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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 isolate and purify a protease having the property of poly-arabinonase produced by the fermentation of bacteria of the genus Cellulomonose sp. Methods: The product of enzymatic reaction - oligosaccharide was separated by TLC. Quanti-Scan software was used to calculate the area gray value of the strip, and then the enzyme activity was calculated. The protein content was determined by the Bradford method. The crude enzyme was separated and purified by DEAE-Sepharose ion exchange chromatography, Sephacryl S-300 molecular sieve and Blue-Dye reactive dye affinity chromatography. The purity of the crude enzyme was determined by SDS-PAGE. SDS-PAGE and gel chromatography Determination of relative molecular mass. Results: The electrophoretically pure arabinose endonuclease was isolated and its relative molecular mass was 45kD. The protease specific activity was 15.42 × 10 (3U / ug) and the purification fold was 77.1. CONCLUSION: This enzyme is an arabinose endonuclease that can be used as a novel tool enzyme in the structural analysis of Mycobacterium tuberculosis cell wall and the target of searching anti-TB drugs.