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

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

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-乙酰-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pH为5.63,最适温度为55℃.该酶在pH 4～8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Km为0.16 mmol/L,最大反应速度Vm为10.73 μmol·L-1·min-1.酶催化pNP-β-D-GlcNAc反应的活化能为66.24 kJ/mol.

β-N-acetyl-D-glucosaminidase （EC3.2.1.52） was purified from the pupae of Helicoverpa armigera bya mmonium sulfate fractionation and chromatography on Sephadex G-200 and DEAE-cellulose. The purified enzymep reparation was homogeneous as judged by polyacrylamide gel electrophoresis. It was found that the specific activity of thee nzyme 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 10.73μmol· L^-1· min^-1, respectively. The activationen ergy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was 66.24 kJ/mol。