Redox enzymes are widely used as powerful biocatalysts owing to their high selectivity and sustainability. Since most of the enzymatic reactions require the expensive coenzyme (the reduced form of nicotinamide adenine dinucleotide, NADH) in a stoichiometric amount, efficient NADH regeneration is essential for various biocatalytic syntheses. Even though an electroenzymatic recycling method has many advantages, it requires low-molecular-weight redox mediators to transfer electrons from electrodes to an enzymes' active site, which are generally toxic and make it difficult to purify products after the reaction. In the present study, we have conjugated a mediator to enzyme catalyst to solve the problem of the electroenzymatic NADH regeneration. Mediators bound to enzymes can be separated from the reaction medium and then reused readily. Moreover, the conjugation can promote a faster electron transfer between mediators and enzymes. For the conjugation, a viologen with a carboxyl group, ethyl carboxyethyl viologen (ECV) was synthesized and covalently linked to diaphorase (DI) using a cross-linker. Cyclic voltammetry showed that the ECV conjugated to DI displayed electrochemical behavior similar to free ECV. In marked contrast to native DI, the DI-ECV conjugate reduced NAD + to NADH without any mediator, and this bioelectrocatalyst regenerated NADH successfully for enzymatic lactate synthesis.Redox enzymes are widely used as selective and sustainable catalysts not only in various organic synthesis processes but also in electrochemical applications such as biosensors and biofuel cells. 1,2 About 90% of them require nicotinamide coenzymes [e.g., nicotinamide adenine dinucleotide (NAD + ) and its reduced form, NADH], which form reversible redox pairs to accept or donate electrons in redox reactions. 1,3 In particular, NADH participates in important and challenging reactions including CO 2 fixation, alkane hydroxylations, and chiral alcohol syntheses in stoichiometric quantities; 1,4 however, NADH is too expensive to use on a large scale. Hence, the efficient regeneration of NADH from NAD + is essential for industrial applications of these biocatalysts. 3,[5][6][7][8] There are three popular methods to recycle NADH: enzymatic, electrochemical, and electroenzymatic. Even though enzymatic regeneration of NADH where a second enzyme (e.g., formate dehydrogenase) and an auxiliary substrate (e.g., formate) are used as a catalyst and a sacrificial donor, respectively, is the most practical method owing to its high total turnover number and selectivity, the co-substrate (e.g., formate) makes it difficult to isolate reaction products. 3,6 In this respect, an electrochemical method in which water is the sole electron and proton source for NAD + reduction has been intensively investigated. 7,8 Although electron supply from water electrolysis is attractive, a direct NAD + reduction on bare electrodes yields enzymatically inactive forms such as NAD dimers and 1,6-NADH isomer due to the formation of NAD free radicals followed by radical c...
