Abstract
<jats:p> Nicotinamide adenine dinucleotide cofactor (NAD+/NADH) is considerably involved in many important biocatalysis processes, especially the reductive biotransformation relying on NADH dependent enzymes. NADH cofactor is consumed stoichiometrically and expensive, making it necessary to regenerate NADH from an economic perspective. Electroenzymatic NADH regeneration is of great interest given its advantages, including elimination of sacrificial substrates, simplified downstream process and less non-active NADH byproduct generation. This approach relies on the establishment of electron communication between a solid electrode surface and the flavin active site of a diaphorase like enzyme. Direct electron transfer-type bioelectrocatalysis of the β subunit of <jats:italic toggle="yes">Methylorubrum extorquens</jats:italic> AM1 formate dehydrogenase 1 (FoDH1B) for NAD <jats:sup>+</jats:sup> /NADH interconversion has been recently reported. Herein, the mediated electron transfer-type reaction of FoDH1B was thoroughly examined by using different freely diffusing mediators, including organic molecules and metal complexes. Mediators with redox potentials in a range between ca. –0.55 and –0.85 V vs. Ag/AgCl at pH 7 are suggested suitable for NAD <jats:sup>+</jats:sup> reduction when catalyzed by FoDH1B. Unlike diaphorase with only a flavin active site, NAD <jats:sup>+</jats:sup> /NADH interconversion seems to be impossible if the same mediator is used for FoDH1B, as two different Fe-S clusters with a potential difference of 100 mV are suggested to be separately involved for either oxidation or reduction. Mediator grafted redox active polymers were then synthesized. Molecular weight of the polymer and protection layer were found to be two important means to improve the mechanical stability of the redox polymer/enzyme coating layer. Electroenzymatic regeneration of bioactive 1,4-NADH was finally demonstrated using compact bioelectrodes. </jats:p>