赤子爱胜蚓碱性磷酸酶的酶学性质研究

Enzymatic Properties of Alkaline Phosphatase from Eisenia Foetide Sarigng

以赤子爱胜蚓(Eiseniafoetidasarigng)整体为材料,采用正丁醇抽提、硫酸铵分级沉淀、DEAE 纤维素柱层析、葡聚糖凝胶过滤等方法分离纯化得到蚓体中的一种碱性磷酸酶(AKP)并对其性质进行研究.经测定其分子量为126000Da,富含谷氨酸、脯氨酸.以磷酸苯二钠为底物,测其最适温度为37℃,最适pH为10.10,Km值为1.15mmol/L,Mg2+、Ca2+、Mn2+对酶有激活作用,KH2PO4、ME、EDTA、L Cys、DFP则对AKP有抑制作用.

Alkaline Phosphatase (AKP)is purified from Eisenia foetide sarigng using n-butanol extraction, ammonium sulfate fractional precipitation, DEAE-cellulose chromatography and Sephadex gel filtration. The purified AKP is found to be homogeneous as judged by polyacrylamide gel electrophoresis. The AKP′s molecular weight is about 126000Da and it contains two equally sized subunits, its amino acid component analysis shows that the relative proportion of glutamic acid and proline is the largest.The Michaelis-Menten contant Km of the AKP for the hydrolysis of disodium phenyl phosphate is 1.15mmol/L. The optimum temperature is 37℃ and the optimum pH is 10.10. The AKP is activated by Mg^(2+), Ca^(2+), Mn^(2+) and inhibited by KH_(2)PO_(4), ME, EDTA, L-Cys and DFP.