Optimization of the mediated electrocatalytic reduction of NAD⁺ by cyclic voltammetry and construction of electrochemically driven enzyme bioreactor

The optimal concentrations of diaphorase, methyl viologen (MV²⁺) and NAD²⁺ in the mediated electrocatalytic reduction of NAD⁺ were decided by applying cyclic voltammetry. The steady-state catalytic current was achieved under the conditions of 1.5 U diaphorase ml^-1, 0.2 mM MV²⁺, and 4.8 mM NAD⁺ at the scan rate of 2 mV s^-1, suggesting that the rate of the electrocatalytic reaction is the highest under the former conditions. However, NAD⁺ was effective at 0.3 mM as it was at 4.8 mM when the electrocatalysis is coupled with an enzymatic synthesis requiring NADH. In effect, the electrochemical procedure under the conditions of 1.5 U diaphorase ml^-1, 0.2 mM MV²⁺, and 0.3 mM NAD⁺ worked satisfactorily to drive an enzymatic reduction of pyruvate to D-lactate in the presence of D-lactate dehydrogenase.