对Pleurotus eryngii Co60-7菌株发酵液中木质素降解酶的分离纯化过程进行了研究,设计了一条主要由加盐沉淀和离子交换组成的分离纯化路线。通过35%硫酸铵饱和度的初沉和75%硫酸铵饱和度二沉可最大程度去除发酵液中的杂蛋白并获得目标蛋白,蛋白回收率为90.8%,纯化因子为3.81。用DEAE-SepharoseTMFast Flow进行三步洗脱层析,可较好地实现发酵液中目标蛋白的捕获。经过该纯化路线,木质素降解酶蛋白回收率达75.42%,纯化因子为10.55。经SDS-PAGE和Native-PAGE电泳分析,洗脱液DEⅡ中含有3种木质素降解酶组分。用该酶液进行溴酚蓝、亚甲基蓝和刚果红降解实验,结果表明该木质素降解酶液对3种染料都有不同程度的降解,染料间结构的差异会影响其降解效率。

The ligninolytic enzymes were separated and purified from the culture broth of Pleurotus eryngii Co60-7 strain.An efficient separation process was proposed,which was mainly composed of the precipitation of ammonium sulfate and ion exchange chromatography.In the precipitation of ammonium sulphate,two-stage precipitations were applied.In the first-stage precipitation,35% saturation of ammonium sulfate was used to remove the hybridproteins and in second-stage of precipitation,75% saturation of ammonium sulfate was used to capture the ligninolytic enzymes in activity yield of 90.8.% and purification factor 3.81.The ligninlolytic enzymes were purified by DEAE-SepharoseTM Fast Flow ion-exchange chromatography under three-step elution.The total purification process attained 75.42% activity yield with a purification factor of 10.55.Ligninolytic enzymes produced in this work were used to decolorize three dyes,i.e.bromchlorphenol blue,methylene blue and Congo red,which could be classified as triphenylmethane,heterocyclic and disazo dyes.The results showed that the structures of dyes can significantly affect decolorization.