源于纤维单胞菌的聚阿拉伯糖内切酶的分离纯化

Purification of Endo-arabinase Secreted from Cellulomonose Bacterial Strain

目的:分离纯化一种由纤维单胞菌属(Cellulomonose sp.)的细菌发酵产生的具有聚阿拉伯糖内切酶的性质的蛋白酶。方法:用薄板层析法(TLC)分离酶促反应的产物-寡糖,以Quanti-Scan软件计算薄板上条带的面积灰度值,以此定量,计算酶活力。用Bradford法测定蛋白含量。通过DEAE-Sepharose离子交换层析,SephacrylS-300分子筛和Blue-Dye活性染料亲和层析三种手段串联,对粗酶进行分离纯化,用SDS-PAGE测定纯度,SDS-PAGE和凝胶层析测定相对分子质量。结果:分离得到了电泳纯的阿拉伯糖内切酶,该酶的相对分子质量为45kD,蛋白酶比活性为15.42×10(3U/ug),纯化倍数为77.1。结论:该酶是一种阿拉伯糖内切酶,可以作为一种新型的工具酶,应用于结核分枝杆菌细胞壁的结构分析及寻找抗结核药物作用的靶点。

Objective： To purify the endo-arabinase with endo-arabinase,secreted from Cellulomonose strain. Methods： The product was separated from substrate based on the thin layer chromatography （TLC）, quantifying the scanned colorful product bands by Quanti-Scan software to calculate the enzyme activity. The protein concentration was determined by Bradford method. The endo-arabinase was purified from the crude enzyme by different chromatography methods：ion-change chromatography （DEAE-Sepharose）, Sephacryl S-300 chromatography and Blue-Dye affinity chromatography. The molecular mass was determined by SDS- PAGE and gel filtration. Results： The endo-arabinase was separated. The relative molecular mass, specific activity and purification ofendo-arabinase were 45 kD, 15.42×10 （U/ug）and 77.1. Conclusion： The enzyme is endoarabinase used for a new toolenzyme to research the structure of Mycobacterial cell wall and look for the exploiture of new anti-TB drugs.