Diaphorase-viologen conjugates as bioelectrocatalysts for NADH regeneration

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.