猪精液酸性N-乙酰-β-D-氨基葡萄糖苷酶的纯化及性质

Purification and Properties of the Acidic N-acetyl-β-D-glucosaminidase from Porcine Semen

以杜洛克猪精液为材料,采用硫酸铵分级沉淀、DEAE-32阴离子交换柱层析和SephadexG-100分子筛柱层析纯化,获得纯化倍数为27.64、比活力为1773.25Umg-1的酸性N-乙酰-β-D-氨基葡萄糖苷酶纯酶制剂.经SDS-聚丙烯酰胺凝胶电泳法测定,纯酶两种亚基相对分子质量分别为129.13×103和62.24×103.对其酶学性质的研究结果表明,酶的等电点为5.10,最适pH值为5.5,最适温度为60℃.酶在pH3.6～9.2、温度10℃～55℃的范围内较稳定.以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-NAG)为底物,测得米氏常数Km为0.455mmolL-1,最大反应速度Vm为17.34μmolL-1min-1|活化能为41.70kJmol-1.

The acidic N-acetyl-β-D-glucosaminidase （EC3.2.1.52） was purified from the porcine semen by ammonium sulfate fractionation,chromatography on DEAE-32 and Sephadex G-100. The specific activity of the enzyme was 1 773.25 U mg-1. The purified enzyme preparation was homogeneous judged by polyacrylamide gel electrophoresis. The molecular weights of two subunits of the purified enzyme were determined to be 129.13×103 and 62.24×103,respectively. The pI value was calculated to be 5.10 by isoelectric focusing. The optimum temperature and pH of the enzyme were 60℃ and 5,5. The enzyme was stable in the pH ranges from 3.6 to 9.2 under 37℃ and at temperature ranges from 10 to 55℃. The K and Vm values were determined to be 0.455 mmol/L and 17.34μmol L-1min-1 at pH 5.6 and at 37 ℃, respectively. The activation energy of the enzyme for hydrolysis of the substrate was 41.70 kJ mol-1. Fig 8, Tab 1, Ref 15