPDILAIADIYLNQHNWFY 
               
               
                   
               
               
                 GGGLSKKRVIGYGDYPDEPYTGIKNGDYHKKILWHSNGVVEDFYKGVEKAKFYNLEGKDF 
               
               
                   
               
               
                 TDPEQIQEFVTHSWYKYPDETKGLHPWDGITEPNYTGPKEGTKTHWKYLDENGKYSWIKA 
               
               
                   
               
               
                 PRWRGKACEVGPLARYIIVYTKVKQGHIKPTWVDELIVNQIDTVSKILNLPPEKWLPTTV 
               
               
                   
               
               
                 GRTIARALEAQMSAHTNLYWMKKLYDNIKAGDTSVANMEKWDPSTWPKEAKGVGLTEAPR 
               
               
                   
               
               
                 GALGHWVIIKDGKVANYQCVVPTTWNGSPKDPKGQHGAFEESMIDTKVKVPEKPLEVLRG 
               
               
                   
               
               
                 IHSFDPCLACSTHLYNEKGEEIASVRVQGVVHV 
               
               
                   
               
               
                 SEQ ID NO: 11 -  Aquifex aeolicus  hydrogenase 1 (small subunit). 
               
               
                 METFWEVFKRHGVSRRDFLKFATTITGLMGLAPSMVPEVVRAMETKPRVPVLWIHGLECT 
               
               
                   
               
               
                 CCSESEIRSATPLASDVVLSMISLEYDDTLSAAAGEAVEKHRERIIKEYWGNYILAVEGN 
               
               
                   
               
               
                 PPLGEDGMYCIIGGRPFVEILKESAEGAKAVIAWGSCASWGCVQAAKPNPTTAVPIDKVI 
               
               
                   
               
               
                 KDKPIIKVPGCPPIAEVMTGVIMYMVLFDRIPPLDSQGRPKMFYGNRIHDTCYRRSFFNA 
               
               
                   
               
               
                 GQFVEQFDDEGAKKGWCLYKVGCRGPTTYNSCGNMRWYNGLSYPIQSGHGCIGCAENNFW 
               
               
                   
               
               
                 DNGPFYERIGGIPVPGIESKADKVGAIAAAAAAGGAIIHGIASKIRKSGEKEE 
               
               
                   
               
               
                 SEQ ID NO: 12 -  Hydrogenovibrio marinus  hydrogenase (large 
               
               
                 subunit). 
               
               
                 MSVLNTPNHYKMDNSGRRVVIDPVTRIEGHMRCEVNVDENNVIQNAVSTGTMWRGLEVIL 
               
               
                   
               
               
                 RGRDPRDAWAFVERICGVCTGCHALASVRAVEDALDIKIPHNATLIREIMAKTLQIHDHI 
               
               
                   
               
               
                 VHFYHLHALDWVNPVNALKADPQATSELQKLVSPHHPMSSPGYFKDIQIRIQKFVDSGQL 
               
               
                   
               
               
                 GIFKNGYWSNPAYKLSPEADLMAVTHYLEALDFQKEIVKIHAIFGGKNPHPNYMVGGVPC 
               
               
                   
               
               
                 AINIDGDMAAGAPINMERLNFVKSLIEQGRTFNTNVYVPDVIAIAAFYRDWLYGGGLSAT 
               
               
                   
               
               
                 NVMDYGAYPKTPYDKSTDQLPGGAIINGDWGKIHPVDPRDPEQVQEFVTHSWYKYPDETK 
               
               
                   
               
               
                 GLHPWDGITEPNYELGSKTKGSRTNIIEIDESAKYSWIKSPRWRGHAVEVGPLARYILAY 
               
               
                   
               
               
                 AQGVEYVKTQVHTSLNRFNAVCRLLDPNHKDITDLKAFLGSTIGRTLARALESEYCGDMM 
               
               
                   
               
               
                 LDDFNQLISNIKNGDSSTANTDKWDPSSWPEHAKGVGTVAAPRGALAHWIVIEKGKIKNY 
               
               
                   
               
               
                 QCVVPTTWNGSPRDPKGNIGAFEASLMGTPMERPDEPVEVLRTLHSFDPCLACSTHVMSE 
               
               
                   
               
               
                 EGEEMATVKVR 
               
               
                   
               
               
                 SEQ ID NO: 13 -  Hydrogenovibrio marinus  hydrogenase (small 
               
               
                 subunit). 
               
               
                 MSSQVETFYEVMRRQGITRRSFLKYCSLTAAALGLSPAYANKIAHAMETKPRTPVIWLHG 
               
               
                   
               
               
                 LECTCCSESFIRSAHPLAKDVVLSMISLDYDDTLMAASGHAAEAILDEIKEKYKGNYILA 
               
               
                   
               
               
                 VEGNPPLNQDGMSCIIGGRPFSEQLKRMADDAKAIISWGSCASWGCVQAAKPNPTQATPV 
               
               
                   
               
               
                 HKFLGGGYDKPIIKVPGCPPIAEVMTGVITYMLTFDRIPELDRQGRPKMFYSQRIHDKCY 
               
               
                   
               
               
                 RRPHFDAGQFVEEWDDEGARKGYCLYKVGCKGPTTYNACSTVRWNGGTSFPIQSGHGCIG 
               
               
                   
               
               
                 CSEDGFWDKGSFYSRDTEMNAFGIEATADDIGKTAIGVVGAAVVAHAAISAVKAAQKKGD 
               
               
                   
               
               
                 K 
               
               
                   
               
               
                 SEQ ID NO: 14 -  Thiocapsa roseopersicina  hydrogenase HupL. 
               
               
                 MSVTTANGFELDTAGRRLVVDPVTRIEGHLRCEVNLDENNVIRNAVSTGTMWRGLEVILR 
               
               
                   
               
               
                 GRDPRDAWAETERICGVCTGTHALTSVRAVEDALGIPIPENANSIRNIMHVTLQAHDHLV 
               
               
                   
               
               
                 HFYHLHALDWVDVVSALGADPKATSALAQSISDWPKSSPGYFRDVQNRLKRFVESGQLGP 
               
               
                   
               
               
                 FMNGYWGSPAYKLPPEANLMAVTHYLEALDFQKEIVKIHTVYGGKNPHPNWLVGGMPCAI 
               
               
                   
               
               
                 NVDGTGAVGAINMERLNLVSSIIDQTIAFIDKVYIPDLIAIASFYKDWTYGGGLSSQAVM 
               
               
                   
               
               
                 SYGDIPDHANDMSSKNLLLPRGAIINGNLNEIHEIDLRNPEEIQEFVDHSWFSYKDETRG 
               
               
                   
               
               
                 LHPWDGVTEPNFVLGPNAVGSRTRIEALDEQAKYSWIKAPRWRGHAMEVGPLARYVIGYA 
               
               
                   
               
               
                 KGIPEFKEPVDKVLTDLGQPLEAIFSTLGRTAARGLEASWAAHKMRYFQDKLVANIRAGD 
               
               
                   
               
               
                 TATANVDNWDPKTWPKEARGVGTTEAPRGALGHWIVIKDGKIDNYQAVVPTTWNGSPRDP 
               
               
                   
               
               
                 AGNIGAFEASLLNTPLAKADEPLEILRTLHSFDPCLACATHIMGPDGEELTRIKVR 
               
               
                   
               
               
                 SEQ ID NO: 15 -  Thiocapsa roseopersicina  hydrogenase HupS. 
               
               
                 MPTTETYYEVMRRQGITRRSFLKFCSLTATALGLSPTFAGKIAHAMETKPRIPVVWLHGL 
               
               
                   
               
               
                 ECTCCSESFIRSAHPLVSDVILSMISLDYTILIMAAAGHQAEAILEEVRHKHAGNYILAV 
               
               
                   
               
               
                 EGNPPLNQDGMSCIIGGRPFLEQLLEMADSCKAVISWGSCASWGCVQAARPNPTRATPVH 
               
               
                   
               
               
                 EVIRDKPVIKVPGCPPIAEVMTGVLTYILTFDRLPELDRQGRPLMFYGQRIHDKCYRRPH 
               
               
                   
               
               
                 FDAGQFVESWDDEGARRGYCLYKVGCKGPTTYNACSTIRWNGGVSFPIQSGHGCIGCSED 
               
               
                   
               
               
                 GFWDKGSFYQHVTDTHAFGIEANADRTGIAVATRRGAAHRAHAAVSVVKRVQQKKEEDQS 
               
               
                   
               
               
                 SEQ ID NO: 16 -  Alteromonas macleodii  hydrogenase small subunit 
               
               
                 MALPTLNKQLQASGISRRTFLKFCATTASLLALPQSAVADLATALGNARRPSVIWLPFQE 
               
               
                   
               
               
                 CTGCTEAILRSHAPTLESLIFDHISLDYQHTIMAAAGEQAEDARRAAMNAHKGQYLLLVD 
               
               
                   
               
               
                 GSVPVGNPGYSTISGMSNVDMLRESAKDAAGIIAIGTCASFGGIPKANPNPTGAVAVSDI 
               
               
                   
               
               
                 ITDKPIVNISGCPPLPIAITAVLVHYLTFKRFPDLDELQRPLAFFGESIHDRCYRRPFFE 
               
               
                   
               
               
                 QRKFAKSFDDEGAKNGWCLFELGCKGPETFNACATVKWNQGTSFPIESGHPCLGCSEPDF 
               
               
                   
               
               
                 WDKSSFYQALGPWEWYKSKPGKGAQKHAGKNS 
               
               
                   
               
               
                 SEQ ID NO: 17 -  Alteromonas macleodii  hydrogenase large subunit. 
               
               
                 MENTASNNRLVVDPITRIEGHLRIEAEMDGNTIKQAFSSGTSVRGIELILQGRDPRDAWA 
               
               
                   
               
               
                 FAQRICGVCTLVHGMASVRAVEDAIRKAWRSNAKLGVAIGKPSMTSMPKGPMQHGKKGHR 
               
               
                   
               
               
                 QSRTSIGVLSEAEMAIPQNAQLIRNIMIATQYVHDHVMHFYHLHALDWVDVVSALDADPT 
               
               
                   
               
               
                 RTATLAGQLSDYPRSSPGYFKDVKQKVKTLVESGQLGIFSNAYWGHPGYKLPPEVNLMAL 
               
               
                   
               
               
                 AHYLDALTWQREVVKVHTIFGGKNPHPNFVVGGVPSPINLNASTGINTSRLVQLQDAITQ 
               
               
                   
               
               
                 MKSFVDQVYYPDIVAIAGYYKEWGTRGEGLGNFLTYGDLPMTSMDDPDSFLFPRGAILGR 
               
               
                   
               
               
                 DLSKVHDLDLDDPSEIQEFVSSSWYRYSGGNASGLHPFNGQTTLEYTGPKPPYKHLNVGA 
               
               
                   
               
               
                 EYSWLKSPRWKGHAMEVGPLARVLMMYAKKDAAAQDIVNRSLSILDLETSALFSTLGRTL 
               
               
                   
               
               
                 ARAVETKIVVNQLQSWYDQLLDNIAKGDTDTFNPLYFDPTNWPIKGQGVGVMEAPRGALG 
               
               
                   
               
               
                 HWLVMQNGKIENYQCVVPTTWNAGPRDPNSQAGAYEAALQDKHTLHDPDQPLEILRTLHS 
               
               
                   
               
               
                 FDPCLACAVHVMDETGEERLRLKVR 
               
               
                   
               
               
                 SEQ ID NO: 18 -  Allochromatium vinosum  Membrane Bound Hydrogenase 
               
               
                 large subunit. 
               
               
                 MSERIVVDPVTRIEGHLRIEAQMDGENIAQAYSSGTSVRGLETILKGRDPRDAWAFAQRI 
               
               
                   
               
               
                 CGVCTLVHGIASVRSVEDALKIELPPNAQLIRNLMISSQFVHDHVMHFYHLHALDWVDVV 
               
               
                   
               
               
                 SALSADPKATSDLAQSISSWPKSSPGYFADTQKRIKTFVESGQLGIFANGYWGHPAYKLP 
               
               
                   
               
               
                 PEANLMAVAHYLEALAWQRDVARLHAIFGGKNPHPNFVVGGVPSPIDIDSDSAINAKRLA 
               
               
                   
               
               
                 EVQQILQSMQTFVDQVYVPDTLAIASFYKDWGERGEGLGNFMSYGDLPATGTMDPAQFLF 
               
               
                   
               
               
                 PRGVILNRDLSTIHEIDLHDAGQIQEYVAHSWYEYSGGNDQGLHPYDGETNLEYDARGGV 
               
               
                   
               
               
                 KPPYTQLDVNDGYSWMKAPRWKGHAMEVGPLARVLLLYASGHEQTKELVEMTLTTLDLPV 
               
               
                   
               
               
                 RALYSTLGRTAARTLETKILTDTAQDWYNQLIANIKAGDSRTFNETLWEPSSWPAEARGA 
               
               
                   
               
               
                 GYMEAPRGALGHWIVIKDRKIANYQAVVPSTWNAGPRDPSDQPGAYEAALQDNHQLVDVK 
               
               
                   
               
               
                 QPIEILRTIHSFDPCIACAVHLTDPETGEQMEIKIT 
               
               
                   
               
               
                 SEQ ID NO: 19 -  Allochromatium vinosum  Membrane Bound Hydrogenase 
               
               
                 small subunit. 
               
               
                 PSVVWLSFQECTGCTESLTRAHAPTLEDLILDFISLDYHHTLQAASGEAAEAARLQAMDE 
               
               
                   
               
               
                 NRGQYLVIVDGSIPGPDANPGFSTVAGHSNYSILMETVEHAAAVIAVGTCAAFGGLPQAR 
               
               
                   
               
               
                 PNPTGAMSVMDLVRDKPVINVPGCPPIPMVITGVIAHYLVFGRLPEVDGYGRPLAFYGQS 
               
               
                   
               
               
                 IHDRCYRRPFYDKGLFAESFDDEGAKQGWCLYRLGCKGPTTYNACATMKWNDGTSWPVEA 
               
               
                   
               
               
                 GHPCLGCSEPQFWDAGGFYEPVSVPLTLGPATLLGAGAAGAVVGGGLAALSRKKGRDAAA 
               
               
                   
               
               
                 TRQPVTVDELEQKL 
               
               
                   
               
               
                 SEQ ID NO: 20 -  Salmonella enterica  serovar Typhimurium LT2 
               
               
                 nickel-iron hydrogenase 5 Large subunit. 
               
               
                 MAYPYQTQGFTLDNSGRRIVVDPVTRIEGHMRCEVNIDSNNVITNAVSTGTMWRGLEVIL 
               
               
                   
               
               
                 KGRDPRDAWAFVERICGVCTGTHALTSIRAVENALGIAIPDNANCIRNMMQATLHVHDHL 
               
               
                   
               
               
                 VHFYHLHALDWVDVVAALKADPHQTSAIAQSLSAWPLSSPGYFRDLQNRLKRFIESGQLG 
               
               
                   
               
               
                 PFRNGYWGHPAMKLPPEANLLAVAHYLEALDFQKEIVKIHTVFGGKNPHPNWLVGGVPCA 
               
               
                   
               
               
                 INLDETGAVGAVNMERLNLVSSIIQKARQFCEQVYLPDVLLIASYYKDWAKIGGGLSSMN 
               
               
                   
               
               
                 LLAYGEFPDNPNDYSASNLLLPRGAIINGRFDEIHPVDLTAPDEIQEFVTHSWYTYGNGN 
               
               
                   
               
               
                 NDKGLHPWDGLTEPQLVMGEHYKGTKTFIEQVDESAKYSWIKSPRWKGHAMEVGPLARYL 
               
               
                   
               
               
                 IGYHQNKPEFKEPVDQLLSVLKLPKEALFSTLGRTAARALESVWAGNTLQYFFDRLMRNL 
               
               
                   
               
               
                 KSGDTATANVTLWEPDTWPTSAKGVGFSEAPRGALGHWIKIANQKIDSYQCVVPTTWNAG 
               
               
                   
               
               
                 PRDDKGQIGAYEAALMGTKLAVPDQPLEILRTLHSFDPCLACSTH 
               
               
                   
               
               
                 SEQ ID NO: 21 -  Salmonella enterica  serovar Typhimurium LT2 
               
               
                 nickel-iron hydrogenase 5 Small subunit. 
               
               
                 LENKPRTPVIWLHGLECTCCTESFIRSAHPLAKDAILSLISLDYDDTIMAAAGQQAEQAL 
               
               
                   
               
               
                 ADVMREYKGNYIVAVEGNAPLNEDGMFCILAGEPFLEKLKRVSADAKAIIAWGSCASWGC 
               
               
                   
               
               
                 VQAARPNPTKATPVHKLITDKPIIKVPGCPPIPEVMSAVITYMLAFDRIPPLDRLGRPKM 
               
               
                   
               
               
                 FYGQRIHDKCYRRAHFDAGQFVEAWDDEGARKGYCLYKMGCKGPTTYNACSTVRWNDGVS 
               
               
                   
               
               
                 FPIQSGHGCLGCSEDGFWDYGSFYSRATGSRSHHHHHHH 
               
               
                   
               
               
                 SEQ ID NO: 22   Escherichia coli  cytochrome HyaC. 
               
               
                 MQQKSDNVVSHYVFEAPVRIWHWLTVLCMAVLMVTGYFIGKPLPSVSGEATYLFYMGYIRLIHFSA 
               
               
                   
               
               
                 GMVFTVVLLMRIYWAFVGNRYSRELFIVPVWRKSWWQGVWYEIRWYLFLAKRPSADIGHNPIAQAA 
               
               
                   
               
               
                 MFGYFLMSVFMIITGFALYSEHSQYAIFAPFRYVVEFFYWTGGNSMDIHSWHRLGMWLIGAFVIGH 
               
               
                   
               
               
                 VYMALREDIMSDDTVISTMVNGYRSHKFGKISNKERS 
               
               
                   
               
               
                 SEQ ID NO: 23 -  Desulfovibrio vulgaris  Miyazaki F hydrogenase 
               
               
                 (large subunit). 
               
               
                 SSYSGPIVVDPVTRIEGHLRIEVEVENGKVKNAYSSSTLFRGLEIILKGRDPRDAQHFTQRTCGVC 
               
               
                   
               
               
                 TYTHALASTRCVDNAVGVHIPKNATYIRNLVLGAQYLHDHIVHFYHLHALDFVDVTAALKADPAKA 
               
               
                   
               
               
                 AKVASSISPRKTTAADLKAVQDKLKTFVETGQLGPFTNAYFLGGHPAYYLDPETNLIATAHYLEAL 
               
               
                   
               
               
                 RLQVKAARAMAVFGAKNPHTQFTVVGGVTCYDALTPQRIAEFEALWKETKAFVDEVYIPDLLVVAA 
               
               
                   
               
               
                 AYKDWTQYGGTDNFITFGEFPKDEYDLNSRFFKPGVVFKRDFKNIKPFDKMQIEEHVRHSWYEGAE 
               
               
                   
               
               
                 ARHPWKGQTQPKYTDLHGDDRYSWMKAPRYMGEPMETGPLAQVLIAYSQGHPKVKAVTDAVLAKLG 
               
               
                   
               
               
                 VGPEALFSTLGRTAARGIETAVIAEYVGVMLQEYKDNIAKGDNVICAPWEMPKQAEGVGFVNAPRG 
               
               
                   
               
               
                 GLSHWIRIEDGKIGNFQLVVPSTWTLGPRCDKNKLSPVEASLIGTPVADAKRPVEILRTVHSFDPC 
               
               
                   
               
               
                 IACGVH 
               
               
                   
               
               
                 SEQ ID NO: 24 -  Desulfovibrio vulgaris  Miyazaki F hydrogenase 
               
               
                 (small subunit). 
               
               
                 LMGPRRPSVVYLHNAECTGCSESVLRAFEPYIDTLILDTLSLDYHETIMAAAGDAAEAALEQAVNS 
               
               
                   
               
               
                 PHGFIAVVEGGIPTAANGIYGKVANHTMLDICSRILPKAQAVIAYGTCATFGGVQAAKPNPTGAKG 
               
               
                   
               
               
                 VNDALKHLGVKAINIAGCPPNPYNLVGTIVYYLKNKAAPELDSLNRPTMFFGQTVHEQCPRLPHFD 
               
               
                   
               
               
                 AGEFAPSFESEEARKGWCLYELGCKGPVTMNNCPKIKFNQTNWPVDAGHPCIGCSEPDFWDAMTPF 
               
               
                   
               
               
                 YQN 
               
               
                   
               
               
                 SEQ ID NO: 31 - Chromate Reductase, ‘TsOYE’ from  Thermus   
               
               
                   scotoductus . 
               
               
                 MALLFTPLELGGLRLKNRLAMSPMCQYSATLEGEVTDWHLLHYPTRALGGVGLILVEATA 
               
               
                   
               
               
                 VEPLGRISPYDLGIWSEDHLPGLKELARRIREAGAVPGIQLAHAGRKAGTARPWEGGKPL 
               
               
                   
               
               
                 GWRVVGPSPIPFDEGYPVPEPLDEAGMERILQAFVEGARRALRAGFQVIELHMAHGYLLS 
               
               
                   
               
               
                 SFLSPLSNQRTDAYGGSLENRMRFPLQVAQAVREVVPRELPLFVRVSATDWGEGGWSLED 
               
               
                   
               
               
                 TLAFARRLKELGVDLLDCSSGGVVLRVRIPLAPGFQVPFADAVRKRVGLRTGAVGLITTP 
               
               
                   
               
               
                 EQAETLLQAGSADLVLLGRVLLRDPYFPLRAAECALGVAPEVPPQYQRGF 
               
               
                   
               
               
                 SEQ ID NO: 32 - NADPH Dehydrogenase 1, ‘OYE-1’,  Saccharomyces   
               
               
                   pastorianus . 
               
               
                 MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRALHPGNIPNRDWAVEYYTQ 
               
               
                   
               
               
                 RAQRPGTMIITEGAFISPQAGGYDNAPGVWSEEQMVEWTKIFNAIHEKKSFVWVQLWVLG 
               
               
                   
               
               
                 WAAFPDNLARDGLRYDSASDNVFMDAEQEAKAKKANNPQHSLTKDEIKQYIKEYVQAAKN 
               
               
                   
               
               
                 SIAAGADGVEIHSANGYLLNQFLDPHSNTRTDEYGGSIENRARFTLEVVDALVEAIGHEK 
               
               
                   
               
               
                 VGLRLSPYGVFNSMSGGAETGIVAQYAYVAGELEKRAKAGKRLAFVHLVEPRVTNPFLTE 
               
               
                   
               
               
                 GEGEYEGGSNDFVYSIWKGPVIRAGNFALHPEVVREEVKDKRTLIGYGRFFISNPDLVDR 
               
               
                   
               
               
                 LEKGLPLNKYDRDTFYQMSAHGYIDYPTYEEALKLGWDKK 
               
               
                   
               
               
                 SEQ ID NO: 33: NADPH Dehydrogenase 2, ‘OYE-2’,  Saccharomyces   
               
               
                   cerevisiae  strain ATCC 204508/S288c. 
               
               
                 MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQ 
               
               
                   
               
               
                 RAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLG 
               
               
                   
               
               
                 WAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKN 
               
               
                   
               
               
                 SIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEK 
               
               
                   
               
               
                 VGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE 
               
               
                   
               
               
                 GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDR 
               
               
                   
               
               
                 LEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN 
               
               
                   
               
               
                 SEQ ID NO: 34: NADPH Dehydrogenase, ‘YqjM’,  Bacillus subtilis . 
               
               
                 MARKLFTPITIKDMTLKNRIVMSPMCMYSSHEKDGKLTPFHMAHYISRAIGQVGLIIVEASAVNPQ 
               
               
                   
               
               
                 GRITDQDLGIWSDEHIEGFAKLTEQVKEQGSKIGIQLAHAGRKAELEGDIFAPSAIAFDEQSATPV 
               
               
                   
               
               
                 EMSAEKVKETVQEFKQAAARAKEAGFDVIEIHAAHGYLIHEFLSPLSNHRTDEYGGSPENRYRFLR 
               
               
                   
               
               
                 EIIDEVKQVWDGPLFVRVSASDYTDKGLDIADHIGFAKWMKEQGVDLIDCSSGALVHADINVFPGY 
               
               
                   
               
               
                 QVSFAEKIREQADMATGAVGMITDGSMAEEILQNGRADLIFIGRELLRDPFFARTAAKQLNTEIPA 
               
               
                   
               
               
                 PVQYERGW 
               
               
                   
               
               
                 SEQ ID NO: 35: Xenobiotic Reductase A, ‘XenA’,  Pseudomonas putida . 
               
               
                 MSALFEPYTLKDVTLRNRIAIPPMCQYMAEDGMINDWHHVHLAGLARGGAGLLVVEATAVAPEGRI 
               
               
                   
               
               
                 TPGCAGIWSDAHAQAFVPVVQAIKAAGSVPGIQIAHAGRKASANRPWEGDDHIAADDTRGWETIAP 
               
               
                   
               
               
                 SAIAFGAHLPKVPREMTLDDIARVKQDFVDAARRARDAGFEWIELHFAHGYLGQSFFSEHSNKRTD 
               
               
                   
               
               
                 AYGGSFDNRSRFLLETLAAVREVWPENLPLTARFGVLEYDGRDEQTLEESIELARRFKAGGLDLLS 
               
               
                   
               
               
                 VSVGFTIPDTNIPWGPAFMGPIAERVRREAKLPVTSAWGFGTPQLAEAALQANQLDLVSVGRAHLA 
               
               
                   
               
               
                 DPHWAYFAAKELGVEKASWTLPAPYAHWLERYR 
               
               
                   
               
               
                 SEQ ID NO: 36: NADPH dehydrogenase, ‘FOYE-1’, ‘ Ferrovum ’ strain 
               
               
                 JA12. 
               
               
                 MSLLFSPYQLGSLSLANRLVIAPMCQYSAVDGIAQDWHLMHLGRLAISGAGLVIVEATGV 
               
               
                   
               
               
                 NPEGRITPFCLGLYNDEQEAALGRIVAFAREFGQAKMAIQLAHAGRKASTRRPWDPGSPY 
               
               
                   
               
               
                 SPEEGGWQTWAPSAIKFYEESLTPHPMSIEDLETVKQDFVNSAIRAERAGFKAIELHGAH 
               
               
                   
               
               
                 GYLIHQFLSPLSNQRQDQYGGSLENRMRYPLEILSAVKHALSAEMVVGMRISAVDWAPGG 
               
               
                   
               
               
                 LTIEESITFSQECEKRGAGFIHVSTGGLVAHQQIPVGPGYQVEHAQAIKQNVNIPTMAVG 
               
               
                   
               
               
                 LITHSAQAETILKSEQADMIAIARAALKNPHWPWTAALELGDKPFAPPQYQRAR 
               
               
                   
               
               
                 SEQ ID NO: 37: Oxidored_FMN domain-containing protein, ‘MgER’, 
               
               
                   Meyerozyma guilliermondii . 
               
               
                 MSVNINPLGETQVFQPIKLGKNTLSHRVFFPPTTRTRSLEDHTPSNLAYKYYDERSKFPG 
               
               
                   
               
               
                 TLIISEGTFPSAQAGLYEGVPGIWTERQTKTWKHIIDKIHENKSFASIQLWNLGRTGDPA 
               
               
                   
               
               
                 LLKKAGKPFLAPSAIYFDEESKKAAEKAGNPLRAMTEEEIKDMIYEQYTIAAKNALEAGF 
               
               
                   
               
               
                 DYIELHSAHGYLLHEFLEESSNKRTDKYGGSIENRARFVLELVDHMISIVGAERLGIRIS 
               
               
                   
               
               
                 PWATFQGMKSVHGEVHPLTTYSYLVNELEKRAQAGNRLAYISLVEPRVDGINSVEKKDQT 
               
               
                   
               
               
                 GNNDFVKDLWKGTILKAGNYTYDAPKFGQLLDDVSDGRTLVGFSRYFISNPDLISRLEKG 
               
               
                   
               
               
                 HQLAPYERETFYGRSDFGYNDYPKYGEKREDAEVAKKRVPEELVV 
               
               
                   
               
               
                 SEQ ID NO: 38: Oxidored_FMN domain-containing protein, ‘ClER’, 
               
               
                   Clavispora  ( Candida )  lusitaniae . 
               
               
                 MVAVKPLKDTEIFKPTKVGNHELSNKIVYAPTTRMRAIADHTPSDLAYKYYDDRTKYPGS 
               
               
                   
               
               
                 LVITEATLMSPKTGLYDRVPGIYTDEHVAGWKKITDKIHANGSKVSMQLWPLGRVADPVA 
               
               
                   
               
               
                 TKKAGYPLVAPSLIYPSEEAKKAAEEAGNPIHVLTTEEVEDLVNDFVHAAKKAVAAGVDY 
               
               
                   
               
               
                 VEVHGAHGYLVDTFFQVSTNKRTDKYGGSIENRARFALEILDRLIEEIGAERVAIRISPW 
               
               
                   
               
               
                 AKFQGILAEEGEVNPVAQFGYFLSELENRARAGKRIAYVSIVEPRVSGVIDVAGEDIQGD 
               
               
                   
               
               
                 NSFVRSVWKGIVIKAGNYTYDAPEFKTLLQDVSDGKTLVAFARYFTSNPDLVQRLHDGAD 
               
               
                   
               
               
                 LTPYKRELFYAPSNWGYNTFTNAGETKTFSEEEESKRLPAPIDTKA 
               
               
                   
               
               
                 SEQ ID NO: 39: Tryptophan 2-Halogenase, ‘CmdE’,  Chondromyces   
               
               
                   crocatus . 
               
               
                 MQLPSSTKILVVGGGPAGSTAATLLAREGFEVTLVEKAIFPRYHIGESLLISVQPIIDLL 
               
               
                   
               
               
                 GAREAVEAHGFQRKKGVLWEWGGERWLFDWKKLRYDYTFHVKREEFDEILLRNAQKNGVK 
               
               
                   
               
               
                 VFEGIDISRLEFDGERPVAAKWSKSSTGESGTIQFEFLLDASGRAGLMATQYLRSRMFMK 
               
               
                   
               
               
                 AFQNVATWGYWKGATIPEVEVEGPITVGSIPYGWIWGIPLRDQTMSVGLVIHQELFKEKR 
               
               
                   
               
               
                 ATQSVEEIYHEGLKASPLFQDVVLKGATLEPQIRTETDYSYISRTLAGPGFFLVGDSGAE 
               
               
                   
               
               
                 IDPLLSSGVHLAMHSALLAAASVKSIIAGEVDMASATEFYQRCYQGHFLRWALIVASFYE 
               
               
                   
               
               
                 VNARKETYFWTAQQLAHEELGVFNMSQADMKDVFATMVSGVVDLGDAQNAGRLQKGAERV 
               
               
                   
               
               
                 HQYLDDDGREEDVTALLQKSKQRIFEYLDRVKNRDSRAAMQRYKAGGTETFSMGLDADGA 
               
               
                   
               
               
                 VGGLYVTTEPRLGLLRKVVEERAEAATEAPAPAAPPPAVAEV 
               
               
                   
               
               
                 SEQ ID NO: 40: Tryptophan 5-Halogenase, ‘PyrH’,  Streptomyces   
               
               
                   rugosporus . 
               
               
                 MIRSVVIVGGGTAGWMTASYLKAAFDDRIDVTLVESGNVRRIGVGEATFSTVRHFFDYLG 
               
               
                   
               
               
                 LDEREWLPRCAGGYKLGIRFENWSEPGEYFYHPFERLRVVDGFNMAEWWLAVGDRRTSFS 
               
               
                   
               
               
                 EACYLTHRLCEAKRAPRMLDGSLFASQVDESLGRSTLAEQRAQFPYAYHFDADEVARYLS 
               
               
                   
               
               
                 EYAIARGVRHVVDDVQHVGQDERGWISGVHTKQHGEISGDLFVDCTGFRGLLINQTLGGR 
               
               
                   
               
               
                 FQSFSDVLPNNRAVALRVPRENDEDMRPYTTATAMSAGWMWTIPLFKRDGNGYVYSDEFI 
               
               
                   
               
               
                 SPEEAERELRSTVAPGRDDLEANHIQMRIGRNERTWINNCVAVGLSAAFVEPLESTGIFF 
               
               
                   
               
               
                 IQHAIEQLVKHFPGERWDPVLISAYNERMAHMVDGVKEFLVLHYKGAQREDTPYWKAAKT 
               
               
                   
               
               
                 RAMPDGLARKLELSASHLLDEQTIYPYYHGFETYSWITMNLGLGIVPERPRPALLHMDPA 
               
               
                   
               
               
                 PALAEFERLRREGDELIAALPSCYEYLASIQ 
               
               
                   
               
               
                 SEQ ID NO: 41: Flavin-Dependent Tryptophan Halogenase, ‘RebH’, 
               
               
                   Lentzea aerocolonigenes  ( Lechevalierla aerocolonigenes ) 
               
               
                 ( Saccharothrix aerocolonigenes ). 
               
               
                 MSGKIDKILIVGGGTAGWMAASYLGKALQGTADITLLQAPDIPTLGVGEATIPNLQTAFF 
               
               
                   
               
               
                 DFLGIPEDEWMRECNASYKVAIKFINWRTAGEGTSEARELDGGPDHFYHSFGLLKYHEQI 
               
               
                   
               
               
                 PLSHYWFDRSYRGKTVEPFDYACYKEPVILDANRSPRRLDGSKVTNYAWHFDAHLVADFL 
               
               
                   
               
               
                 RRFATEKLGVRHVEDRVEHVQRDANGNIESVRTATGRVFDADLFVDCSGFRGLLINKAME 
               
               
                   
               
               
                 EPFLDMSDHLLNDSAVATQVPHDDDANGVEPFTSAIAMKSGWTWKIPMLGRFGTGYVYSS 
               
               
                   
               
               
                 RFATEDEAVREFCEMWHLDPETQPLNRIRFRVGRNRRAWVGNCVSIGTSSCFVEPLESTG 
               
               
                   
               
               
                 IYFVYAALYQLVKHFPDKSLNPVLTARFNREIETMFDDTRDFIQAHFYFSPRTDTPFWRA 
               
               
                   
               
               
                 NKELRLADGMQEKIDMYRAGMAINAPASDDAQLYYGNFEEEFRNFWNNSNYYCVLAGLGL 
               
               
                   
               
               
                 VPDAPSPRLAHMPQATESVDEVFGAVKDRQRNLLETLPSLHEFLRQQHGR 
               
               
                   
               
               
                 SEQ ID NO: 42: Flavin-Dependent Tryptophan Halogenase, ‘PrnA’, 
               
               
                   Pseudomonas fluorescens . 
               
               
                 MNKPIKNIVIVGGGTAGWMAASYLVRALQQQANITLIESAAIPRIGVGEATIPSLQKVFF 
               
               
                   
               
               
                 DFLGIPEREWMPQVNGAFKAAIKFVNWRKSPDPSRDDHFYHLFGNVPNCDGVPLTHYWLR 
               
               
                   
               
               
                 KREQGFQQPMEYACYPQPGALDGKLAPCLSDGTRQMSHAWHFDAHLVADFLKRWAVERGV 
               
               
                   
               
               
                 NRVVDEVVDVRLNNRGYISNLLTKEGRTLEADLFIDCSGMRGLLINQALKEPFIDMSDYL 
               
               
                   
               
               
                 LCDSAVASAVPNDDARDGVEPYTSSIAMNSGWTWKIPMLGRFGSGYVFSSHFTSRDQATA 
               
               
                   
               
               
                 DFLKLWGLSDNQPLNQIKFRVGRNKRAWVNNCVSIGLSSCFLEPLESTGIYFIYAALYQL 
               
               
                   
               
               
                 VKHFPDTSFDPRLSDAFNAEIVHMFDDCRDFVQAHYFTTSRDDTPFWLANRHDLRLSDAI 
               
               
                   
               
               
                 KEKVQRYKAGLPLTTTSFDDSTYYETFDYEFKNFWLNGNYYCIFAGLGMLPDRSLPLLQH 
               
               
                   
               
               
                 RPESIEKAEAMFASIRREAERLRTSLPTNYDYLRSLRDGDAGLSRGQRGPKLAAQESL 
               
               
                   
               
               
                 SEQ ID NO: 43: Thermophilic Tryptophan Halogenase, ‘Th-Hal’, 
               
               
                   Streptomyces violaceusnige . 
               
               
                 LNNVVIVGGGTAGWMTASYLKAAFGDRIDITLVESGHIGAVGVGEATFSDIRHFFEFLGLKEKDWM 
               
               
                   
               
               
                 PACNATYKLAVRFENWREKGHYFYHPFEQMRSVNGFPLTDWWLKQGPTDRFDKDCFVMASVIDAGL 
               
               
                   
               
               
                 SPRHQDGTLIDQPFDEGADEMQGLTMSEHQGKTQFPYAYQFEAALLAKYLTKYSVERGVKHIVDDV 
               
               
                   
               
               
                 REVSLDDRGWITGVRTGEHGDLTGDLFIDCTGFRGLLLNOALEEPFISYODTLPNDSAVALQVPMD 
               
               
                   
               
               
                 MERRGILPCTTATAQDAGWIWTIPLTGRVGTGYVYAKDYLSPEEAERTLREFVGPAAADVEANHIR 
               
               
                   
               
               
                 MRIGRSRNSWVKNCVAIGLSSGFVEPLESTGIFFTHHAIEQLVKNFPAADWNSMHRDLYNSAVSHV 
               
               
                   
               
               
                 MDGVREFLVLHYVAAKRNDTQYWRDTKTRKIPDSLAERIEKWKVQLPDSETVYPYYHGLPPYSYMC 
               
               
                   
               
               
                 ILLGMGGIELKPSPALALADGGAAQREFEQIRNKTQRLTEVLPKAYDYFTQ 
               
               
                   
               
               
                 SEQ ID NO: 44: Tryptophan 6-Halogenase, ‘SttH’,  Streptomyces   
               
               
                   toxytricini . 
               
               
                 MNTRNPDKVVIVGGGTAGWMTASYLKKAFGERVSVTLVESGTIGTVGVGEATFSDIRHFF 
               
               
                   
               
               
                 EFLDLREEEWMPACNATYKLAVRFQDWQRPGHHFYHPFEQMRSVDGFPLTDWWLQNGPTD 
               
               
                   
               
               
                 RFDRDCFVMASLCDAGRSPRYLNGSLLQQEFDERAEEPAGLTMSEHQGKTQFPYAYHFEA 
               
               
                   
               
               
                 ALLAEFLSGYSKDRGVKHVVDEVLEVKLDDRGWISHVVTKEHGDIGGDLFVDCTGFRGVL 
               
               
                   
               
               
                 LNQALGVPFVSYQDTLPNDSAVALQVPLDMEARGIPPYTRATAKEAGWIWTIPLIGRIGT 
               
               
                   
               
               
                 GYVYAKDYCSPEEAERTLREFVGPEAADVEANHIRMRIGRSEQSWKNNCVAIGLSSGFVE 
               
               
                   
               
               
                 PLESTGIFFIHHAIEQLVKHFPAGDWHPQLRAGYNSAVANVMDGVREFLVLHYLGAARND 
               
               
                   
               
               
                 TRYWKDTKTRAVPDALAERIERWKVQLPDSENVFPYYHGLPPYSYMAILLGTGAIGLRPS 
               
               
                   
               
               
                 PALALADPAAAEKEFTAIRDRARFLVDTLPSQYEYFAAMGQRV 
               
               
                   
               
               
                 SEQ ID NO: 45: KtzQ, ‘KtzQ’,  Kutzneria  sp. 744. 
               
               
                 MDDNRIRSILVLGGGTAGWMSACYLSKALGPGVEVTVLEAPSISRIRVGEATIPNLHKVF 
               
               
                   
               
               
                 FDFLGIAEDEWMRECNASYKAAVRFVNWRTPGDGQATPRRRPDGRPDHFDHLFGQLPEHE 
               
               
                   
               
               
                 NLPLSQYWAHRRLNGLTDEPFDRSCYVQPELLDRKLSPRLMDGTKLASYAWHFDADLVAD 
               
               
                   
               
               
                 FLCRFAVQKLNVTHVQDVFTHADLDQRGHITAVNTESGRTLAADLFIDCSGFRSVLMGKV 
               
               
                   
               
               
                 MQEPFLDMSKHLLNDRAVALMLPHDDEKVGIEPYTSSLAMRSGWSWKIPLLGRFGSGYVY 
               
               
                   
               
               
                 SSQFTSQDEAAEELCRMWDVDPAEQTFNNVRFRVGRSRRAWVRNCVAIGVSAMFVEPLES 
               
               
                   
               
               
                 TGLYFSYASLYQLVKHFPDKRFRPILADRFNREVATMYDDTRDFLQAHFSLSPRDDSEFW 
               
               
                   
               
               
                 RACKELPFADGFAEKVEMYRAGLPVELPVTIDDGHYYGNFEAEFRNFWTNSNYYCIFAGL 
               
               
                   
               
               
                 GFLPEHPLPVLEFRPEAVDRAEPVFAAVRRRTEELVATAPTMQAYLRRLHQGT 
               
               
                   
               
               
                 SEQ ID NO: 46: Monodechloroaminopyrrolnitrin halogenase, ‘PrnC’, 
               
               
                   Pseudomonas fluorescens . 
               
               
                 MTQKSPANEHDSNHFDVIILGSGMSGTQMGAILAKQQFRVLIIEESSHPRFTIGESSIPE 
               
               
                   
               
               
                 TSLMNRIIADRYGIPELDHITSFYSTQRYVASSTGIKRNFGFVFHKPGQEHDPKEFTQCV 
               
               
                   
               
               
                 IPELPWGPESHYYRQDVDAYLLQAAIKYGCKVHQKTTVTEYHADKDGVAVTTAQGERFTG 
               
               
                   
               
               
                 RYMIDCGGPRAPLATKFKLREEPCRFKTHSRSLYTHMLGVKPFDDIFKVKGQRWRWHEGT 
               
               
                   
               
               
                 LHHMFEGGWLWVIPFNNHPRSTNNLVSVGLQLDPRVYPKTDISAQQEFDEFLARFPSIGA 
               
               
                   
               
               
                 QFRDAVPVRDWVKTDRLQFSSNACVGDRYCLMLHANGFIDPLFSRGLENTAVTIHALAAR 
               
               
                   
               
               
                 LIKALRDDDFSPERFEYIERLQQKLLDHNDDFVSCCYTAFSDFRLWDAFHRLWAVGTILG 
               
               
                   
               
               
                 QFRLVQAHARFRASRNEGDLDHLDNDPPYLGYLCADMEEYYQLFNDAKAEVEAVSAGRKP 
               
               
                   
               
               
                 ADEAAARIHALIDERDFAKPMFGFGYCITGDKPQLNNSKYSLLPAMRLMYWTQTRAPAEV 
               
               
                   
               
               
                 KKYFDYNPMFALLKAYITTRIGLALKK 
               
               
                   
               
               
                 SEQ ID NO: 47: FADH2-dependent halogenase, ‘PltA’,  Pseudomonas   
               
               
                   protegens  Pf-5. 
               
               
                 GPHMSDHDYDVVIIGGGPAGSTMASYLAKAGVKCAVFEKELFEREHVGESLVPATTPVLLEIGVME 
               
               
                   
               
               
                 KIEKANFPKKFGAAWTSADSGPEDKMGFQGLDHDFRSAEILFNERKQEGVDRDFTFHVDRGKFDRI 
               
               
                   
               
               
                 LLEHAGSLGAKVFQGVEIADVEFLSPGNVIVNAKLGKRSVEIKAKMVVDASGRNVLLGRRLGLREK 
               
               
                   
               
               
                 DPVFNQFAIHSWFDNFDRKSATQSPDKVDYIFIHFLPMTNTWVWQIPITETITSVGVVTQKQNYTN 
               
               
                   
               
               
                 SDLTYEEFFWEAVKTRENLHDALKASEQVRPFKKEADYSYGMKEVCGDSFVLIGDAARFVDPIFSS 
               
               
                   
               
               
                 GVSVALNSARIASGDIIEAVKNNDFSKSSFTHYEGMIRNGIKNWYEFITLYYRLNILFTAFVQDPR 
               
               
                   
               
               
                 YRLDILQLLQGDVYSGKRLEVLDKMREIIAAVESDPEHLWHKYLGDMQVPTAKPAFEND 
               
               
                   
               
               
                 SEQ ID NO: 48: Halogenase, ‘PltM’,  Pseudomonas fluorescens  (strain 
               
               
                 ATCC BAA-477/NRRL B-23932/Pf-5). 
               
               
                 MNQYDVIIIGSGIAGALTGAVLAKSGLNVLILDSAQHPRFSVGEAATPESGFLLRLLSKR 
               
               
                   
               
               
                 FDIPEIAYLSHPDKIIQHVGSSACGIKLGFSFAWHQENAPSSPDHLVAPPLKVPEAHLFR 
               
               
                   
               
               
                 QDIDYFALMIALKHGAESRQNIKIESISLNDDGVEVALSNAAPVKAAFIIDAAAQGSPLS 
               
               
                   
               
               
                 RQLGLRTTEGLATDTCSFFTHMLNVKSYEDALAPLSRTRSPIELFKSTLHHIFEEGWLWV 
               
               
                   
               
               
                 IPFNNHPQGTNQLCSIGFQFNNAKYRPTEAPEIEFRKLLKKYPAIGEHFKDAVNAREWIY 
               
               
                   
               
               
                 APRINYRSVQNVGDRFCLLPQATGFIDPLFSRGLITTFESILRLAPKVLDAARSNRWQRE 
               
               
                   
               
               
                 QFIEVERHCLNAVATNDQLVSCSYEAFSDFHLNNVWHRVWLSGSNLGSAFLQKLLHDLEH 
               
               
                   
               
               
                 SGDARQFDAALEAVRFPGCLSLDSPAYESLFRQSCQVMQQAREQARPVAETANALHELIK 
               
               
                   
               
               
                 EHEAELLPLGYSRISNRFILKV 
               
               
                   
               
               
                 SEQ ID NO: 49: Flavin-Dependent Halogenase, ‘Clz5’,  Streptomyces   
               
               
                 sp. CNH-287. 
               
               
                 MSEVDFDIGIIGGGPAGSAIASYLAKAGLNCVIFEGDIFPREHVGESLIPATTPVLDDIG 
               
               
                   
               
               
                 FLPQMETAGFPKKYGAAWTSASDQNIPTMGFEGMSHGFRAAEIEFHEREQLGVTQDYTYH 
               
               
                   
               
               
                 VDRAKFDLMLLQHAASLGAKVYQGVRVRRVDFSETNPRIIFPIGKTETSVRVRMVVDASG 
               
               
                   
               
               
                 RNTFLGRQLKLKVSDPVFNQYAVHTWFDNLDRTALSVNKEQADYIFIHFLPVTDTWVWQI 
               
               
                   
               
               
                 PITDTITSIGVVTQKEQFKASKEDLDGFFWECVGSRPELKEALEKSDQVRPFKTEGDYSY 
               
               
                   
               
               
                 AMKQIVGDQWATIGDAARFVDPIFSSGVSVALNSARLISGEIIAAAEQGDFRKEMFSNYE 
               
               
                   
               
               
                 GKIRRAVSNWYEFISIYYRLNILFTAFVQDPRYRLDVLKMLQGDVYDDEEPKALAAMREI 
               
               
                   
               
               
                 TKQVEENPDHLWHKHLGSLRAPSAAPMF 
               
               
                   
               
               
                 SEQ ID NO: 50: Pyrrole Halogenase, ‘Bmp2’,  Pseudoalteromonas   
               
               
                   piscicida . 
               
               
                 MNGFTHYDVVIIGSGPAGSLCGIECRKKGMSVLCIEKEQFPRFHIGESLTGNAGQIIRDL 
               
               
                   
               
               
                 GLADAMDAAGFPDKPGVNVIGSLSKNEFFIPILAPTWQVRRSDFDNMIKQKAVEHGVEYQ 
               
               
                   
               
               
                 LGMVTDVIREGDKVVGAVYKADGIEHQVRSKVLVDASGQNTFLSRKGIAGKRQIEFFSQQ 
               
               
                   
               
               
                 IASFAHYKGVERDLPPFSTNTTILYSKQYHWSWIIPISPDTDSLGVVIPKDLYYKECKNP 
               
               
                   
               
               
                 DDAIEWGMEHISPELKRRFKNAERQGESQSMADFSYRIEPFVGDGWLCIGDAHRFLDPIF 
               
               
                   
               
               
                 SYGVSFAMKEGIRAAEAIEQVVNGQDWKAPFYAYRDWSNGGQQIAADLIRYFWIYPIFFG 
               
               
                   
               
               
                 YQMQNPDLRDEVIRLLGGCCFDCKDWKAPAIFRNAIEEYDRKQMAS 
               
               
                   
               
               
                 SEQ ID NO: 51: Non-Heme Halogenase, ‘Rdc2’,  Metacordyceps   
               
               
                   chlamydosporia  ( Pochonia chlamydosporia ). 
               
               
                 MSVPKSCTILVAGGGPAGSYAAAALAREGNDVVLLEADQHPRYHIGESMLPSLRPLLRFI 
               
               
                   
               
               
                 DLEDKFDAHGFQKKLGAAFKLTSKREGSHGPRGYSWNVVRSESDEILFNHARESGAQAFQ 
               
               
                   
               
               
                 GIKINAIEFEPYEEEYPNGEKVANPGKPTSAKWSSKDGSSGDIAFKYLVDATGRIGIMST 
               
               
                   
               
               
                 KYLKNRHYNEGLKNLAIWGYYKNNIPWAQGTPRENQPFFEGMRDGAGWCWTIPLHNGTVS 
               
               
                   
               
               
                 VGAVMRKDLFFEKKKSLGENATNTQIMAECMKLCPTIGELLAPAELVSDIKQATDYSYSA 
               
               
                   
               
               
                 TAYAGPNFRIVGDAGCFIDPFFSSGHHLAVAGALAAAVSINASIKGDCTEYEASRWHAKK 
               
               
                   
               
               
                 VDEGYTLFLLVVMAALKQIRMQENPVLSDVDEDGFDRAFQFLRPEAVKKFTKEDVAQTID 
               
               
                   
               
               
                 FAVHALNNMAELDMDIPEHMINGDKEGENGVTNGNNGAAKTAGLASNMEKLTNDEEKVLN 
               
               
                   
               
               
                 GLRILGKAAPGGTLADFEGTAIDGLMPRLEHGKLGLNKV 
               
               
                   
               
               
                 SEQ ID NO: 52: Tryptophan 6-Halogenase, ‘BorH’, uncultured 
               
               
                 bacteria. 
               
               
                 MDNRINRIVILGGGTAGWMTASYLAKALGDTVTITLLEAPAIGRIGVGEATVPNLQRVFF 
               
               
                   
               
               
                 DFLGLREEEWMPECNAAFKTAVKFINWRTPGPGEAKARTIDGRPDHFYHPFGLLPEHGQV 
               
               
                   
               
               
                 PLSHYWAYNRAAGTTDEPFDYACFAETAAMDAVRAPKWLDGRPATRYAWHFDAHLVAEFL 
               
               
                   
               
               
                 RRHATERLNVEHVQGEMQQVLRDERGFITALRTVEGRDLEGDLFIDCSGFRGLLINKAME 
               
               
                   
               
               
                 EPFIDMNDQLLCNRAVATAIKHDDDAHGVEPYTSAIAMRSGWSWKIPMLGRFGTGYVYSS 
               
               
                   
               
               
                 RFAEKDEATLDFCRMWGLDPENTPLNQVAFRVGRNRRAWVKNCVSIGLASCFLEPLESTG 
               
               
                   
               
               
                 IYFITAAIYQLTQHFPDRTFALALSDAFNHEIEAMFDDTRDFIQAHFYVSPRTDTPFWKA 
               
               
                   
               
               
                 NKDLHLPEQMREKIAMYKAGLPINAPVTDESTYYGRFEAEFRNFWTNGSYYCIFAGLGLR 
               
               
                   
               
               
                 PDNPLPMLRHRPEQVREAQALFAGVKDKQRELVETLPSNLEFLRSLHGK 
               
               
                   
               
               
                 SEQ ID NO: 53: Styrene Monooxygenase,‘StyA’, Pseudomonas sp. 
               
               
                 MKKRIGIVGAGTAGLHLGLELRQHDVDVTVYTDRKPDEYSGLRLLNTVAHNAVTVQREVA 
               
               
                   
               
               
                 LDVNEWPSEEFGYFGHYYYVGGPQPMRFYGDLKAPSRAVDYRLYQPMLMRALEARGGKFC 
               
               
                   
               
               
                 YDAVSAEDLEGLSEQYDLLVVCTGKYALGKVFEKQSENSPFEKPQRALCVGLFKGIKEAP 
               
               
                   
               
               
                 IRAVTMSFSPGHGELIEIPTLSFNGMSTALVLENHIGSDLEVLAHTKYDDDPRAFLDLML 
               
               
                   
               
               
                 EKLGKHHPSVAERIDPAEFDLANSSLDILQGGVVPAFRDGHATLNNGKTIIGLGDIQATV 
               
               
                   
               
               
                 DPVLGQGANMASYAAWILGEEILAHSVYDLRFSEHLERRRQDRVLCATRWTNFTLSALSA 
               
               
                   
               
               
                 LPPEFLAFLQILSQSREMADEFTDNFNYPERQWDRFSSPERIGQWCSQFAPTIAA 
               
               
                   
               
               
                 SEQ ID NO: 54: 4-Nitrophenol 2-Monooxygenase Oxygenase Component, 
               
               
                 ‘PheA1’,  Rhodococcus erythropolis  ( Arthrobacter picolinophilus ). 
               
               
                 MTTTEIPPTGVDPADSGAPQVNPAADSEANRTKNFATRPMTGDEYISSLQDGREIWLHGD 
               
               
                   
               
               
                 RVKDVTTHPAFRNPIRMTARLYDALHTGEHVDALTVPTDTGNGGVTMPFFRTPTSSADLL 
               
               
                   
               
               
                 KERDAIATWARMTYGWMGRSPDYKASFLGTLHANKELYAPFQDNAERWYRESQEKVLYWN 
               
               
                   
               
               
                 HAIINPPVDRQLPPDEVGDVFMKVEKETDAGLIVSGAKVVATGSAITNYNFIAHYGLPIK 
               
               
                   
               
               
                 KKQFALICTVPMDAPGVKLICRTSYTEHAAVMGSPFDYPLSSRMDENDTIFVFDKVLVPW 
               
               
                   
               
               
                 ENVFMYGDVDRINAFFPQSGFLPRFTFQGCTRLAVKLDFIAGLLMKALEATGAGGFRGVQ 
               
               
                   
               
               
                 TRVGEVIGWRNLFWSLTESMARDPEQWVGDSVIPKLEYGLTYRMFMIQGYPRIKEIIEQD 
               
               
                   
               
               
                 VASGLIYLPSSSLDFKSPDVRPYLDKYVRGSDGITAVDRVKVMKALWDSIGTEFGGRHEL 
               
               
                   
               
               
                 YERNYSGNHENVKAELLFAAQNRGHASSMKGLAEQCLSEYDLDGWTVPDLIGNDDVSFFK 
               
               
                   
               
               
                 NR 
               
               
                   
               
               
                 SEQ ID NO: 55: 4-Hydroxyphenylacetate 3-Monooxygenase Oxygenase 
               
               
                 Component, ‘HpaB’,  Klebsiella oxytoca . 
               
               
                 MKPENFRADTKRPLTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASVAQLYDALH 
               
               
                   
               
               
                 NPELQNTLCWGTDTGSGGYTHKFFRVAKSADDLRQQRDAIAEWSRLSYGWMGRTPDYKAA 
               
               
                   
               
               
                 FGGGLGANPGFYGQFEQNARDWYTRIQETGLYFNHAIVNPPIDRHKPADEVKDVYIKLEK 
               
               
                   
               
               
                 ETDAGIIVSGAKVVATNSALTHYNMIGFGSAQVMGENPDFALMFVAPMDAEGDKLISRAS 
               
               
                   
               
               
                 YELVAGATGSPYDYPLSSRFDENDAILVMDNVLIPWENVLIYRDFDRCRRWTMEGGFARM 
               
               
                   
               
               
                 YPLQACVRLAVKLDFITALLKRSLECTGTLEFRGVQAELGEVVAWRNMFWALSDSMCAEA 
               
               
                   
               
               
                 TPWVNGAYLPDHAALQTYRVMAPMPYAKIKNIIERSVTSGLIYLPSSARDLNNPQINDTL 
               
               
                   
               
               
                 AKYVRGSNGMDHVERIKILKLMWDAIGSEFGGCHELYEINYSGSQDEIRLQCLRQAQSSG 
               
               
                   
               
               
                 NMDKMMAMVDRCLSEYDQNGWTVPHLHNNTDINMLDKLLK 
               
               
                   
               
               
                 SEQ ID NO: 56: Chlorophenol Monooxygenase, ‘HadA’,  Ralstonia   
               
               
                   pickettii  ( Burkholderia pickettii ). 
               
               
                 MIRTGTQYLESLNDGRNVWVGNEKIDNVATHPKTRDYAQRHADFYDLHHRPDLQDVMTYI 
               
               
                   
               
               
                 DEGGQRRAMQWFGHRDKEQLRRKRKYHETVMREMAGASFPRTPDVNNYVLTTYIDDPAPW 
               
               
                   
               
               
                 ETQSIGDDGHIKAGKIVDFIRYAREHDLNCAPQFVDPQMDRSNPDAQERSPGLRVVEKNE 
               
               
                   
               
               
                 KGIVVNGVKAIGTGVAFADWIHIGVFFRPGIPGDQVIFAATPVNTPGVTIVCRESLVKDD 
               
               
                   
               
               
                 KVEHPLAAQGDELDGMTVFENVFIPWSHVFHIGNPNHAKLYPQRVFDWLHYHALIRQMVR 
               
               
                   
               
               
                 AELVAGLAVLITEHIGTNKIPAVQTRVAKLIGFHQAMLAHLIASEELGFHTPGGHYKPNI 
               
               
                   
               
               
                 LIYDFGRALYLENFSQMIYELVDLSGRSALIFASEDQWNDDKLNGWFERMNNGPVGRPHD 
               
               
                   
               
               
                 RVKIGRVIRDLFLTDWGSRLFVFENFNGTPLQGIRMLTMQRAEFSGSGPYGKLARQVCGI 
               
               
                   
               
               
                 DSAVTDDTEYRKTADYAKALDAARHQEEVALAGAMAI 
               
               
                   
               
               
                 SEQ ID NO: 57: Tetrachlorobenzoquinone Reductase, ‘PcpD’, 
               
               
                   Sphingobium chlorophenolicum . 
               
               
                 MTNPVSTIDMTVTQITRVAKDINSYELRPEPGVILPEFTAGAHIGVSLPNGIQRSYSLVN 
               
               
                   
               
               
                 PQGERDRYVITVNLDRNSRGGSRYLHEQLRVGQRLSIVPPANNFALVETAPHSVLFAGGI 
               
               
                   
               
               
                 GITPIWSMIQRLRELGSTWELHYACRGKDFVAYRQELEQAAAEAGARFHLHLDEEADGKF 
               
               
                   
               
               
                 LDLAGPVAQAGQDSIFYCCGPEAMLQAYKAATADLPSERVRFEHFGAALTGEPADDVFTV 
               
               
                   
               
               
                 VLARRSGQEFTVEPGMTILETLLQNGISRNYSCTQGVCGTCETKVLEGEPDHRDWVLSDE 
               
               
                   
               
               
                 KKASNSTMLICCSLSKSPRLVLDI 
               
               
                   
               
               
                 SEQ ID NO: 58: 2-Methyl-6-ethyl-4-monooxygenase Oxygenase 
               
               
                 Component,‘MeaX’,  Sphingobium baderi . 
               
               
                 MTQAIDAGERVVSLDLETHGPEALIKRAAELVPLLRENAVRCHNERRIPSENLSAMRQAG 
               
               
                   
               
               
                 LLRMSRPRRYGGHEAMVATKTAVFGELARGCGSTAWVTTLYEDAAFLISLFPDEVQDEVF 
               
               
                   
               
               
                 ADQDTLITATLIPAGRAQPQGEGFLVNGRWPFNTGCLDATYVVEPAVVELSRGAPEVCLF 
               
               
                   
               
               
                 LMPYSELVIEDDWSPVGLRGTGSNSVRAKDVYVRPERMLRLADVMRGIYGTKLNKGPLYR 
               
               
                   
               
               
                 VPPIPYIVSSAGGTFPGLAQSAFELVTERLVGRPITYTLYTDRAQAPVTHLQLGEAALRI 
               
               
                   
               
               
                 KAANHLMTEVADRLDRRALNNEALTPDEGPMIWGIIGYTSRIYAEVIESLRQMSGASALS 
               
               
                   
               
               
                 ETSAIQAVARNAQALATHAMLIPTTGIEHYGRAICGLPPNTPFLSS 
               
               
                   
               
               
                 SEQ ID NO: 59: Alkanesulfonate Monooxygenase, ‘SsuD’,  Escherichia   
               
               
                   coli  (strain K12). 
               
               
                 MSLNMFWFLPTHGDGHYLGTEEGSRPVDHGYLQQIAQAADRLGYTGVLIPTGRSCEDAWL 
               
               
                   
               
               
                 VAASMIPVTQRLKFLVALRPSVTSPTVAARQAATLDRLSNGRALFNLVTGSDPQELAGDG 
               
               
                   
               
               
                 VFLDHSERYEASAEFTQVWRRLLQRETVDFNGKHIHVRGAKLLFPAIQQPYPPLYFGGSS 
               
               
                   
               
               
                 DVAQELAAEQVDLYLTWGEPPELVKEKIEQVRAKAAAHGRKIRFGIRLHVIVRETNDEAW 
               
               
                   
               
               
                 QAAERLISHLDDETIAKAQAAFARTDSVGQQRMAALHNGKRDNLEISPNLWAGVGLVRGG 
               
               
                   
               
               
                 AGTALVGDGPTVAARINEYAALGIDSFVLSGYPHLEEAYRVGELLFPLLDVAIPEIPQPQ 
               
               
                   
               
               
                 PLNPQGEAVANDFIPRKVAQS 
               
               
                   
               
               
                 SEQ ID NO: 60: p-Hydroxyphenylacetate 3-Hydroxylase, Oxygenase 
               
               
                 Component, ‘C2-HpaH’,  Acinetobacter baumannii . 
               
               
                 MENTVLNLDSDVIHACEAIFQPIRLVYTHAQTPDVSGVSMLEKIQQILPQIAKNAESAEQ 
               
               
                   
               
               
                 LRRVPDENIKLLKEIGLHRAFQPKVYGGLEMSLPDFANCIVTLAGACAGTAWAFSLLCTH 
               
               
                   
               
               
                 SHQIAMFSKQLQDEIWLKDPDATASSSIAPFGKVEEVEGGIILNGDYGWSSGCDHAEYAI 
               
               
                   
               
               
                 VGFNRFDADGNKIYSFGVIPRSDYEIVDNWYAQAIKSSGSKMLKLVNVFIPEYRISKAKD 
               
               
                   
               
               
                 MMEGKSAGFGLYPDSKIFYTPYRPYFASGFSAVSLGIAERMIEAFKEKQRNRVRAYTGAN 
               
               
                   
               
               
                 VGLATPALMRIAESTHQVAAARALLEKTWEDHRIHGLNHQYPNKETLAFWRTNQAYAVKM 
               
               
                   
               
               
                 CIEAVDRLMAAAGATSFMDNSELQRLFRDAHMTGAHAYTDYDVCAQILGRELMGMEPDPT 
               
               
                   
               
               
                 MV 
               
               
                   
               
               
                 SEQ ID NO: 61: FADH(2)-Dependent Monooxygenase, ‘TftD’, 
               
               
                   Burkholderia cepacia  ( Pseudomonas cepacia ). 
               
               
                 MRTGKQYLESLNDGRVVWVGNEKIDNVATHPLTRDYAERVAQFYDLHHRPDLQDVLTFVD 
               
               
                   
               
               
                 ADGVRRSRQWQDPKDAAGLRVKRKYHETILREIAAGSYGRLPDAHNYTFTTYADDPEVWE 
               
               
                   
               
               
                 KQSIGAEGRNLTQNIHNFLKLLREKDLNCPLNFVDPQTDRSSDAAQARSPNLRIVEKTDD 
               
               
                   
               
               
                 GIIVNGVKAVGTGIAFGDYMHIGCLYRPGIPGEQVIFAAIPTNTPGVTVFCRESTVKNDP 
               
               
                   
               
               
                 AEHPLASQGDELDSTTVFDNVFIPWEQVFHIGNPEHAKLYPQRIFDWVHYHILIRQVLRA 
               
               
                   
               
               
                 ELIVGLAILITEHIGTSKLPTVSARVAKLVAFHLAMQAHLIASEETGFHTKGGRYKPNPL 
               
               
                   
               
               
                 IYDFGRAHFLQNQMSVMYELLDLAGRSSLMIPSEGQWDDSQSGQWFVKLNNGPKGNPRER 
               
               
                   
               
               
                 VQIGRVIRDLYLTDWGGRQFMFENFNGTPLFAVFAATMTRDDMSAAGTYGKFASQVCGIE 
               
               
                   
               
               
                 FGGAEPTAYAATADYARALDRGLAPEPAAAESATS 
               
               
                   
               
               
                 SEQ ID NO: 62: 4-Nitrophenol 2-Monooxygenase, Oxygenase Component, 
               
               
                 ‘NphA1’,  Rhodococcus  sp. 
               
               
                 MTTSAFVDDRVGVPNDVRPMTGDEYLESLRDGREVYFRGERVDDVTTHPAFRNSARSVAR 
               
               
                   
               
               
                 MYDALHQPEQEGVLAVPTDTGNGGFTHPFFRTARSADDLVLSRDAIVAWQREVYGWLGRS 
               
               
                   
               
               
                 PDYRASFLGTLGANADFYGPYRDNALRWYRHAQERMLYLNHAIVNPPIDRDRPADETADV 
               
               
                   
               
               
                 CVHVVEETDAGLIVSGAKVVATGSAITNANFIAHYGLLRKKEYGLIFTVPMDSPGLKLFC 
               
               
                   
               
               
                 RTSYEMNAAVMGTPFDYPLSSRFDENDAIMVFDRVLVPWENVFAYDTDTANGFVMRSGFL 
               
               
                   
               
               
                 SRFMFHGCARLAVKLDFIAGCVMKGVEMTGSAGFRGVQMQIGEILNWRDMFWGLSDAMAK 
               
               
                   
               
               
                 SPEQWVNGAVQPNLNYGLAYRTFMGVGYPRVKEIIQQVLGSGLIYLNSHASDWANPAMRP 
               
               
                   
               
               
                 YLDQYVRGSNGVAAIDRVQLLKLLWDAVGTEFGGRHELYERNYGGDHEAVRFQTLFAYQA 
               
               
                   
               
               
                 TGQDLALKGFAEQCMSEYDVDGWTRPDLIGNDDLRIVRG 
               
               
                   
               
               
                 SEQ ID NO: 63: Putative dehydrogenase/oxygenase subunit, 
               
               
                 ‘VpStyA1’,  Variovorax paradoxus  (strain EPS). 
               
               
                 MKRIAIVGAGQSGLQLGLGLLAAGYEVTMFSNRTGEDIRRGKVMSSQCMFDTSLQIERDL 
               
               
                   
               
               
                 GLDHWASDCPTVDGIGLAVPHPEQKGAKVIDWAARLNASAQSVDQRLKIPAWMDEFQKKG 
               
               
                   
               
               
                 GELVFKDAGIDELEACTQSHDLTLVASGKGEISKLFERDAHKSPYDKPQRALALTYVKGM 
               
               
                   
               
               
                 APREPFSAVCFNLIPGVGEYFVFPALTTTGPCEIMVFEGVPGGPMDCWADVKTPEEHLAR 
               
               
                   
               
               
                 SKWILDTFTPWEAERCKDIELTDDNGILAGRFAPTVRKPVATLPSGRKVLGLADVVVLND 
               
               
                   
               
               
                 PITGQGSNNAAKCADTYLKSILARGDGAADAAWMQQTFDRYWFGYAQWVTQWTNMLLAPP 
               
               
                   
               
               
                 PPHVLNLLGSAGAVPPLASAFANGFDDPRTFFPWFADAAESERYIATCAAVA 
               
               
                   
               
               
                 SEQ ID NO: 64: Oxygenase, ‘RoIndA1’ {from styA1 gene},  Rhodococcus   
               
               
                   opacus  ( Nocardia opaca ). 
               
               
                 MRKITIVGAGQAGLQLAIGLVDAGYDVTVVSNRTPEQIREGKVMSSQCMFGGALARERAV 
               
               
                   
               
               
                 GLDLWSDECPSVEGISFTVADNGNQAFSWASRLDLPAQSVDQRVKMPAWLKEFEARGGTL 
               
               
                   
               
               
                 VLQDAGIGDLEGFAVDSDLVILAAGKGEIAKMFERDATRSVFDRPQRALALTYVNGMVPR 
               
               
                   
               
               
                 EEHSAVAFNMIPGVGEYFAFPALTTSGPCEIMVMEGIPGGPMDCWADVTTPEEHLAKSKW 
               
               
                   
               
               
                 VVETFVPWEAERCADITLTDDNGILAGRFPPTVRKPIGVLPSGANILGLADVVVLNDPLT 
               
               
                   
               
               
                 GQGSNNASKCAASYLASILEHGGRPFDAEFMTDTFERYWDYAQYVASWTNALLSPPPEHV 
               
               
                   
               
               
                 LKLLGAAATEPRIARRFANGFDDPRDFYHWFMTPEAAAQYLTDVAN 
               
               
                   
               
               
                 SEQ ID NO: 65: Smoa_sbd domain-containing protein, ‘AbIndA’, 
               
               
                   Acinetobacter baylyi  (strain ATCC 33305/BD413/ADP1). 
               
               
                 MMRRIAIVGAGQSGLQLGLSLLDTGYDVTIVTNRTADQIRQGKVMSSQCMFHTALQTERD 
               
               
                   
               
               
                 VGLNFWEEQCPAVEGIGFTLVSPETGKPAFSWSARLERYAQSVDQRVKMPYWIEEFERRG 
               
               
                   
               
               
                 GKLIIQDVGIDELEQLTTEYELVLLAAGKGEVVKQFVRDDERSTFDKPQRALALTYVTGM 
               
               
                   
               
               
                 KPMSPYSRVTFNVIPGVGEYFCFPALTVTGPCEIMVFEGIPGGPMDCWQDAKTPEQHLQM 
               
               
                   
               
               
                 SKDILNTYLPWEAERCENIEITDAGGYLAGRFPPSVRKPILTLPSGRQVFGMADALVVND 
               
               
                   
               
               
                 PITGQGSNNAAKCSKIYFDAILAHDTQSFTPEWMQQTFERYWSYAEKVVAWTNSLLVPPQ 
               
               
                   
               
               
                 PQMIDVLAAASQNQAIASTIANNFDDPRNFSPWWFDAEQAQHFIESKSCQKVA 
               
               
                   
               
               
                 SEQ ID NO: 66: 2,5-Diketocamphane 1,2-Monooxygenase 1, ‘CamP’, 
               
               
                   Pseudomonas putida  ( Arthrobacter siderocapsulatus ). 
               
               
                 MKCGFFHTPYNLPTRTARQMFDWSLKLAQVCDEAGFADFMIGEHSTLAWENIPCPEIIIG 
               
               
                   
               
               
                 AAAPLTKNIRFAPMAHLLPYHNPATLAIQIGWLSQILEGRYFLGVAPGGHHTDAILHGFE 
               
               
                   
               
               
                 GIGPLQEQMFESLELMEKIWAREPFMEKGKFFQAGFPGPDTMPEYDVEIADNSPWGGRES 
               
               
                   
               
               
                 MEVAVTGLTKNSSSLKWAGERNYSPISFFGGHEVMRSHYDTWAAAMQSKGFTPERSRFRV 
               
               
                   
               
               
                 TRDIFIADTDAEAKKRAKASGLGKSWEHYLFPIYKKFNLFPGIIADAGLDIDPSQVDMDF 
               
               
                   
               
               
                 LAEHVWLCGSPETVKGKIERMMERSGGCGQIVVCSHDNIDNPEPYFESLQRLASEVLPKV 
               
               
                   
               
               
                 RMG 
               
               
                   
               
               
                 SEQ ID NO: 67: 3,6-Diketocamphane 1,6-Monooxygenase, ‘CamE36’, 
               
               
                   Pseudomonas putida  ( Arthrobacter siderocapsulatus ). 
               
               
                 MAMETGLIFHPYMRPGRSARQTFDWGIKSAVQADSVGIDSMMISEHASQIWENIPNPELL 
               
               
                   
               
               
                 IAAAALQTKNIKFAPMAHLLPHQHPAKLATMIGWLSQILEGRYFLGIGAGAYPQASYMHG 
               
               
                   
               
               
                 IRNAGQSNTATGGEETKNLNDMVRESLFIMEKIWKREPFFHEGKYWDAGYPEELEGEEGD 
               
               
                   
               
               
                 EQHKLADFSPWGGKAPEIAVTGFSYNSPSMRLAGERNFKPVSIFSGLDALKRHWEVYSEA 
               
               
                   
               
               
                 AIEAGHTPDRSRHAVSHTVFCADTDKEAKRLVMEGPIGYCFERYLIPIWRRFGMMDGYAK 
               
               
                   
               
               
                 DAGIDPVDADLEFLVDNVFLVGSPDTVTEKINALFEATGGWGTLQVEAHDYYDDPAPWFQ 
               
               
                   
               
               
                 SLELISKEVAPKILLPKR 
               
               
                   
               
               
                 SEQ ID NO: 68: Alkanal monooxygenase, alpha chain, ‘LuxA’,  Vibrio   
               
               
                   harveyi  ( Beneckea harveyi ). 
               
               
                 MKFGNFLLTYQPPELSQTEVMKRLVNLGKASEGCGFDTVWLLEHHFTEFGLLGNPYVAAA 
               
               
                   
               
               
                 HLLGATETLNVGTAAIVLPTAHPVRQAEDVNLLDQMSKGRERFGICRGLYDKDFRVFGTD 
               
               
                   
               
               
                 MDNSRALMDCWYDLMKEGFNEGYIAADNEHIKFPKIQLNPSAYTQGGAPVYVVAESASTT 
               
               
                   
               
               
                 EWAAERGLPMILSWIINTHEKKAQLDLYNEVATEHGYDVTKIDHCLSYITSVDHDSNRAK 
               
               
                   
               
               
                 DICRNFLGHWYDSYVNATKIFDDSDQTKGYDFNKGQWRDFVLKGHKDTNRRIDYSYEINP 
               
               
                   
               
               
                 VGTPEECIAIIQQDIDATGIDNICCGFEANGSEEEIIASMKLFQSDVMPYLKEKQ 
               
               
                   
               
               
                 SEQ ID NO: 69: Alkanal monooxygenase, beta chain, ‘LuxB’,  Vibrio   
               
               
                   harveyi  ( Beneckea harveyi ). 
               
               
                 MKFGLFFLNFMNSKRSSDQVIEEMLDTAHYVDQLKFDTLAVYENHFSNNGVVGAPLTVAG 
               
               
                   
               
               
                 FLLGMTKNAKVASLNHVITTHHPVRVAEEACLLDQMSEGRFAFGFSDCEKSADMRFFNRP 
               
               
                   
               
               
                 TDSQFQLFSECHKIINDAFTTGYCHPNNDFYSFPKISVNPHAFTEGGPAQFVNATSKEVV 
               
               
                   
               
               
                 EWAAKLGLPLVFRWDDSNAQRKEYAGLYHEVAQAHGVDVSQVRHKLTLLVNQNVDGEAAR 
               
               
                   
               
               
                 AEARVYLEEFVRESYSNTDFEQKMGELLSENAIGTYEESTQAARVAIECCGAADLLMSFE 
               
               
                   
               
               
                 SMEDKAQQRAVIDVVNANIVKYHS 
               
               
                   
               
               
                 SEQ ID NO: 70: Alkanal monooxygenase, alpha chain,‘LuxA’, 
               
               
                   Photorhabdus luminescens  ( Xenorhabdus luminescens ). 
               
               
                 MKFGNFLLTYQPPELSQTEVMKRLVNLGKASEGCGFDTVWLLEHHFTEFGLLGNPYVAAA 
               
               
                   
               
               
                 HLLGATETLNVGTAAIVLPTAHPVRQAEDVNLLDQMSKGRFRFGICRGLYDKDFRVFGTD 
               
               
                   
               
               
                 MDNSRALMDCWYDLMKEGFNEGYIAADNEHIKFPKIQLNPSAYTQGGAPVYVVAESASTT 
               
               
                   
               
               
                 EWAAERGLPMILSWIINTHEKKAQLDLYNEVATEHGYDVTKIDHCLSYITSVDHDSNRAK 
               
               
                   
               
               
                 DICRNFLGHWYDSYVNATKIFDDSDQTKGYDFNKGQWRDFVLKGHKDTNRRIDYSYEINP 
               
               
                   
               
               
                 VGTPEECIAIIQQDIDATGIDNICCGFEANGSEEEIIASMKLFQSDVMPYLKEKQ 
               
               
                   
               
               
                 SEQ ID NO: 71: Alkanal monooxygenase, beta chain, ‘LuxB’, 
               
               
                   Photorhabdus luminescens  ( Xenorhabdus luminescens ). 
               
               
                 MKFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHFSDNGVVGAPLTVSG 
               
               
                   
               
               
                 FLLGLTEKIKIGSLNHIITTHHPVAIAEEACLLDQLSEGRFILGFSDCEKKDEMHFFNRP 
               
               
                   
               
               
                 VEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYTPGGPRKYVTATSHHIV 
               
               
                   
               
               
                 EWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEIDHQLMILVNYNEDSNKAK 
               
               
                   
               
               
                 QETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITAAKLAIEKCGAKSVLLSFE 
               
               
                   
               
               
                 PMNDLMSQKNVINIVDDNIKKYHMEYT 
               
               
                   
               
               
                 SEQ ID NO: 72: Alkane Monooxygenase, ‘LadA’,  Geobacillus   
               
               
                   thermodenitrificans . 
               
               
                 MTKKIHINAFEMNCVGHIAHGLWRHPENQRHRYTDLNYWTELAQLLEKGKFDALFLADVVGIYDVY 
               
               
                   
               
               
                 RQSRDTAVREAVQIPVNDPLMLISAMAYVTKHLAFAVTFSTTYEHPYGHARRMSTLDHLTKGRIAW 
               
               
                   
               
               
                 NVVTSHLPSADKNFGIKKILEHDERYDLADEYLEVCYKLWEGSWEDNAVIRDIENNIYTDPSKVHE 
               
               
                   
               
               
                 INHSGKYFEVPGPHLCEPSPQRTPVIYQAGMSERGREFAAKHAECVFLGGKDVETLKFFVDDIRKR 
               
               
                   
               
               
                 AKKYGRNPDHIKMFAGICVIVGKTHDEAMEKLNSFQKYWSLEGHLAHYGGGTGYDLSKYSSNDYIG 
               
               
                   
               
               
                 SISVGEIINNMSKLDGKWFKLSVGTPKKVADEMQYLVEEAGIDGFNLVQYVSPGTFVDFIELVVPE 
               
               
                   
               
               
                 LQKRGLYRVDYEEGTYREKLFGKGNYRLPDDHIAARYRNISSNV 
               
               
                   
               
               
                 SEQ ID NO: 73: EDTA Monooxygenase, ‘EmoA’,  Chelativorans   
               
               
                   multitrophicus . 
               
               
                 PAVAANYGAAIATHDNRYERAEEFLEVVHGLWNSWKFPWDEAIGPNPNPFGEVMPINHEG 
               
               
                   
               
               
                 KYFKVAGPLNVPLPPYGPPVVVQAGGSDQGKRLASRFGEIIYAFLGSKPAGRRFVAEARA 
               
               
                   
               
               
                 AARAQGRPEGSTLVLPSFVPLIGSTEAEVKRLVAEYEAGLDPAEQRIEALSKQLGIDLER 
               
               
                   
               
               
                 INVDQVLQEKDFNLPKESATPIGILKSMVDVALDEKLSLRQLA 
               
               
                   
               
               
                 SEQ ID NO: 74: Isobutylamine N-hydroxylase, ‘IBAH’,  Streptomyces   
               
               
                   viridifaciens . 
               
               
                 MRSLDAARDTCERLHPGLIKALEELPLLEREAEGSPVLDIFRAHGGAGLLVPSAYGGHGA 
               
               
                   
               
               
                 DALDAVRVTRALGACSPSLAAAATMHNFTAAMLFALTDRVIPPTDEQKKLLARVAPEGML 
               
               
                   
               
               
                 LASGWAEGRTQQDILNPSVKATPVDDGFILNGSKKPCSLSRSMDILTASVILPDETGQQS 
               
               
                   
               
               
                 LAVPLIMADSPGISVHPFWESPVLAGSQSNEVRLKDVHVPEKLIIRGTPDDPGRLDDLQT 
               
               
                   
               
               
                 ATFVWFELLITSAYVGAASALTELVMERDRGSVTDRAALGIQLESAVGLTEGVARAVRDG 
               
               
                   
               
               
                 VFGEEAVAAALTARFAVQKTLAAISDQAIELLGGIAFIKSPELAYLSSALHPLAFHPPGR 
               
               
                   
               
               
                 TSSSPHLVEYFSGGPLEI 
               
               
                   
               
               
                 SEQ ID NO: 75: ActVA 6 Protein, ‘ActVA-Orf6’,  Streptomyces   
               
               
                   coelicolor . 
               
               
                 MAEVNDPRVGFVAVVTFPVDGPATQHKLVELATGGVQEWIREVPGFLSATYHASTDGTAV 
               
               
                   
               
               
                 VNYAQWESEQAYRVNFGADPRSAELREALSSLPGLMGPPKAVFMTPRGAILPS 
               
               
                   
               
               
                 SEQ ID NO: 76: Pyrimidine Monooxygenase, ‘RutA’,  Escherichia coli   
               
               
                 (strain K12). 
               
               
                 MQDAAPRLTFTLRDEERLMMKIGVFVPIGNNGWLISTHAPQYMPTFELNKAIVQKAEHYH 
               
               
                   
               
               
                 FDFALSMIKLRGFGGKTEFWDHNLESFTLMAGLAAVTSRIQIYATAATLTLPPAIVARMA 
               
               
                   
               
               
                 ATIDSISGGRFGVNLVTGWQKPEYEQMGIWPGDDYFSRRYDYLTEYVQVLRDLWGTGKSD 
               
               
                   
               
               
                 FKGDFFTMNDCRVSPQPSVPMKVICAGQSDAGMAFSARYADFNFCFGKGVNTPTAFAPTA 
               
               
                   
               
               
                 ARMKQAAEQTGRDVGSYVLFMVIADETDDAARAKWEHYKAGADEEALSWLTEQSQKDTRS 
               
               
                   
               
               
                 GTDTNVRQMADPTSAVNINMGTLVGSYASVARMLDEVASVPGAEGVLLTFDDFLSGIETF 
               
               
                   
               
               
                 GERIQPLMQCRAHLPALTQEVA 
               
               
                   
               
               
                 SEQ ID NO: 77: p-Hydroxyphenylacetate 2-Hydroxylase Reductase 
               
               
                 Component, ‘C1-HpaH’,  Acinetobacter baumannii . 
               
               
                 MNQLNTAIVEKEVIDPMAFRRALGNFATGVTIMTAQTSSGERVGVTANSFNSVSLDPALV 
               
               
                   
               
               
                 LWSIDKKSSSYRIFEEATHFGVNILSAAQIELSNRFARRSEDKFANIEFDLGVGNIPLFK 
               
               
                   
               
               
                 NCSAAFECERYNIVEGGDHWIIIGRVVKFHDHGRSPLLYHQGAYSAVLPHPSLNMKSETA 
               
               
                   
               
               
                 EGVFPGRLYDNMYYLLTQAVRAYQNDYQPKQLASGFRTSEARLLLVLESKTASSKCDLQR 
               
               
                   
               
               
                 EVAMPIREIEEATKILSEKGLLIDNGQHYELTEQGNACAHMLYKIAESHQEEVFAKYTVD 
               
               
                   
               
               
                 ERKLFKNMLKDLIGI 
               
               
                   
               
               
                 SEQ ID NO: 78: FMN_red Domain-Containing Protein, ‘YdhA’,  Bacillus   
               
               
                   subtilis  subsp. natto (strain BEST195). 
               
               
                 MLVINGTPRKHGRTRIAASYIAALYHTDLIDLSEFVLPVFNGEADQSELLKVQELKKRVT 
               
               
                   
               
               
                 KADAIVLLSPEYHSGMSGALKNALDFLSSEQFKYKPVALLAVAGGGKGGINALNNMRTVM 
               
               
                   
               
               
                 RGVYANVIPKQLVLDPVHIDVENATVAENIKESIKELVEELSMFAKTGNPGV 
               
               
                   
               
               
                 SEQ ID NO: 79: NAD(P)H-Flavin Reductase, ‘Fre’,  Escherichia coli   
               
               
                 (strain K12). 
               
               
                 MTTLSCKVTSVEAITDTVYRVRIVPDAAFSFRAGQYLMVVMDERDKRPFSMASTPDEKGF 
               
               
                   
               
               
                 IELHIGASEINLYAKAVMDRILKDHQIVVDIPHGEAWLRDDEERPMILIAGGTGFSYARS 
               
               
                   
               
               
                 ILLTALARNPNRDITIYWGGREEQHLYDLCELEALSLKHPGLQVVPVVEQPEAGWRGRTG 
               
               
                   
               
               
                 TVLTAVLQDHGTLAEHDIYIAGRFEMAKIARDLFCSERNAREDRLFGDAFAFI 
               
               
                   
               
               
                 SEQ ID NO: 80: 4-hydroxyphenylacetate 3-monooxygenase reductase 
               
               
                 component, ‘HpaC’,  Escherichia coli . 
               
               
                 MQLDEQRLRFRDAMASLSAAVNIITTEGDAGQCGITATAVCSVTDTPPSLMVCINANSAM 
               
               
                   
               
               
                 NPVFQGNGKLCVNVLNHEQELMARHFAGMTGMAMEERFSLSCWQKGPLAQPVLKGSLASL 
               
               
                   
               
               
                 EGEIRDVQAIGTHLVYLVEIKNIILSAEGHGLIYFKRRFHPVMLEMEAAI 
               
               
                   
               
               
                 SEQ ID NO: 81: nitroreductase ‘NfsB’,  Escherichia coli  (strain 
               
               
                 K12). 
               
               
                 MDIISVALKRHSTKAFDASKKLTPEQAEQIKTLLQYSPSSTNSQPWHFIVASTEEGKARV 
               
               
                   
               
               
                 AKSAAGNYVFNERKMLDASHVVVFCAKTAMDDVWLKLVVDQEDADGRFATPEAKAANDKG 
               
               
                   
               
               
                 RKFFADMHRKDLHDDAEWMAKQVYLNVGNFLLGVAALGLDAVPIEGFDAAILDAEFGLKE 
               
               
                   
               
               
                 KGYTSLVVVPVGHHSVEDFNATLPKSRLPQNITLTEV 
               
               
                   
               
               
                 SEQ ID NO: 82: vanadium chloroperoxidase ‘CPO’ or ‘CiVHPO’, 
               
               
                   Curvularia inaequalis . 
               
               
                 MGSVTPIPLPKIDEPEEYNTNYILFWNHVGLELNRVTHTVGGPLTGPPLSARALGMLHLAIHDAYF 
               
               
                   
               
               
                 SICPPTDFTTFLSPDTENAAYRLPSPNGANDARQAVAGAALKMLSSLYMKPVEQPNPNPGANISDN 
               
               
                   
               
               
                 AYAQLGLVLDRSVLEAPGGVDRESASFMFGEDVADVFFALLNDPRGASQEGYHPTPGRYKFDDEPT 
               
               
                   
               
               
                 HPVVLIPVDPNNPNGPKMPFRQYHAPFYGKTTKRFATQSEHFLADPPGLRSNADETAEYDDAVRVA 
               
               
                   
               
               
                 IAMGGAQALNSTKRSPWQTAQGLYWAYDGSNLIGTPPRFYNQIVRRIAVTYKKEEDLANSEVNNAD 
               
               
                   
               
               
                 FARLFALVDVACTDAGIFSWKEKWEFEFWRPLSGVRDDGRPDHGDPFWLTLGAPATNTNDIPFKPP 
               
               
                   
               
               
                 FPAYPSGHATFGGAVFQMVRRYYNGRVGTWKDDEPDNIAIDMMISEELNGVNRDLRQPYDPTAPIE 
               
               
                   
               
               
                 DQPGIVRTRIVRHFDSAWELMFENAISRIFLGVHWRFDAAAARDILIPTTTKDVYAVDNNGATVFQ 
               
               
                   
               
               
                 NVEDIRYTTRGTREDPEGLFPIGGVPLGIEIADEIFNNGLKPTPPEIQPMPQETPVQKPVGQQPVK 
               
               
                   
               
               
                 GMWEEEQAPVVKEAP 
               
               
                   
               
               
                 SEQ ID NO: 83: aromatic unspecified peroxygenase ‘APO1’ or 
               
               
                 ‘AaeUPO’,  Agrocybe aegerita  (Black poplar mushroom) ( Agaricus   
               
               
                   aegerita ). 
               
               
                 MKYFPLFPTLVFAARVVAFPAYASLAGLSQQELDAIIPTLEAREPGLPPGPLENSSAKLVNDEAHP 
               
               
                   
               
               
                 WKPLRPGDIRGPCPGLNTLASHGYLPRNGVATPVQIINAVQEGLNFDNQAAVFATYAAHLVDGNLI 
               
               
                   
               
               
                 TDLLSIGRKTRLTGPDPPPPASVGGLNEHGTFEGDASMTRGDAFFGNNHDFNETLFEQLVDYSNRF 
               
               
                   
               
               
                 GGGKYNLTVAGELRFKRIQDSIATNPNFSFVDFRFFTAYGETTFPANLFVDGRRDDGQLDMDAARS 
               
               
                   
               
               
                 FFQFSRMPDDFFRAPSPRSGTGVEVVIQAHPMQPGRNVGKINSYTVDPTSSDFSTPCLMYEKFVNI 
               
               
                   
               
               
                 TVKSLYPNPTVQLRKALNTNLDFFFQGVAAGCTQVFPYGRD