摘要
经Tris-HCl缓冲液(pH 8.6)抽提,正丁醇脱脂、硫酸铵分级沉淀分离,DEAE-琼脂糖离子交换柱和Sephacryl S-200分子筛纯化,从黄鳝肝脏组织中获得单一电泳纯碱性磷酸酶制剂。该酶提纯倍数为123.12倍,比活力2957.34 U/mg。酶学性质和动力学性质研究表明,该酶在260 nm处有一特征吸收峰,等电点为5.21;该酶催化磷酸苯二钠水解反应,初速度为6.02μmol/(L.min),米氏常数为1.17 mmol/L,最大反应速度为13.75μmol/(L.min),反应活化能为52.6 kJ.mol;最适pH为10.4,pH>11不稳定;最适温度为40℃,温度高于50℃不稳定。
A hepatic alkaline phosphatase(ALP) in ricefield eel(Monopterus aIbus) was prepared and purified by means of the following techniques: n-butanol extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200. Then the ALP obtained by Sephacryl S-200 gel chromatography was dialysed and de- hydrated,then identified the purification. The pure hepatic ALP had one protein strip by PAGE, IEF- PAGE, indicating that pure ALP was homogeneous. Finally 29.51% of the ALP activity was recovered, and the purified 123.12-fold. the pure ALP had the specific activity of 2957.34 U/mg protein, isoelectrie point of 5.21 by IEF-PAGE. The optimum pH and optimum temperature for the enzyme to catalyze the hydrolysis of phenylphosphoric acid disodium salt were pH 10. 4 and 40 ℃. The Michaelis constant was 1.17 mmol/L and the maximum velocity 13. 75μmol/(L·min) with the initial velocity of 6. 02μmol/(L· min). The activation energy of the enzyme was 52. 6 kJ · mol.
出处
《水产科学》
CAS
北大核心
2009年第6期312-316,共5页
Fisheries Science
基金
重庆市科委科技基金资助项目(2003-7852)
关键词
黄鳝
肝脏
碱性磷酸酶
分离纯化
alkaline phosphatase
Monospters albus
isolation and purification
dynamics