Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Abstract

Recycling of NADP(+) using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP(+) was recycled more than 10-fold.We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation. 1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm. 2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration. 3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2). o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity.Protection against heat inactivation was obtained for the two activities with NAD(+), NADP(+), NADH, and NADPH.All these results suggested the possibility of recycling of NADP(+) under similar conditions to those previously described for NAD(+) (Burstein et al., 1981). It becomes thus possible to use various NAD(+) and NADP(+)-dependent dehydrogenases in enzyme technology.