摘要
经Tris-HCl缓冲液(pH8.6)抽提,正丁醇处理,30%—75%硫酸铵分级沉淀分离,DEAE-Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从黄鳝内脏组织中分离纯化出电泳纯的碱性磷酸酶。该酶提纯倍数为564倍,比活力达到3015U/mg。酶学性质和动力学性质研究表明,该酶催化磷酸苯二钠的水解反应,最适pH值为10.2,pH小于7和大于12均不稳定;最适温度为40℃,温度高于50℃不稳定;米氏常数Km值为1.17mmo1/L。金属离子对该酶的催化活力有不同的影响,K+对该酶活力无影响,Mg2+对该酶有激活作用,Zn2+对该酶有抑制作用。
Alkaline phosphatase(AKP)is one kind of enzyme, which is not absolutely specific.It could hydrolise lots of phosphoric acid ester chemical compound in alkaline environment while the activator Mg 2+ and Mn 2+ are needed.It's a high valuable tool enzyme.It's mainly used in studying nucleric acid,analysising nucleoticle sequences and the isolation, recombination of genes.The merchant reagent of AKP is very expensive.Comparing with activity of AKP in many species,we found that there is high activity in the viscera of Monopterus. albus.So this paper mainly deals with isolating,purifying AKP from viscera of Monopterus albus,studying its characters and preparing for its application.It was prepared and purified by means of the following techniques:n-butanol extraction,amonanium sulfate precipitation,ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200.The preparation was shown to be homogenous on polyacrylamide gel electrophoresis.The specific activity was 3015 unit/mg protein.Its molecular weight was determined to be about 133.4 kD on gel filtration chromatography and submit is 66.8 kD on SDS-PAGE,so we speculate this enzyme has two submits.The kinetic characters of the enzyme have been studied.The optimum pH and optimum temperature for the enzyme to catalyze the hydrolysis of phenylphosphoric acid disodium salt are pH10.2 and 40℃,and this enzyme activity is not stable at high temperature and low pH,the Michaelis constant(Km)is 1.17mmol/L.The positive monovalent alkali ions(K +,Na +)have no effects on the activity of the enzyme;Magnesium ion activates the enzyme activity and Zinc ion inhibit the enzyme activity.
出处
《水生生物学报》
CAS
CSCD
北大核心
2005年第3期323-328,共6页
Acta Hydrobiologica Sinica
基金
西南师范大学博士基金和重庆市科委(2003-7852)资助
关键词
黄鳝
碱性磷酸酶
分离
纯化
酶学性质
Alkaline phosphatase
Isolation
Purification
Properties
Monopterus albus
