Coupling of the electroenzymatic reduction of NAD(+) with a synthesis reaction

Hydrogenase from Alcaligenes eutrophus H16 (E.C. 1.12.1.2) catalyzes the electrochemical reduction of the pyridine cofactor NAD(+). Previous studies emphasized the kinetics and thermodynamics of the reaction. We applied hydrogenase to regenerate NADH for the transformation of alpha-ketoglutarate into L-glutamate catalyzed by an L-glutamate dehydrogenase. The system was first studied in an electrochemical thin-layer cell. A turnover number (TN) of 450 h(-1) was obtained The influence of the ratio of L-glutamate dehydrogenase and hydrogenase activities is discussed. The separate analyses of the glutamate dehydrogenase catalysis on the one hand and the hydrogenase electrocatalysis on the other led to the hypothesis that the behavior of the coupled process was controlled by the NADH concentration level. We exploited the qualitative conclusions drawn from the analytical study to test the process on a preparative scale-batch reactor. A TN of 207 h(-1) was reached. The poor stability of the hydrogenase was the major hindrance to the development of the reactor, but a possible solution was discussed by means of the stabilizing effect of the electrochemical conditions.