莆田黑猪精液中NAGase酶学特性研究

试验对以莆田黑猪精液为材料分离纯化得到的N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)进行了理化特性研究。经硫酸铵分级沉淀、DEAE Sepharose Fast Flow离子交换层析和Sephadex G100分子筛层析获得PAGE电泳纯化的NAGase酶制剂。以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-GlcNAc)为底物,研究酶催化水解的相关性质。分离纯化获得的酶制剂比活力为1561.42 U/mg,分子质量为58 ku,只有1个亚基,等电点pI为9.13。酶的最适pH为5.6,最适温度为45 ℃,酶在pH 3.6～7.8之间稳定,当pH>8时迅速失活,在50 ℃以下处理30 min酶活力保存稳定,高于50 ℃时,酶活力迅速降低。酶促反应动力学符合米氏双曲线方程,米氏常数K_m为0.82 mmol/L,最大反应速度V_m为39.23 μmol/(L·min)。催化pNP-GlcNAc反应的活化能为27.30 kJ/mol。金属离子中Na⁺、K⁺、Mg²⁺、Ca²⁺对酶活力无明显影响,Zn²⁺、Cu²⁺、Pb²⁺对酶有抑制作用。

Study on Characterization of NAGase from the Sperm of Putian Black Pig

This paper studied the character of N-acetyl-β-D-glucosaminidase (NAGase) from the sperm of Putian Black pig. The purification steps involved the following procedures: Ammonium sulfate precipitation, anion-exchange chromatography on DEAE Sepharose Fast Flow, gel filtration chromatography on Sephadex G100. The purified enzyme preparation was homogeneous judged by polyacrylamide gel electrophoresis. It was found that the specific activity of the enzyme was 1561.42 U/mg. The enzyme molecular weight was estimated as 58 ku. The optimal pH value was 5.6 and the optimal temperature was 45 ℃. The enzyme was stable in the pH ranges of 3.6 to 7.8. The enzyme followed typical Michaelis-Menten kinetics for the hydrolysis of pNP-GlcNAc and the K_m and V_m values were determined to be 0.82 mmol/L and 39.23 μmol/(L·min), respectively. The activation energy of the enzyme for hydrolysis of pNP-GlcNAc was 27.30 kJ/mol. The effects of some metal ions on the enzyme activity were determined, Na⁺, K⁺, Mg²⁺, Ca²⁺ had no effects on the enzyme activity, while Zn²⁺, Cu²⁺, Pb²⁺ had inhibitory effect on the enzyme activity.