Unconventional and effective methods for the regeneration of NAD(P) H in microorganisms or crude extracts of cells

Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of 4S-²H]NADH and 4S-²H]NADPH using ²H₂O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction