猪精液碱性N-乙酰-β-D-氨基葡萄糖苷酶的纯化与酶学性质研究

Study on Purification and Properties of the Alkaline N-Acetyl-β-D-glucosaminidase from Porcine Semen

【目的】分离纯化猪精液碱性N-乙酰-β-D-氨基葡萄糖苷酶制剂,研究其理化性质。【方法】采用硫酸铵沉淀分级分离、DEAE-32阴离子交换柱层析、Sephadex G-100分子筛柱层析和CM-52阳离子交换柱层析方法,分离纯化猪精液的N-乙酰-β-D-氨基葡萄糖苷酶;采用聚丙烯酰胺凝胶电泳和SDS-聚丙烯酰胺凝胶电泳法进行纯度鉴定和酶分子量测定;采用聚丙烯酰胺等电聚焦圆盘电泳法测定酶的等电点。【结果】经分离纯化获得比活力为17606.15U·mg-1、纯化倍数为270.53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。酶催化对-硝基苯-N-乙酰-β-D-氨基葡萄糖(pNP-NAG)水解的最适pH为5.5,最适温度为50℃。该酶在pH 3.0～8.9区域较稳定;在45℃以下处理30min,酶活力保持稳定。酶分子中亚基的分子量为58.03 kD,等电点为9.42。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为1.94mmol·L-1,最大反应速度Vm为27.53 μmol·L-1·min-1。酶催化pNP-NAG反应的活化能为88.73 kJ·mol-1。【结论】本试验选用的分离纯化的方法是可行的。

[Objective] The purpose of this experiment was to separate and purify the N-Acetyl-β-D-Glucosaminidase （EC3.2.1.30,NAGase） from the porcine semen. The physicochernical properties of the enzyme were studied, [Method] The NAGase was purified by ammonium sulfate fractionation, chromatography on DEAE-32, Sephadex G-100 and CM-52. The purity of the enzyme was identified by polyacrylamide gel electrophoresis. The pI value was determined by isoelectric focusing. The molecular weight of the subunit of the enzyme was determined by SDS-polyacrylarnide gel electrophoresis. [Result] The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. The specific activity of the enzyme was of 17 606.15 U·mg^-1. The molecular weight of the subunit of the enzyme was determined to be 58.03 kD. The pI value was calctflated to be 9.42. The optimum temperature and pH of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-fl-D-glucosaminide （pNP- NAG） were determined to be at 50℃ and at pH 5.5, respectively. The enzyme was stable in the pH ranges of 3.0 to 8.9 under 37℃ and at temperatures below 45℃, The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-NAG. The Km and Vm values were determined to be 1.94 mmol·L^-1 and 27.53 μmol·L^-l·min^-1 at pH 5.6 and 37℃, respectively, The activation energy of the enzyme for the hydrolysis of pNP-NAG was to be 88.73 kJ·mol^-1. [Conclusion] The method used to separate and purify the enzyme in this experiment was feasible.