›› 2005, Vol. 48 ›› Issue (4): 498-502.

• 研究论文 • 上一篇    下一篇

棉铃虫N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质

黄小红,陈清西,尤民生,王君,关雄   

  • 出版日期:2005-09-07 发布日期:2005-08-20
  • 通讯作者: 关雄

Isolation, purification and partial characterization of the β-N-acetyl-D-glucosaminidase from the pupae of Helicoverpa armigera

HUANG Xiao-Hong, CHEN Qing-Xi, YOU Min-Sheng, WANG Jun, GUAN Xiong   

  1. Fujian Agriculture and Forestry University
  • Online:2005-09-07 Published:2005-08-20

摘要: 以棉铃虫Helicoverpa armigera蛹为材料,通过硫酸铵沉淀分级分离、Sephadex G-200分子筛柱层析和DEAE-32离子交换柱层析纯化,获得聚丙烯酰胺凝胶电泳纯的N-乙酰-β-D-氨基葡萄糖苷酶酶制剂。纯酶的比活力为2 678.79 U/mg。以对硝基苯-N-乙-β-D-氨基葡萄糖苷(pNP-β-D-GlcNAc)为底物,研究酶催化底物水解的反应动力学。结果表明:酶的最适pH5.63,最适温度为55℃。该酶在pH 48区域较稳定,而在pH>8时能迅速失去活力;在50℃以下处理30 min,酶活力仍保持稳定,高于50℃,酶很快失去活力。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km0.16 mmol/L,最大反应速度Vm10.73 μmol·L-1·min-1。酶催化pNP-β-D-GlcNAc反应的活化能为66.24 kJ/mol

关键词: 棉铃虫, N-乙酰-β-D-氨基葡萄糖苷酶, 分离纯化, 动力学, 稳定性

Abstract:

β-N-acetyl-D-glucosaminidase (EC3.2.1.52) was purified from the pupae of Helicoverpa armigera by ammonium sulfate fractionation and chromatography on Sephadex G-200 and DEAE cellulose. The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. It was found that the specific activity of the enzyme was 2 678.79 U/mg. The optimal pH value was 5.63 and the optimal temperature 55℃. The enzyme was stable in the pH ranges of 4 to 8 under 37℃. The enzyme follows typical Michaelis Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc and the Km and Vm values were 0.16 mmol/L and 10.73 μmol·L-1·min-1, respectively. The activation energy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was 66.24 kJ/mol.

Key words: Helicoverpa armigera, β-N-acetyl-D-glucosaminidase, isolation and purification, kinetics, stability