초록
The electrocatalytic reduction of NAD$^{+}$ using diaphorase was studied. methyl viologen (MV$^{2+}$) mediator between an electrode and the enzyme. Steady-state currents could be obtained under the conditions of slow scan rate, low MV$^{2+}$concentration, and high NAD$^{+}$ concentration as the electrode reaction was converted to an electrochemical-catalytic (EC') reaction. The biomecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5$\times$10$^3$M$^{-1}$ s$^{-1}$ from the slope of the current versus [MV$^{2+}$] plot. And the optimal concentrations of diaphorase, MV$^{2+}$ and NAD$^{+}$ in the mediated electrocatalytic reduction of NAD$^{+}$ were decided by applying the cyclic voltammetry. The optimal concentrations of the species were obtained by finding the conditions which gave the highest and steady-state current at a gold-amalgam electrode. The highest and steady-state catalytic current was achieved under the conditions of 1.5 U/ml diaphorase, 0.2 mM MV$^{2+}$, and 4.8 mM NAD$^{+}$ at the scan rate of 2 mV s$^{-1}$ , suggesting that the rate of the electrocatalytic reation is the higest under the former conditions. The electrochemical procedure under the conditions of 1.5 U/ml diaphorase,0.2 mM MV$^{2+}$, and 4.8 mM NAD$^{+}$ was used favorably to drive an enzymatic reduction of pyruvate to D-lactate.