日本沼虾(Macrobrachium nipponense)N-乙酰-β-D-氨基葡萄糖苷酶初步纯化及部分性质

Preliminary Purification and Some Properties of β-N-Acetyl-D-glucosaminidase from Macrobrachium nipponense

以日本沼虾内脏为材料，通过硫酸铵沉淀分级分离、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3000Umg^-1、纯化倍数为8．88倍的N-乙酰-β-D-氨基葡萄糖苷酶制剂（NAGase）．以对-硝基苯-N-乙酰-β-D-氨基葡萄糖为底物，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6．0，最适温度为53℃．该酶在pH4．5—9．3区域较稳定，当pH〉9．3很快失活；在50℃以下处理30min，酶活力保持稳定，高于50℃，酶稳定性较差，75℃酶完全失活．酶促反应动力学符合米氏双曲线方程，测得米氏常数Km为0．165mmolL^-1，最大反应速度‰为6．55μmolL^-1min^-1．酶催化pNP-β-D-GlcNAc反应的活化能为63．55kJmol^-1．

A β-N-Acetyl-D-glucosaminidase （EC3. 2. 1. 30, NAGase ） was purified from the viscera of Macrobrachium nipponense by ammonium sulfate fractionation, chromatography on DEAE-32 and Sephadex G-100. The specific activity of the enzyme was 3 000 U mg^-1. The optimum pH and temperature were determined to be at pH 6.0 and 53 ℃, respectively. The enzyme was stable at pH ranging from 4.5 to 9.3 under 37 ℃, which was followed by typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc. The Km and Vm values were determined to be 0. 165 mmol L^-1 and 6. 554 μmol L^-1 min^-1 , respectively. The activation energy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was detemined to be 63.55 kJ mol^-1. Fig 5, Tab 1, Ref 16