【作者】 樊少华；

【导师】 缪晓青；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 以中华蜜蜂的工蜂成体为提酶材料，经正丁醇提取、硫酸铵分级沉淀，0.15％NaCl溶液透析，得粗酶液。用Sephadex G-150葡聚糖层析柱纯化粗酶，获得提纯的碱性磷酸酶，提纯倍数为16.79，比活力达到135.85 U/mg。 以对硝基苯磷酸二钠（PNPP）为底物，测定提纯后的碱性磷酸酶的理化性质。结果表明：该酶的最适温度为45℃，最适pH值为8.6，米氏常数（Km值）为0.97×10^-3mol／L。 HPO₄^2-是中华蜜蜂工蜂碱性磷酸酶催化pNPP水解的产物之一，WO₄^2-、MoO₄^2-是产物类似物，它们对酶均起了较强的抑制作用，并且抑制类型均是竞争性抑制。Na⁺、K⁺和Li⁺等正一价金属离子对酶活力无影响。Mg²⁺、Ca²⁺、Ba²⁺、Ni²⁺、Mn²⁺、Co²⁺、Zn²⁺对酶均为激活作用，Cu²⁺、Hg²⁺、Cd²⁺及Ag⁺为抑制作用，并且Cu²⁺对酶的抑制类型为非竞争抑制类型。甲醛、甲醇、乙醇和乙二醇对碱性磷酸酶均有抑制作用。脲对AKP的变性失活作用，按脲浓度可分为低于3mol／L和高于3mol／L两种类型。 在一定条件下，分别采用二硫代二硝基苯甲酸（DTNB）、N—溴代琥珀酰亚胺（NBS）、苯甲基磺酰氟（PMSF）、溴乙酸（BrAc）、乙酰丙酮、巯基乙醇（ME）等化学修饰剂选择修饰中华蜜蜂碱性磷酸酶多种氨基酸残基，并测定其酶活力变化。结果表明，DTNB、PMSF的修饰不表现对酶的抑制作用，而NBS、BrAc、ME能显著抑制酶的活力，乙酰丙酮也能抑制酶的活力，但是不太明显。因此认为Trp、His是中华蜜蜂工蜂碱性磷酸酶的必需功能基团，部分二硫键对保护酶的催化功能也是必需的。更多还原

【Abstract】 In this paper, I isolated and purified a kind of alkaline phosphatase (AKP) from Apis cerana cerana Fabricius by n-butanol extraction, ammonium sulfate fractionation and sodium chloride dialyzation. The purified enzyme was gained by means of gel filtration on Sephadex G-150 column. The purification attained to 16. 79 folds and the specific activity was 135. 85 U/mg.Some properties of the enzyme were undertaken. The optimum temperature for the enzyme to catalyze the hydrolysis of p-nitrophenyl phosphate(pNPP) is at 45℃. If the pH value of the assay system was equal to 8. 6, the enzyme would become the most active. The Michaelis-Menten constant(Km) of the hydrolysis pNPP catalyzed by the enzyme is 0. 97 mmol/L.The product HPO42- and the product-analog WO42- Mo042- could competitively inhibit the activity of the enzyme. The survey of effects of metal ions on the AKP activity shows that Na+ K+ and Li+ have not any effect on the enzyme activity, Mg2+ Ca2+ Ba2+ Ni2+ Mn2+ Co2+ and Zn2+ activate the enzyme, Cu2+ Hg2+ Cd2+ and Ag inhibit the enzyme. On the other hand, the AKP is also inhibited by formaldehyde carbinol ethanol and glycol. The AKP is inactivated by urea, and the inactivation can be divided into two kinds according to the concentration of the urea: lower than 3mol/L and higher than 3mol/L.The alkaline phosphatase was selectively modified by DTNB NBS PMSF BrAc acetyl acetone and ME, and changes in the activities ofthe enzyme have been detected. The reaction of alkaline phosphatase with NBS BrAc and ME resulted in a strong inhibition of the enzyme activities which decreased with the increase of modifier concentration. The DTNB PMSF were found without any inhibition effect. The acetyl acetone was found to have little inhibition effect on AKP activity. All of these suggested that Trp and His residues should considered as indispensable functional groups of AKP , and partial disulfide bonds as essential for the function of the enzyme.更多还原