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目的研究玉米细菌性枯萎病菌EGase内切葡聚糖酶蛋白纯化的方法。方法以玉米细菌性枯萎病菌(Pantoea stewartii subsp.stewartii)菌株为材料,分离纯化其内切葡聚糖酶Egase。制备EGase浓缩液,浓缩液经Sephradex~(TM)G-75凝胶过滤层析和DEAE-Sephorose Fast Flow阴离子交换柱层析等提纯步骤,获得了凝胶电泳均一的内切葡聚糖酶。结果经变性聚丙烯酰胺凝胶电泳检测为一条电泳带,纯化后的EGase是单体蛋白,分子量约为72.3 k Da。Egase酶反应的最适温度是60℃,最适pH为5.0。结论本研究从玉米细菌性枯萎病菌中分离得到了一种新的内切葡聚合糖酶,对其部分性质进行了表述,为后续EGase基因的克隆及表达研究提供了研究基础。
Objective To study the purification of EGase endoglucanase from corn bacterial wilt pathogen. Methods The endoglucanase Egase was isolated and purified from a strain of Pantoea stewartii subsp. Stewartii. The EGase concentrate was prepared. The concentrate was purified by Sephradex TM G-75 gel filtration chromatography and DEAE-Sephorose Fast Flow anion exchange column chromatography to obtain homogeneous endoglucanase by gel electrophoresis. Results The denatured polyacrylamide gel electrophoresis was used as an electrophoresis band. The purified EGase was a monomeric protein with a molecular weight of about 72.3 kDa. The optimum temperature for Egase enzyme reaction was 60 ° C and the optimum pH was 5.0. Conclusion In this study, a new endoglucanase was isolated from bacterial wilt pathogen of corn and some of its properties were described. It provided the basis for the cloning and expression of subsequent EGase genes.