Cofactor regeneration system

1. A method for manufacturing a cofactor regeneration system, the method comprising:

a. identifying:

i. a first electron transfer component comprising a NADH: acceptor oxido-reductase that has activity in an assay which comprises:

a. adsorbing the NADH: acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte;

b. holding the electrode at a constant potential of −412 mV while an increasing concentration of NAD + is injected into said electrochemical cell solution; and

c. recording a change in the electrocatalytic current magnitude at the electrode; or

ii. a first electron transfer component comprising a NADPH: acceptor oxido-reductase that has activity in an assay which comprises:

a. adsorbing the NADPH: acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte;

b. holding the electrode at a constant potential of −412 mV while an increasing concentration of NADP+ is injected into said electrochemical cell solution; and

c. recording a change in the electrocatalytic current magnitude at the electrode; and

iii. a second electron transfer component comprising a hydrogenase moiety that has an electrocatalytic H + reduction current, electrocatalytic H 2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H + /H 2 couple under the experimental conditions, when tested in an assay comprising:

a. attaching the hydrogenase to an electrode such that the hydrogenase can exchange electrons directly with the electrode;

b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with H 2 ; and

c. cycling the electrode potential between lower and upper limiting potentials; or

iv. a second electron transfer component comprising a non-biological nanoparticle that has an electrocatalytic H + reduction current, electrocatalytic H 2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H + /H 2 couple under the experimental conditions, when tested in an assay comprising:

a. attaching the non-biological nanoparticle to an electrode such that the non-biological nanoparticle can exchange electrons directly with the electrode;

b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with H 2 ; and

c. cycling the electrode potential between lower and upper limiting potentials; and

b. providing the first electron transfer component, the second electron transfer component, and an electronically conducting surface;

wherein the first and second electron transfer components do not occur together in nature as an enzyme complex; and

c. immobilising the first and second electron transfer components on the electronically conducting surface so that in use electrons flow:

from the first electron transfer component via the electronically conducting surface to the second electron transfer component; or

from the second electron transfer component via the electronically conducting surface to the first electron transfer component.

2. The method according to claim 1 , wherein the first electron transfer component is a diaphorase.

3. The method according to claim 1 , wherein the first electron transfer component comprises an amino acid sequence having at least 90% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65; or wherein the second electron transfer component comprises an amino acid sequence having at least 90% sequence identity to one or more sequences selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.

4. A cofactor regeneration system comprising:

a. a first electron transfer component comprising:

i) a NADH: acceptor oxido-reductase that has activity in an assay which comprises:

a. adsorbing the NADH: acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte;

b. holding the electrode at a constant potential of −412 mV while an increasing concentration of NAD + is injected into said electrochemical cell solution; and

c. recording a change in the electrocatalytic current magnitude at the electrode; or

ii) a NADPH acceptor: oxido-reductase that has activity in an assay which comprises:

a. adsorbing the NADPH: acceptor oxido-reductase onto an electrode which is immersed in an electrochemical cell solution containing buffered electrolyte;

b. holding the electrode at a constant potential of −412 mV while an increasing concentration of NADP + is injected into said electrochemical cell solution; and

c. recording a change in the electrocatalytic current magnitude at the electrode; and

b. a second electron transfer component comprising:

i) a hydrogenase moiety that has an electrocatalytic H + reduction current, electrocatalytic H 2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H + /H 2 couple under the experimental conditions, when tested in an assay comprising:

a. attaching the hydrogenase to an electrode such that the hydrogenase can exchange electrons directly with the electrode;

b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with H 2 ; and

c. cycling the electrode potential between lower and upper limiting potentials; or

ii) a non-biological nanoparticle that has an electrocatalytic H + reduction current, electrocatalytic H 2 oxidation current, or a combination thereof, within 100 mV below or above the thermodynamic potential of the 2H + /H 2 couple under the experimental conditions, when tested in an assay comprising:

a. attaching the non-biological nanoparticle to an electrode such that the non-biological nanoparticle can exchange electrons directly with the electrode;

b. immersing the electrode in an electrochemical cell solution containing buffered electrolyte saturated with H 2 ; and

c. cycling the electrode potential between lower and upper limiting potentials; and

c. an electronically conducting surface; and

wherein the first and second electron transfer components are immobilised on the electronically conducting surface; and

wherein the first and second electron transfer components do not occur together in nature as an enzyme complex; and

wherein the cofactor regeneration system is configured so that in use electrons flow:

from the first electron transfer component via the electronically conducting surface to the second electron transfer component; or

from the second electron transfer component via the electronically conducting surface to the first electron transfer component.

5. The method according to claim 1 , wherein the first electron transfer component comprises an amino acid sequence having at least 90% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 66 and SEQ ID NO: 67; or wherein the second electron transfer component comprises an amino acid sequence having at least 90% sequence identity to one or more sequences selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72.