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Updated: May 15, 2026

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Direct electron transfer-type NADH regeneration flow reactor with recombinant diaphorase subunit of formate
Reiji Kinosada1, Sachika Takebe1, Taiki Adachi1
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
Abstract:
Nicotinamide adenine dinucleotide redox couple (NAD+/NADH) plays important roles in vivo as a redox cofactor, making NADH regeneration systems valuable to the biochemical industry. Among various methods, electrochemical NADH regeneration employing direct electron transfer (DET)-type bioelectrocatalysis offers low overpotentials and high specificity without the need for mediators. A recombinant β subunit (diaphorase subunit) of formate dehydrogenase 1 (FoDH1B) from Methylorubrum extorquens AM1 has reportedly shown DET-type activity in NAD+/NADH interconversion. In this study, a heterologous expression system for FoDH1B in Escherichia coli (EcFoDH1B) was constructed to improve protein expression efficiency, and its electrochemical properties were elucidated, exhibiting 10-fold higher DET-type activity than that expressed in M. extorquens AM1. In addition, appropriate conditions for the DET-type reaction were investigated. Furthermore, a bioelectrochemical NADH regeneration flow reactor was constructed using an EcFoDH1B-modified carbon felt electrode, achieving a turnover frequency of 10,000 h-1 and a Faradaic efficiency over 90% at a low overpotential (0.10 V).
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