食品科学 ›› 2010, Vol. 31 ›› Issue (19): 231-235.doi: 10.7506/spkx1002-6630-201019050

• 生物工程 • 上一篇    下一篇

鸭肝谷氨酸脱氢酶的纯化与酶学性质研究

朱 鸿,李想韵,王 松,付伟丽,唐靓婷,诰赵伟,唐云明*   

  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
  • 收稿日期:2010-01-25 修回日期:2010-09-03 出版日期:2010-10-15 发布日期:2010-12-29
  • 通讯作者: 唐云明* E-mail:tbright@swu.edu.cn
  • 基金资助:

    重庆市科委科技攻关项目(CSCT,2004AC1012)

Separation, Purification and Characterization of Glutamate Dehydrogenase from Duck Liver

ZHU Hong,LI Xiang-yun,WANG Song,FU Wei-li,TANG Liang-ting,GAO Zhao-wei,TANG Yun-ming*   

  1. School of Life Science, Southwest University, Key Laboratory of Eco-environments in Three Gorges Reservoir Region,
    Ministry of Education, Chongqing Sweetpotato Engineering Research Center, Chongqing 400715, China
  • Received:2010-01-25 Revised:2010-09-03 Online:2010-10-15 Published:2010-12-29
  • Contact: TANG Yun-ming* E-mail:tbright@swu.edu.cn

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。

关键词: 鸭肝, 谷氨酸脱氢酶, 分离纯化, 性质

Abstract:

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.

Key words: duck liver, glutamate dehydrogenase (GDH), purification, characterization

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