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.
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 Sephaeryl S-300. The specific activities of the enzymes were determined to be 5537.00 and 85.30 U · mg-l. The purified enzyme from testes was homoge- neous as judged by isoelectrie focusing. The pl 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-fl-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 tempera- tures 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 V,, 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.
出处
《福建农林大学学报（自然科学版）》
CSCD
北大核心
2009年第1期49-54,共6页
Journal of Fujian Agriculture and Forestry University:Natural Science Edition
基金
福建省教育厅资助项目(2006F5004)
作者简介
王长康（1962-），男，副教授．研究方向：家禽营养与繁殖．
通讯作者黄小红（1966-），女，博士，教授．研究方向：动物生物化学．Email：xhhuang@rip．sina．com
