摘要
以蝾螺内脏为材料,通过硫酸铵沉淀分级分离、SephadexG 200分子筛柱层析和两次DEAE SephadexA 50离子交换柱层析纯化,获得聚丙烯酰胺凝胶电泳单一纯的N 乙酰 β D 氨基葡萄糖苷酶酶制剂.纯酶的比活力为1448U·mg-1.酶的紫外特征吸收峰在275nm处,内源荧光发射峰在340nm处.以对 硝基苯 N 乙酰 β D 氨基葡萄糖为底物,研究酶催化底物水解的反应动力学,结果表明:酶的最适pH为4.5,最适温度为45℃.该酶在pH3.5～6.0区域较稳定,而在pH>7能很快失活;在40℃以下处理30min,酶活力保持稳定,高于40℃,酶稳定性较差,很快失活.酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为3.13mmol·L-1,最大反应速度Vm为17.68μmol·L-1·min-1.
The β-N-Acetyl-D-glucosaminidase (NAGase,EC 3.2.1.30) can catalyzes the cleavage of N-acetylglucosamine polymers.It is one of the chitinases and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine.The NAGase was purified from the viscera of Turbo cornutus Solander by ammonium sulfate fractionation and gel filtration column chromatography on Sephadex G-200 and twice ion exchange column chromatography on DEAE-Sephadex A-50.The purified enzyme preparation was homogeneous as judged by FPLC and polyacrylamide gel electrophoresis.The specific activity of the enzyme was 1 448 U·mg^(-1).The characteristic peak of UV-absorption spectrum of the enzyme was found at 275 nm and that of the intrinsic fluorescence emission was at 340 nm.The optimum pH was determined to be at pH 4.5 and the optimum temperature was at 45℃.The enzyme was stable in the pH ranges from 3.5 to 6.0,the enzyme activity rapidly decreased while the pH value was lower than 3.5 or higher than 6.0.The enzyme has good thermal stability under 40℃.The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc in 0.1 mol·L^(-1)Citric-Na_2HPO_4 buffer at 37℃.The K_m and V_m values were determined to be 3.13 mmol·L^(-1) and 17.68 μmol·L^(-1)·min^(-1),respectively by plot of Lineweaver-Burk.
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
2004年第5期697-701,共5页
Journal of Xiamen University：Natural Science
基金
厦门大学细胞生物学与肿瘤细胞工程教育部重点实验室开放课题
