染料亲和层析分离纯化酵母两种脱氢酶

目的探讨三嗪活性染料Cibacron Blue F3GA和KE-3B艳红制备的亲和层析柱用于建立分离纯化面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的方法。方法在琼脂糖凝胶Sepharose 6B上,分别偶联三嗪活性染料Cibacron Blue F3GA和KE-3B艳红,制成染料亲和层析柱。将面包酵母经超声破碎、高速离心、硫酸铵分级沉淀、透析、冰冻干燥等步骤得到脱氢酶粗品,再经染料亲和层析柱进一步纯化。结果经过固定有Cibacron Blue F3GA的亲和层析柱,乙醇脱氢酶的纯化倍数为2.0,酶活性回收率为30.8%;经过固定有KE-3B艳红的亲和层析柱,葡萄糖-6-磷酸脱氢酶的纯化倍数为23.2,酶活性回收率为66.0%。所得的乙醇脱氢酶在SDS-聚丙烯酰胺凝胶电泳为1条带。结论染料配基亲和层析可作为面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的精制纯化方法。

PURIFICATION OF TWO KINDS OF DEHYDROGENASE FROM BAKER'S YEAST BY DYE AFFINITY CHROMATOGRAPHY

Objective To evaluate the purification of ethanol dehydrogenase(EDH) and glucose-6-phosphate dehydrogenase(G-6-PDH) from baker's yeast by dye affinity chromatography column made by Cibacron Blue F3GA and Red KE-3B.Methods Two kinds of triazine dye,Cibacron Blue F3GA and Red KE-3B,were immobilized on Sepharose 6B respectively to make dye affinity chromatography column.Baker's yeast were treated with ultrasonic crash,high speed centrifugation,ammonium sulfate precipitation,dialysis and freeze-drying to gain the raw dehydrogenases,which then were purified by dye affinity chromatography.Results Through dye affinity chromatography column immobilized with Cibacron Blue F3GA,EDH purification factor was 2.0,activity recovery was 30.8%;and through dye affinity chromatography column immobilized with Red KE-3B,G-6-PDH purification factor was 23.2,activity recovery was 66.0%.EDH from Blue-Sepharose 6B showed one strip on SDS-PAGE electrophoresis.Conclusion Dye affinity chromatography can be used as a refine purification method for purification of EDH and G-6-PDH from baker's yeast.