鸭肝谷氨酸脱氢酶的纯化与酶学性质研究  

Separation,Purification and Characterization of Glutamate Dehydrogenase from Duck Liver

作　　者：朱鸿[1] 李想韵[1] 王松[1] 付伟丽[1] 唐靓婷[1] 诰赵伟[1] 唐云明[1] 

机构地区：[1]西南大学生命科学学院,三峡库区生态环境教育部重点实验室,重庆市甘薯工程研究中心,重庆400715

出　　处：《食品科学》2010年第19期231-235,共5页Food Science

基　　金：重庆市科委科技攻关项目(CSCT;2004AC1012)

摘　　要：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SephacrylS-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。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 53.19 μ mol/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.

关 键 词：鸭肝 谷氨酸脱氢酶 分离纯化 性质 

分 类 号：Q554.9[生物学—生物化学]