摘要
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%。
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＆#183;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.
出处
《海洋科学》
CAS
CSCD
北大核心
2014年第8期52-58,共7页
Marine Sciences
基金
福建省高校服务海西重点建设资助项目(2008HX02)
福建省教育厅科技项目(JB04175)
莆田学院科研基金资助项目(2004031)
