中华绒螯蟹(Eriocheir sinensis)N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及性质的初步研究

PRELIMINARY STUDIES ON ISOLATION,PURIFICATION AND SOME PROPERTIES OF THE β-N-ACETYL-D-GLUCOSAMINIDASE FROM ERIOCHEIR SINENSIS

本研究以中华绒螯蟹内脏为材料,经过硫酸铵沉淀分级分离、两次DEAE-32离子交换柱层析和Sephadex G-100分子筛柱层析纯化,获得比活力为4490.79U/mg、纯化倍数为28.07倍的聚丙烯酰胺凝胶电泳纯的N-乙酰-β-D-氨基葡萄糖苷酶制剂。酶分子中各亚基的分子量分别为121.219、8.63和73.48 kD,等电点为4.5。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖为底物,进行酶催化底物水解的反应动力学研究,结果表明:酶催化底物反应的最适pH为5.5,最适温度为45℃。该酶在pH4.9—9.3区域或40℃以下处理30min,酶活力保持稳定。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为0.357 mmol/L,最大反应速度Vm为10.41μmol/L.min。酶催化pNP-β-D-GlcNAc反应的活化能为76.50kJ/mol。金属离子对酶的效应试验表明:Mg2+、Ca2+和Ba2+对酶活力没有影响。Na+对酶有激活作用,Li+、K+、Zn2+、Hg2+、Pb2+、Cu2+和Al3+对酶活力表现出不同程度的抑制作用。

A β-N-Acetyl-D-glucosaminidase （EC3.2.1.30） was purified from the viscera of Edocheir sinensis by ammonium sulfate fractionation,chromatography on DEAE-32, Sephadex G-100 and DEAE-32. The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. The specific activity of the enzyme was 4490.79U/rag. The molecular weight of the subunits was determined to be 121.21,98.63 and 73.48kD,respectively. The pI value was calculated to be 4.5 by isoelectric focusing. The optimum temperature and pH of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide （pNP-NAG） were determined to be at 45℃and at pHS.5,respectively. The enzyme was stable in the pH ranges of 4.9 to 9.3 under 37℃ and at temperatures below 40℃. The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc.The Km and Vm values were determined to he 0.357mmo/L and 10.41μmol/L min at pH5.6 and 37℃, respectively .The activation energy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was to be 76.50kJ/mol .The effects of metal ions on the enzyme were studied. Mg^2＋ , Ca^2＋ and Ba^2＋ had not influenced the enzyme activity. Na^＋ activated the enzyme, while, Li^＋ , K^＋ , Zn^2＋ , Hg^2＋ , Pb^2＋ , Cu^2＋ , and Al^3＋ showed various degrees of inhibitory effects on the enzyme.