蚯蚓GSH-Px提取与纯化的研究

Extraction and Purification of GSH-Px from Earthworm

以蚯蚓为原材料提取纯化了GSH-Px,通过比较超声波提取法、组织匀浆法和闪式提取法,得出闪式提取法提取效果最佳。通过正交实验优选得到蚯蚓GSH-Px的最佳闪式提取条件为:料液比1:6(m/V)、提取液pH值8.0、提取时间1 min。在最佳条件下得到的粗提液经硫酸铵沉淀、选择性酸碱变性、DEAE-cellulose和Sephadex-G100柱层析,最终得到纯化的GSH-Px,比活达到157.08 U/mg,活性回收率达到12.32%。产物在SDS-PAGE上为单一条带,其亚基分子量约为26 kD。

In this study, glutathione pemxldase （GSH-Px） was extracted and purified from the earthworm. The extraction technologies were investigated. Compared with ultrasonic extraction and tissue homogenate extraction, flash extraction method showed higher extract efficiency. The flash extraction conditions were developed by an orthogonal test design. The optimal flash extraction conditions were determined as follows：1：6 （m/V） of the solid/solvent ratio, pH 8.0, and 1 rain of extraction time. The crude extract obtained from the above conditions was precipitated by solid ammonium sulfate, ad- justed the pH to 5.0 and fractionated by cohmm chromatography （ DEAE-Cellulose and Sephadex G-100）. The specific activity and yield of the purified GSH-Px were 157.8 U/rag and 12.32% ,respectively. The SDS-PAGE showed a single band approximate 26 kD.