菲律宾蛤仔碱性磷酸酶的分离纯化及部分性质研究

Purification and Partial Characterization of Alkaline Phosphatase from Ruditapes philippinarum

经Tris-HCl缓冲液浸提、硫酸铵分级沉淀盐析、DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-75凝胶过滤层析等步骤,从菲律宾蛤仔内脏团中分离得到碱性磷酸酶,SDS-PAGE显示为单一条带,提纯倍数为14.42,比活力达到131.08U/mg。酶学性质研究表明:该酶亚基分子量约44kD,最适反应温度为45℃,最适pH值为9.0,米氏常数Km为0.098mmol/L,最大反应速度Vmax为1.01μmol/(mL·min)。Ca2+、Mg2+对酶活力表现出显著的激活作用。

An alkaline phosphatase from the visceral mass of Ruditapes Philippinarum was purified by the following procedures： Tris-HCl buffer extraction, ammonium sulfate precipitation, anion exchange chromatography of DEAE Sepharose Fast Flow, and gel filtration chromatography of Sephadex G-75. The purification factor of the enzyme was 14. 42. The specific activity was 131.08U/mg. The purified enzyme showed a single band by SDS-PAGE, with a relative molecular mass of 44 kD.＇The enzyme exhitited its maximal actitity at pH 9.0 and 45 ℃. The Km and Vma. of the enzyme for PNPP were 0. 098 mmol/L and 1.01 μmol/（mL · min） respectively. Ca2＋and Mg2＋ had stimulating effects on enzyme activity.