中华根瘤菌L03几丁质酶纯化及其酶学性质研究

Purification and Characterization of Chitinase Produced by Sinorhizobium sp. Strain L03

中华根瘤菌Sinorhizobium sp.菌株L03是1株能产生几丁质酶的生防细菌。采用90%饱和度硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等方法获得了菌株L03的几丁质酶。SDS-PAGE检测发现该酶已被纯化,其分子量约为41.3kD。该酶反应的最适温度为45℃;最适反应的溶液pH值为6;酶液在pH5~8条件下和40℃下分别保存1h,酶活力基本保持稳定;Mg2＋和Ba2＋能显著促进几丁质酶活性上升,而Zn2＋、Cu2＋和Fe3＋等对酶活力有明显的抑制作用。功能分析结果显示,该几丁质酶对供试的几种病原真菌细胞壁都有明显的降解作用。

Sinorhizobium sp.strain L03 was an antagonistic bacterium which could secrete chitinase when it was cultured in the medium amended with colloid chitin.The chitinase in culture filtrates was purified by salting out with 90% saturation of ammonium sulfate,DEAE-Sepharose Fast flow chromatography and Pheny1-Sepharose Fast Flow chromatography.SDS-PAGE analysis demonstrated that the molecular weight of the purified enzyme was about 41.3 kD.The optimal temperature for the hydrolyzation of chitin was 45 ℃ and the optimal pH value was 6.The chitinase activity was stable when it was heated at 40 ℃ for 1 h and kept in the solution with pH5-8 for 1 h.The enzymatic activity was enhanced evidently by Mg2＋ and Ba2＋,and inhibited by Zn2＋,Cu2＋ and Fe3＋.The inhibition bioassays showed that the chitinase could degrade cell walls of Fusarium moniliforme,Magnaporthe oryaze and F.oxysporum f.sp.vasinfectum etc.