| 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5.00 μmol/(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%。 |
| 关键词: 中国明对虾(Fenneropenaeus chinensis) 体壁 N-乙酰-β-D-氨基葡萄糖苷酶 分离纯化 酶学性质 |
| DOI：10.11759/hykx20130706001 |
| 分类号: |
| 基金项目: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) |
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| Isolation, purification and characterization of β-N-acetyl-Dglucosa-minidase from the integument of Fenneropenaeus chinensis |
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| Abstract: |
| 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. |
| Key words: Fenneropenaeus chinensis integument β-N-acetyl-D-glucosaminidase isolation and purification enzyme properties |
