锯缘青蟹碱性磷酸酶分离纯化及部分理化性质研究  

STUDIES ON ISOLATION, PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF ALKALINE PHOSPHATASE FROM GREENCRAB (SCYLLA SERRATA)

作　　者：陈清西[1] 张喆[1] 庄总来[1] 陈祥仁 

机构地区：[1]厦门大学生物系,厦门361005

出　　处：《海洋与湖沼》1998年第4期362-367,共6页Oceanologia Et Limnologia Sinica

基　　金：国家自然科学基金!39470561

摘　　要：于1995年2月在厦门海域采集锯缘青蟹，取其内脏经正丁醇抽提、硫酸铵分级分离、DEAE-52离子交换柱层析及SephadexG－200凝胶过滤柱层析纯化，获得碱性磷酸酶制剂，以快速蛋白液相色谱及聚丙烯酰胺凝胶电泳检验其纯度，获得单一蛋白纯的酶制剂。研究酶的物理性质得出该酶的紫外特征吸收峰在278nm处，荧光激发光谱特征峰在282nm处，荧光发射光谱特征峰在343nm处，全酶分子量为78kD。研究酶催化对－硝基苯磷酸二钠水解的动力学性质，金属离子及有机溶剂对其活力的影响，得知该酶水解对－硝基苯磷酸二的最适温度为52℃，最适pH为9．2，米氏常数为6．67×10-4mol／L，酶活性中心的转换常数为460min-1；Mg2＋对酶有显著的激活作用；Cn2＋，Hg2＋和甲醇、乙醇、乙二醇对酶有不同程度的抑制，说明在一定条件下，效应物引起酶分子构象发生了不同程度的变化。An alkaline phosphatase from green crab (Scylla serrata) was collected from Xiamen sea water in February, 1995. It was prepared and purified by means of the following techniques:n-butanol extraction, ammanium sulfate precipitation, ion exchange chromatography on DEAE cellulose column (DEAE-52) and gel filtration chromatography on Sephadex G-200. The preparation was shown to be homogeous on FPLC and polyacrylamide gel electroporesis. The characteristic peak of UV-absorption spectrum of the enzyme was found to be at 278 nm, the fluorescence excitation spectrum at 282 nm, and the fluorescence emission spectrum at 343 nm. The molecular weight of the enzyme is 78 kD At pH=9.5, the optimum pH is 9.2, at 37℃. At pH= 9.0, 37℃, the Michaelis constant (KM) is 6.67 × 10-4mol/L; Kcat ,460min-1. Magnesium ion activates the enzyme significantly. Copper ion, mercury ion,methanol, ethanol and ethylene glycol ichibit the enzyme echvity in vareing degrees, which indicates tha the conformation of the enzyme caused by the effectors changes at different levels. The inhibition mechanism had been preliminarily studied.

关 键 词：锯缘青蟹 碱性磷酸酶 分离 纯化 理化性质 

分 类 号：TQ464.8[化学工程—制药化工]