鸭肝谷氨酸脱氢酶的纯化与酶学性质研究

目的：获得鸭肝谷氨酸脱氢酶纯品并对其酶学性质进行研究。方法：采用丙酮脱脂、重金属离子沉淀、硫酸铵分级沉淀、DEAE-Sepharose 离子交换层析和Sephacryl S-200 凝胶层析方法，分离纯化鸭肝谷氨酸脱氢酶，用SDS- 聚丙烯酰胺凝胶电泳法进行纯度鉴定和酶相对分子质量测定。结果：从鸭肝中分离纯化获得电泳纯的谷氨酸脱氢酶，纯化倍数为60.93 倍，酶活力回收率为11.02%，比活力达24.37U/mg。酶相对分子质量为371.41，亚基相对分子质量为61.60。推测该酶由6 个相同亚基构成。该酶对NADH 的Km 为53.19μmol/L，最适pH 值为10.0，最适反应温度为35℃。该酶在pH8.0 左右较稳定；在40℃以下酶活力保持稳定。Zn2+、Li+ 和Cu2+ 对该酶具有显著的抑制作用。结论：分离纯化获得谷氨酸脱氢酶，该酶具有较高应用价值。

Separation,Purification and Characterization of Glutamate Dehydrogenase from Duck Liver

Objective: To obtain high-purity glutamate dehydrogenase (GDH) from duck liver and characterize this enzyme. Methods: Crude GDH solution was obtained from duck liver after acetone defatting, addition of MnCl2 for impurity precipitation, ammonium sulfate salting-out and DEAE-Sepharose ion exchange and Sephacryl S-200 gel permeation chromatographic fractionation. Purity identification and relative molecular mass determination were conducted using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: A GDH enzyme of electrophoretic homogeneity was obtained, with a purification fold of 60.93, an activity recovery of 11.02% and a specific activity of 24.37 U/mg. This enzyme had a relative molecular mass of 371.41 and a subunit relative molecular mass of 61.60. It was deduced that this enzyme was composed of the same six subunits. Its apparent Km towards NADH was 53.19μmol/L, and the optimal reaction pH and temperature 10.0 and 35 ℃, respectively. This enzyme had excellent stability at around pH 8.0 and at a temperature below 40 ℃. Zn2+, Li+ and Cu2+ had significant inhibition on this enzyme. Conclusion: A high-purity GDH enzyme has been successfully prepared. This enzyme greatly deserves to be developed and utilized.