【作者】 石艳；

【导师】 陈清西；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 比较菜青虫不同部位及不同生长期的菜青虫N-乙酰-β-D-氨基葡萄糖苷酶（NAGase,EC3.2.1.52）的比活力,发现五龄幼虫表皮来源的NAGase比活力最高。通过解剖获得五龄菜青虫表皮,用0.05 mol/L PBS含0.1 mol/L NaCl缓冲液（pH=6.8）抽提、硫酸铵分级分离获得粗酶,并进一步通过Sephadex G-200凝胶过滤柱层析和羟基磷灰石柱层析（CHT II柱）纯化获得比活力为8715 U/mg的聚丙烯酰胺凝胶电泳单一纯NAGase酶制剂。采用凝胶过滤柱层析法测定该酶的分子量约为106 kD。通过SDS- PAGE方法测定NAGase含两个异型亚基,分子量分别为59.5和57.2 kD。该酶不含寡糖。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖苷（pNP-NAG）为底物,测得该酶在0.1 mol/L PBS缓冲液中催化水解底物pNP-NAG的最适pH为6.2,最适温度为42℃;在37℃、pH 6.2 PBS中,该酶催化pNP-NAG水解的米氏常数Km为0.326 mmol/L,反应的活化能为83.86 KJ/mol。该酶在45℃以下,pH 4-9范围内稳定。以动力学方法测定酶活性功能基团的解离性质,39℃下测得菜青虫NAGase酶活性中心可解离基团解离常数pK_e值为5.20,标准解离热焓ΔH^o为2.18 kcal/mol,表明该酶的活性中心的可解离基团是谷氨酸或天冬氨酸的侧链羧基。化学修饰法研究进一步证明侧链羧基是酶的必需基团,并测定出酶活性必需的羧基数为一个。修饰剂对该酶的化学修饰研究还表明半胱氨酸巯基、色氨酸的吲哚基、组氨酸的咪唑基、二硫键也是酶活性的必需基团。而精氨酸残基及赖氨酸ε-氨基被修饰后对酶活力没有影响,不是酶的必需基团。通过对产物NAG与产物pNP的类似物苯酚对酶的抑制作用研究表明酶催化底物pNP-NAG水解反应属有序双双机理。通过对变性剂盐酸胍和脲的作用下菜青虫NAGase活力和构象变化的比较表明酶的活力变化快于构象变化。说明该酶分子的活性中心处于对变性剂较敏感的区域,即活性部位具有柔曲性。比较酶经热失活后活力与构象变化间的关系,也得到酶活力丧失明显快于其构象的结果。通过失活动力学方法测定热失活速度常数得到自由酶的热失活速度常数（k+0）明显大于酶-底物络合物的失活速度常数(k₊₀’),说明底物与酶的结合对酶的热失活作用有一定的保护作用。更多还原

【Abstract】 A N-Acetyl-β-D-glucosaminidase (NAGase) was purified from the integument of cabbage butterfly (Pieris rapae) by extraction with 50 mmol/L of sodium phosphate buffer (PBS, pH 6.8) containing 0.1 M NaCl and ammonium sulfate fractionation, then chromatography on Sephadex G-200 and Econo-Pac CHT-Пcolumn. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was measured to be 106.0 kD by gel filtration on Sephacry-S 200. The enzyme was determined to be a heterodimer with molecular mass of 59.5 and 57.2 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 oC, respectively and the Michaelis-Menten constant (Km) was determined to be 0.326 mmol/L at pH 6.2 and 37 oC. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 oC. The activation energy was 83.86 kJ/mol.The ionization constant, pKe, of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 oC, and the standard dissociation enthalpy (?Ho) was determined to be 2.18 Kcal/mol. These results showed that the ionizing group of the enzyme active center is the carboxyl group. The enzyme was modified respectively by several chemical modification reagents, such as carbidiimide (EDC), pCMB (p-chloromercuribenzoate), N-Bromosuccinimide (NBS), bromoacetic acid (BrAc), DTT (dithiothreitol), acetic anhydride and acetyl acetone at certain condition, and the residue activity was assayed in normal reaction media. The results showed that the enzyme activity was decreased when the carboxyl group, sulfhydryl group, indolyl group, imidazolyl group and disulfide bond were modified, respectively, which means that these groups were essential for the enzyme catalytic activity. The results also showed that the residues of lysine, arginine were irrespective with the enzyme activity. The reaction mechanism of this enzyme hydrolysis of pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products.Inactivation and unfolding of the NAGase during denaturation in guanidine hydrochloride and urea at different concentrations had been compared, the results更多还原