鲍鱼碱性磷酸酶的分离纯化和性质研究  

Purification and Characterization of Alkaline Phosphatase from Haliotis diversicolor

作　　者：廖金花[1] 陈巧[1] 林丽蓉[1] 洪燕飞[1] 陈清西[1] 

机构地区：[1]厦门大学生命科学学院

出　　处：《厦门大学学报（自然科学版）》2005年第2期272-275,共4页Journal of Xiamen University：Natural Science

基　　金：教育部留学回国人员科研启动基金资助项目;教育部细胞生物学与肿瘤细胞工程重点实验室开发课题

摘　　要：以杂色鲍(Haliotis diversicolor)内脏为原料,经Tris HCl缓冲液(pH 7.5,含0.1mol/L NaCl)抽提,正丁醇处理,硫酸铵分级沉淀分离,DE 32离子交换、Sephadex G 150分子筛、DE 32纯化,得到电泳单一纯的碱性磷酸酶制剂,比活力为1 226 U/mg.酶学性质和动力学性质研究表明:该酶催化对硝基苯磷酸水解反应的最适 pH值为 10.08,最适温度为48℃.米氏常数Km 值为0.80 mmol/L,Vm 值为20.1 mmol/L·min-1(pH 10.0,37℃).酶的热稳定性研究表明:该酶在pH 7~11区间和在温度低于55℃下稳定.金属离子对酶活力的影响结果表明:Li+、Na+和K+对酶活力没有影响,Mg2+、Ca2+、Ba2+、Mn2+和Co2+对酶有不同程度的激活作用,而Cu2+、Cd2+、Zn2+、Hg2+、Ag+、Bi2+和Pb2+等对酶起抑制作用.An alkaline phosphatase was purified from Haliotis diversicolor by following procedures:Tris-HCl buffer (pH 7.5) extraction,n-butanol disposal,ammonium sulfate precipitation,ion-exchange chromatography on DE-32 cellulose,then by gel filtration through SephadexG-150 following again ion-exchange chromatography on DE-32.The final preparation was homogeneous on PAGE and SDS-PAGE.The specific activity of the enzyme was 1 226 U/mg.The optimum pH is 10.08,and the optimum temperature is 48℃ for the hydrolysis of pNPP.Mchaelis-Menten contant (K_m) is 0.8 mmol/L and the maximun velocity (V_m) is 20.1 mmol/L·min^(-1) at pH 10.0 and 37℃.The enzyme is stable in the range of pH from 7 to 11 and in temperature below 55℃. A survey of effects of metal ions on the enzyme showed that the positive monovalent metal ions (Li^+,Na^+ and K^+) have no effect on the enzyme;positive bivalent metal ions (Mg^(2+),Ca^(2+),Ba^(2+),Mn^(2+) and Co^(2+)) activate the enzyme,Cu^(2+)、Cd^(2+)、Zn^(2+)、Hg^(2+)、Ag^+、Bi^(2+) and Pb^(2+) inhibit the enzyme.The inhibition effect of Hg^(2+) is strongest.When the concentration of Hg^(2+)come to 0.2 mmol/L,the ALP is complete inactivation.The result indicates that different metal ions have different effect on the ALP.The study of ALP from Haliotis diversicolor may give some help to Haliotis diversicolor’s culture.

关 键 词：杂色鲍 碱性磷酸酶 分离纯化 酶活性 稳定性 金属离子 鲍鱼 生物体 

分 类 号：Q959.212[生物学—动物学] S944.45[农业科学—水产养殖]