棉花黄萎病拮抗细菌DS45-2菌株抗菌蛋白的分离纯化

枯草芽孢杆菌(Bacillus subtilis) DS45-2菌株产生的抗菌蛋白对棉花黄萎病有较强的抗性。用NB培养基摇床振荡培养DS45-2菌株(30℃，180r/min，48h)，发酵液经硫酸铵盐析得到抗菌蛋白。经分析，抗菌蛋白对热稳定；对胃蛋白酶、胰蛋白酶均不敏感，对蛋白酶K部分敏感；在碱性条件下稳定，酸性条件下抑菌活性减弱。抗菌蛋白经DEAE Sepharose Fast Flow阴离子交换层析和反相层析后，分离纯化出一个抗菌蛋白0组分，经SDS-PAGE检测分子.质量约为23 kDa。

Purification of Antifungal Protein from Antagonistic Bacteria DS45-2 Strain against Verticillium dahliae Kleb

The antagonistic strain (Bacillus subtilis) DS45-2 were cultured in NB medium for 48h at 30℃. The supernatant was collected by centrifugation at 8000r/min for 15 min at 4℃. The crude extracts of antifungal protein was obtained from the cell free supernatant of fermentation liqour of DS45-2 strain by ammonium sulphate precipitation. Assays for the antifungal activity of crude extracts were done on agar plates. The antifungal protein was found to be thermostable, resistant to pepsinum and trypsin, and partially sensitive to proteinase K. Its active pH range was wide, from 4.0 to 10.0. A antifungal protein purified by DEAE Sepharose Fast Flow chromatography and reversion phase chromatography, It’s molecular weight was estimated to be 23 k daltons by SDS-PAGE. It is significant to clone the genes because the antifungal proteins were greatly inhibitory to Verticillium dahliae Kleb.