【摘要】 山麻鸭的睾丸和肝脏经硫酸铵分级沉淀分离、DEAE-32离子交换柱层析,获得的肝脏N-乙酰-β-D-氨基葡萄糖苷酶(NAGase,EC3.2.1.52)比活力为85.30 U.mg-1,纯化倍数为10.48.睾丸NAGase再进一步通过Sephacryl S-300凝胶过滤纯化,获得的NAGase比活力为5537.00 U.mg-1,纯化倍数为59.20.睾丸NAGase经聚丙烯酰胺凝胶等电聚焦电泳,出现单一蛋白带,测定等电点pI为5.05.以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-NAG)为底物研究酶催化底物水解的反应动力学.结果表明:山麻鸭睾丸和肝脏NAGase的最适pH分别为5.70和5.60,酶在pH为3.00-8.43时较稳定,而pH>8.43时很快失活;酶的最适温度分别为45和60℃,当温度为80℃时酶完全失活.睾丸NAGase在40℃下处理30 min,酶活力保持稳定;而肝脏NAGase在30℃下处理30 min,酶活力保持稳定,酶催化pNP-NAG水解反应遵循米氏方程,睾丸和肝脏NAGase水解pNP-NAG的Km分别为0.17和0.21 mmol.L-1,Vm分别为8.82和7.25μmol.L-1.min-1,活化能分别为57.57和79.47 kJ.mol-1.更多还原

【Abstract】 β-N-acetyl-D-glucosaminidase(NAGase,EC3.2.1.52) was purified from the testes and liver of Shanma duck by ammonium sulfate fractionation,chromatography on DEAE-32,and testes′ NAGase was subsequently purified on Sephacryl S-300.The specific activities of the enzymes were determined to be 5537.00 and 85.30 U·mg-1.The purified enzyme from testes was homogeneous as judged by isoelectric focusing.The pI value was determined to be 5.05.The optimum pH and optimum temperature of these two kinds of enzymes for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.70,45 ℃ and pH 5.60,60 ℃, respectively.The enzymes were both stable in the pH range from 3.00 to 8.43 and at temperatures below 45 and 35 ℃.The kinetic behavior of the enzymes in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics.Km and Vm of the enzyme from testes were 0.17 mmol·L-1 and 8.82 μmol·L-1·min-1,while Km and Vm of the enzyme from liver were 0.21 mmol·L-1 and 7.25 μmol·L-1·min-1 at pH 5.63 and 37 ℃,and the activation energy was determined to be 57.57 and 79.47 kJ·mol-1,respectively.更多还原