Method of reducing and recycling oxidized flavin cofactors
AssigneeOxford University Innovation Limited
InventorsKylie VINCENT, Sarah CLEARY, Shiny Joseph SRINIVASAN, Holly REEVE
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Abstract
The invention relates to an enzymatic method for producing a reaction product. A method of recycling a biological cofactor is also provided. The invention also relates to systems and apparatuses for conducting such methods.
Claims32
A method of producing a reaction product, comprising:
- i) contacting an oxidised flavin cofactor and molecular hydrogen (1H2) 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.
A method according to any one of the preceding claims, wherein the oxidised cofactor is selected from flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), riboflavin, or a derivative thereof.
A method according to any one of the preceding claims, wherein the first polypeptide transfers the electrons to the oxidised flavin cofactor via an intramolecular electronically-conducting pathway.
A method according to any one of the preceding claims, wherein the reduction of the oxidised flavin cofactor takes place at an [FeS] cluster within the first polypeptide.
A method according to any one of the preceding claims, wherein said first polypeptide does not comprise a native flavin active site for NAD(P)+ reduction.
A method according to any one of the preceding claims, wherein the first polypeptide is an uptake hydrogenase or a hydrogen-sensing hydrogenase.
A method according to any one of the preceding claims, wherein the first polypeptide is a hydrogenase of class 1 or 2b.
A method according to any one of the preceding claims, wherein 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.
A method according to any one of the preceding claims, wherein the second polypeptide comprises the electron acceptor and/or hydride ion acceptor.
A method according to any one of the preceding claims, wherein the second polypeptide comprises a prosthetic group for oxidising the reduced flavin cofactor.
A method according to any one of the preceding claims, wherein the second polypeptide is a flavin-accepting oxidoreductase, or a functional fragment, derivative or variant thereof.
A method according to any one of the preceding claims, wherein the second polypeptide is a flavin-dependent oxidoreductase, or a functional fragment, derivative or variant thereof.
A method according to any one of the preceding claims, wherein the first polypeptide and/or the second polypeptide are in solution.
A method according to any one of the preceding claims wherein the first polypeptide and the second polypeptide are attached together.
A method according to any one of the preceding claims, wherein said method is carried out under aerobic conditions.
A method according to any one of the preceding claims, wherein said method is carried out at a temperature of from about 20° C. to about 80° C.
A method of reducing an oxidised flavin cofactor, comprising:
- contacting the oxidised flavin cofactor and molecular hydrogen (1H2) 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.
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 (1H2) or an isotope thereof,
- wherein the first polypeptide does not comprise a native flavin active site for NAD(P)+ reduction.
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 (1H2) or an isotope thereof;
- and
- a reactant for conversion to said reaction product.
Dependencies are inferred from the claim text.