摘要
河田鸡和艾维茵肉鸡的睾丸,经硫酸铵分级沉淀、DEAE-32离子交换柱层析,获得比活力为54.84 U.mg-1,纯化倍数为8.86的河田鸡睾丸N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)制剂.艾维茵肉鸡睾丸NAGase经Sephacry S-300凝胶过滤层析柱进一步纯化,获得比活力为171.50 U.mg-1,纯化倍数为19.8倍的NAGase制剂.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:河田鸡和艾维茵肉鸡睾丸NAGase的最适pH分别为5.6、5.7,最适温度分别为55、50℃;NAGase分别在pH为3.0-9.2、4.0-9.2及温度为10-60、10-30℃下能保持稳定;NAGase酶促反应动力学均符合米氏双曲线方程,测得米氏常数(Km)分别为0.223、0.319 mmol.L-1,最大反应速度(Vm)分别为9.438、6.238μmol.L-1.m in-1;NAGase催化pNP-NAG反应的活化能分别为85.74、73.47 kJ.mol-1.
N-acetyl-β-D-glucosaminidase （NAGase） was purified partly from Hetian broiler and Avian broiler by ammonium sulfate fractionation, chromatography on DEAE-32 and Avian broilers＇ NAGase was subsequently purified on Sephacry S-300. The specific activities of the enzymes were determined to be 54.84 and 171.50 U · mg-1 respectively. The optimum pH were found to be at 5.6 and 5.7, respectively. The optimum temperatures were 55 and 50℃. The enzymes from different sources were stable in the pH below 9.2. The enzyme purified from Hetian broiler was stable at temperature below 60 ℃, but the enzyme purified from Avian broiler was stable only at temperature below 30 ℃. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Miehaelis Menten kinetics with Km of 0.223 and 0.319 mmol · L-1, respectively. The maximum velocities （V,） were 9.438 and 6.238 μmol · L-1 · min -1 , respectively. The ativation energies were determined to be 85.74 and 73.47 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3期265-270,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)
