Glucose 6-Phosphate dehydrogenase from $\underline{L}$. $\underline{mesenteroides}$ 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℃ 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 7.46μM; $K_{G6P}$ for NADP-linked reaction was 76.9μM ; $K'_{NADP}$ was 7.14μM ; $K_{NAD}$ was 115.2μM; $K_{G6P}$ for NAD-linked reaction was 53.65μM; $K'_{NAD}$ was 707.2μM. Blue Dextran 2,000 inhibited the enzyme completely with respect to $NAD^+$ and the inhibition constant was 0.98 M. From the studies of the effect of varying pH the optimum pH was 7.8 for NAD-linked reaction. The pH dependent kinetic constants suggests that two groups whose pKb is 7.2 and pKa is 9.0 to 9.6 are involved in the interaction of the enzyme and the substrates. Among these the participation of a group on the enzyme whose pKb is 7.2 in the catalytic function was tested. Evidence was confirmed by the results of photooxidation and carboxymethylation that the group may be the imidazole group of histidine. Practical use of this enzyme with $NAD^+$ is advantageous because of the decreased cost and greater stability of coenzyme. This enzyme coupled with hexokinase was used for the determination of glucose. Coefficient of variation ranged from 0.64 to 2.22% for the range of glucose concentration between 0.4 and 40 mM. Recovery of glucose was determined to be 100.5 ± 3.1% and 104.3 ± 9.6% respectively for the case of glucose dissolved in serum and urine.
$\underline{Leuconostoc}$ mesenteroides No. 20-2-10의 glucose 6-phosphate dehydrogenase 를 생산, 정제 후 생화학적인 특성을 연구하였다. 미생물의 배양 시간의 경과에 따른 배양 상태를 관찰한 결과 생장 초기에는 효소의 생산량이 미생물의 생장과 비례하였으나 생장 말기에는 오히려 감소하였다. 이것은 미생물이 생장하면서 액체배지의 pH 가 낮아지며 이에 따른 효소의 변성에 의한 것으로 밝혀졌다. 또한 미생물의 전 생장 과정을 통해 액체 배지의 pH 를 7.0 으로 조절한 결과 효소의 생산량이 56% 증가하였다.
미생물로 부터 생산된 효소는 간단히 Cibacron Blue F3G-ASepharose 4B affinity chromatography와 hydroxyapatite chromatography 에 의해 결정 정제하였으며 전기 영동 결과 순수함을 알았다. 이 효소는 $NAD^+$ 와 $NADP^+$ 두가지 조효소에 대한 특이성을 보이며 이러한 반응은 동일한 효소에 의함을 알 수 있었다. Sephadex G-200 gel chromatography 에 의해 외형 분자량은 112,000 으로 나타났다. 최적 온도는 50℃ 였으며 활성화 에너지는 8.36 kcal/mole 였으며 변성 에너지는 -58.2 kcal/mole 이었다. 최적 pH 7.8 에서 초기 반응속도를 연구한 결과 반응 속도 상수를 구했으며 pH 의 변화에 따른 반응속도 상수의 변화를 연구 하였다. 결과 pKb = 7.2 인 활성기가 효소작용에 관계하는 것으로 나타났으며 photooxidation 과 carboxymethylation 에 의해 이 활성기는 histidine 의 imidazole group 임이 밝혀졌다. 생산 정제된 효소를 hexokinase 와 결합하여 소변 및 혈청의 glucose 정량에 사용해 본 결과 아주 합리적인 결과를 얻었다.