中国明对虾体壁N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质

摘要

The purpose of this study was to isolate, purify and characterize the β-N-acetyl-D-glucosaminidase (EC3.2.1.52, NAGase) from the integument of Fenneropenaeus chinensis. The NAGase from the integument of F. chinensis was precipitated by ammonium sulfate and then purified by Sephadex G-100 and DEAE-cellulose. The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The specific activity of the purified enzyme was 3938.56 U/mg. The molecular weight of NAGase was 48.88 kD. The optimal pH value was 6.0 and the optimal temperature was 45℃. The enzyme was stable in the pH ranges of 6.0~9.0 with temperature between 20 and 35℃. The enzyme exhibited typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc. The Km and Vmax values were 0.229 mmol/L and 5.00 µmol/(L·min), respectively. Metal ions Na+, Li+ and Ba2+ had no effect on the enzyme activity. Mg2+, Ca2+, Mn2+, Co2+, Fe3+and Al3+activated the enzyme. In contrast, Cu2+, Zn2+ and Pb2+ inhibited the enzyme. Hg2+inhibited the enzyme activity by 42.37%at 10 mmol/L, while it activated the enzyme at 1 mmol/L.%为了探讨对虾 N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52, NAGase)的分离纯化及其酶学性质,作者以中国明对虾(Fenneropenaeus chinensis)体壁为材料,通过硫酸铵沉淀分级分离、SephadexG-100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,纯化酶比活力为3938.56U/mg。通过SDS-PAGE凝胶电泳,测得该酶亚基分子量为48.88 kD。酶的最适pH为6.0,最适温度为45℃；该酶在pH 6.0~9.0区域较稳定,温度稳定性范围是20~35℃,45℃下处理1 h酶活力丧失65.04％。酶水解对硝基苯-N-乙酰-β-D-氨基葡萄糖苷的Km为0.229 mmol/L, Vmax为5.00μmol/(Lmin)。进一步研究金属离子对酶活力的影响,结果表明： Li+、Na+和Ba2+对酶没有明显影响, Mg2+、Ca2+、Mn2+、Co2+、Fe3+和Al3+对酶均有不同程度的激活作用, Cu2+、Zn2+和Pb2+对酶呈抑制作用,1.0 mmol/L Hg2+对酶呈激活作用,而10 mmol/L Hg2+使酶活力降低42.37%。