蛹虫草菌丝体超氧化物歧化酶的制备

Preparation of Superoxide Dismutase from Cordyceps militaris Mycelium

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 10g湿菌丝体，最佳超声破碎功率400W，超声时间15min。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13592．1U·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266nm。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。

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 rain for 10 g wet weight myeelium, respectively.The purification was performed through three steps： （NH4）2 SO4 precipitation （55 % - 99% saturation）, DEAE-sepharoseTM 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.