An enhanced direct electron transfer-type NAD+/NADH regenerating system using the diaphorase subunit of formate dehydrogenase 1

Abstract

Nicotinamide adenine dinucleotide redox couple (NAD+/NADH) is a significant coenzyme in the catabolism of organisms. Coupling NAD-dependent enzymatic reaction and NAD+/NADH regenerating systems can be utilized for biomimetics-associated applications, such as the production of bioreactors, biofuel cells, and biosensors. Direct electron transfer (DET)-type bioelectrocatalysis, in which an enzyme directly exchanges electrons with the electrode, can produce an efficient, simple, and biocompatible system owing to a mediator-free configuration. Formate dehydrogenase 1 from Methylorubrum extorquens AM1 (FoDH1) has been reported to have diaphorase and DET-type activity. The β subunit (diaphorase subunit) of FoDH1 is an attractive variant for constructing an efficient regenerating system due to downsizing effects and efficient interfacial electron transfer. In this study, we attempt to construct a homologous expression system to establish a recombinant FoDH1 (rFoDH1) expression system and to obtain the β subunit variant (FoDH1B). Their bioelectrochemical properties and the ET pathways of DET-type reactions have been elucidated. Finally, we demonstrated the coupling of an efficient DET-type NAD+/NADH regenerating system with NAD-dependent enzymes.