蛹虫草菌丝体超氧化物歧化酶的制备

 目的从蛹虫草(Cordyceps militaris)菌丝体中制备Cu,Zn-超氧化物歧化酶。方法超声破碎、硫酸铵沉淀、离子交换。结果10 g湿菌丝体,最佳超声破碎功率400 W,超声时间15 min。粗酶液经55%～95%硫酸铵沉淀,DEAE-FF阴离子交换柱,CM-52阳离子交换柱纯化,酶蛋白收率22.4%,比活达到13 592.1 U·mg(蛋白)^-1,纯化倍数214.1倍。SDS-PAGE电泳分析呈现均一条带。紫外最大吸收峰为266 nm。该酶对KCN和H₂O₂敏感。氨基酸组成和其他来源Cu,Zn-SOD相似。结论本制备工艺切实可行,能获得理想的电泳纯蛋白。

Preparation of Superoxide Dismutase from Cordyceps militaris Mycelium

OBJECTIVE To prepare Cu,Zn containing superoxide dismutase from Cordyceps militaris Mycelia.METHODS The homogenization of mycelia was prepared by sonicator,(NH₄)₂SO₄ precipitation,ion-exchange chromatography.RESULTS The optimized sonic power and time were 400 W and 14 min for 10 g wet weight mycelium,respectively.The purification was performed through three steps:(NH)₂SO₄ precipitation(55%～99% saturation),DEAE-sepharose^TM fast flow anion-exchange chromatography,CM-52 cation-exchange chromatography.After this procedure,a specific activity of 13 759.1 U·mg(pro)^-1 was reached,corresponding to 214.1 fold purification with a yield of 22.4%.The purity was homogeneous by SDS-PAGE analysis.The purified enzyme was sensitive to H₂O₂ and KCN.The maximum absorption was observed at 266 nm.The amino acid compositions were similar to that of other sources Cu and Zn-SOD.CONCLUSION The method established in this study is effective and feasible,the homogeneous protein is obtained by SDS-PAGE analysis.