谷氨酸生产菌S_(9114)中依赖于NADPH谷氨酸脱氢酶的纯化及性质

Purification and Characterization of NADPH-specific Glutamate Dehydrogenase from Corynebacterium glutamicum S_(9114)

利用DEAE 纤维素离交柱层析、疏水柱层析和凝胶过滤层析等从谷氨酸棒杆菌S9114 中分离纯化出依赖于NADPH的谷氨酸脱氢酶 (GOH NADPH )。经HPLC测定 ,该酶的分子量为 188ku ;经SDS -PAGE电泳测得该酶的亚基分子量为 3 2ku。提示该谷氨酸脱氢酶由 6个亚基组成。该酶对NADPH具有高度专一性 ,在pH 7 5、3 7℃下 ,以α 酮戊二酸、NH4Cl和NADPH为底物时的米氏常数Km 分别为 9 2 2mmol/L、5 2 7mmol/L和 2 3 1× 10 -3mmol/L。最适反应pH为 7 5 ,最适反应温度为 42℃ ,并对热比较稳定。此外 ,KCl对GDH NADPH的活性具有激活作用。

NADPH-specific glutamate dehydrogenase (GDH) from Corynebacterium glutamicum S 9114, a glutamate-producing bacterium, was purified by DEAE-cellulose ion exchange chromatography, hydrophobic interaction chromatography(HIC) and gel filtration chromatography. The native molecular weight estimated by HPLC was 188ku and the molecular weight of the subunit by SDS-PAGE was 32ku. It is shown that the GDH-NADPH is a hexamer with identical subunits. The optimal pH and temperature were pH 7.5 and 42℃ respectively. The enzyme had the property thermal stability. The k m values for NADPH, α-Ketoglutarate and NH 4Cl were 2.31×10 -3?mmol/L ,9.22 ?mmol/L and 52.7?mmol/L respectively. The presence of KCl remarkably activates the enzyme.