日本沼虾肝胰腺碱性磷酸酶的纯化及特性研究

【作者】 张辉；

【导师】 王安利； 王维娜；

【作者基本信息】 河北大学 ， 水生生物学， 2000， 硕士

【摘要】 从白洋淀日本沼虾肝胰腺中提取的碱性磷酸酶，经正丁醇抽提、硫酸铵分级沉淀，0.15％NaCl溶液透析，得粗酶液。分别用Sephadex G-200葡聚糖层析柱、CM-纤维素层析柱、DEAE-纤维素层析柱纯化粗酶，结果表明DEAE-纤维素柱层析效果最好。 以对硝基苯磷酸二钠（PNPP）为底物，测定提纯后酶的理化性质，结果表明：该酶的最适pH值为8.5，最适温度为45℃，Km值为2.8×10^-3mol/L。Mg²⁺、Mn²⁺、Zn²⁺对酶有明显的激活作用，而Cu²⁺则表现出抑制作用。甲醛、乙醇、甲醇、Vc、草酸、酒石酸对碱性磷酸酶均有抑制作用。 在一定条件下分别采用苯甲基磺酰氟（PMSF）、N-溴代琥珀酰亚胺（NBS）、一氯乙酸、乙酚丙酮、巯基乙醇、甲醛等化学修饰剂选择修饰日本沼虾碱性磷酸酶的多种氨基酸残基，并测定其酶活力变化。结果表明，巯基乙醇、甲醛、一氯乙酸能显著抑制酶的活力，活力的下降与修饰剂的浓度相关；PMSF也能抑制酶的活力，活力的下降与修饰时间相关；乙酰丙酮、NBS的修饰不表现对酶的抑制作用。因此认为Lys、Ser、His残基是日本沼虾肝胰腺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】 Study of alkaline phosphatase from hepatopancreas of Macro brachium nipponensis collected from the lake of BaiYang, HeBei province. The alkaline phosphatase was partially purified from the mantle by homogenation, n-butanol extraction and ammonium sulfate fractionation. Gained alkaline phosphatase was filtrated by exchanging chromatography on DEAE-cellulose column (DEAE-52), CM-cellulose column and gel filtration chromatography on Sephadex G-200. The results were shown that the purification multiple of DEAE-cellulose column was the highest. Some properties of the enzyme were undertaken. The results are as follows: The optimum pH of alkaline phosphatase for PNPP is 8.5, and the optimum temperature is 45 0C. The Michaelis-Menten constant (Kin) is 2.8 X i03 molIL. The alkaline phosphatase is activated by Mg2~, Mn2~ and Zn2~ ion, but is inhibited by Cu2~ ion, formaldehyde, ethanol, methanol, oxalic acid and tartaric acid. The alkaline phosphatase was selectively modified by PMSF, NBS, ClAc, formaldehyde, ME and acetyl acetone, and changes in the activities of the enzyme have been detected. The reaction of alkaline phosphatase with ME, formaldehyde, PMSF and C1Ac resulted in a strong inhibition of the enzyme activities which decreased with the increase of modifier concentration. The acetyl acetone and NBS were found without any inhibition effect. Studies of inactivation of alkaline phosphatase by chemical modification demonstrated that Lys, Ser and His residues are indirpensble function groups of the alkaline phosphatase. Partial disulfide bones are also for the function of this enzyme.更多还原