Purification and characterization of glucose 6-phosphate dehydrogenase of leuconostoc mesenteroides

Abstract

Glucose 6-Phosphate dehydrogenase from $\mbox{\underline{L}}$. $\mbox{\underline{mesenteroides}}$ strain No. 20-2-10 which catalyzes the oxidation of glucose 6-phosphate in the presence of either $NADP^+$ or $NAD^+$ was prepared. From the studies of time course of cultivation it was found that the enzyme productivity increased rapidly as cell grows and it was maximum at 15 hrs cultivation where the cell growth reached a stationary phase and thereafter it decreased. This phenomenon was due to the low pH of growth medium, which was associated with the growth of cell producing lactic acid. Controlling pH of culture broth at constant with an automatic titrator enzyme productivity increased by 56\%. Using the combination of Gibacron Blue F3G-A-Sepharose 4B affinity chromatography and hydroxyapatite chromatography the enzyme was purified from the crude extract of cell with 80-fold of purification. Thoroughout the purification steps the ratio of activities with $NADP^+$ to $NAD^+$ remained constant. It appeared that both coenzymes are accepted by the single enzyme. The purified enzyme was proved to be homogeneous by disc gel electrophoresis. Using this enzyme preparation, further studies on kinetics, characterization, chemical modification and glucose determination coupled with hexokinase were carried out. The apparent molecular weight of the enzyme was determined to be 112,000 by using gel filtration on Sephadex G-200 column. The optimum temperature of the NAD-linked reaction was 50$^\circ$C and the activation energy was 8.36 Kcal/mole and the heat of denaturation was -58.2 Kcal/mole. Steady state kinetic constants for the NADP-linked and NAD-linked reaction were obtained. At pH 7.8 the $K_{NADP}$ was 7.46$\mu$M; $K_{G6P}$ for NADP-linked reaction was 76.9$\mu$M ; $K``_{NADP}$ was 7.14$\mu$M ; $K_{NAD}$ was 115.2$\mu$M; $K_{G6P}$ for NAD-linked reaction was 53.65$\mu$M; $K``_{NAD}$ was 707.2$\mu$M. Blue Dextran 2,000 inhibited the enzyme completely with respect to $N...