枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质

PURIFICATION AND PROPERTIES OF ANTIFUNGAL PROTEIN X98Ⅲ FROM AACILLUS SUBTILIS

枯草芽孢杆菌（Bacillussubtilis）BS－98是一株能强烈抑制苹果轮纹病菌（Physalosporapiricola）等植物病原真菌的拮抗菌株。BS－98菌株培养液经硫酸铵分级盐析、SephadexG－100柱层析和DEAE－纤维素（DE32）柱层析后分离纯化出一种抗菌蛋白，命名为X98Ⅲ。蛋白电泳分析结果表明，此蛋白分子量为59000，等电点为4．50．醋酸纤维膜电泳后经特异染色证明X98Ⅲ含糖及胀。用DNS法测其含糖量为6％。此蛋白对热稳定，对蛋白酶部分敏感。氨基酸组分分析表明，该蛋白含11种不同氨基酸，尤富含谷氨酸和半胱氨酸等，而缺少天冬氨酸等。纯化后的X98Ⅲ对苹果轮纹病菌、芦笋茎枯病菌等有很强的抑制作用。X98Ⅲ的抑菌机理主要是溶解细胞壁，造成菌丝畸形、孢子不发芽或发芽异常。

Bacillus subtilis BS-98 is one of the antagonistic strains strongly againstplant fungal pathogens such as Physalospora piricola Nose. The antifungal protein waspurified by ammonium sulphate precipitation and column chromatography on SephadexG-100 and DEAE-cellulose, and it was named X98Ⅲ. Molecular weight of X98Ⅲ is59 000 by SDS-PAGE and PI value is 4.50 by PAG-IEF, respectihvely. X98Ⅲ wasdemonstrated as glycoprotein and lipoprotein by CAM (cellulose acetate membrane)electrophoresis and special staining. We estimated it contains 6% saccharides by usingDN methods. This protein was also found to be thermostabale and partially sensitiveto proteinases. The amino acid analysis of the protein X98Ⅲ showed that it comprisesof 11 different amino acids and Glu, Tyr, Cys are the aboundant amino acids. NoAsp, Phe and Met were found. Purified X98Ⅲ has strong inhibiting activity againstthe pathogens of Physalospora piricola, Phoma asparagi, etc. The antifungalmechanism of X98Ⅲ was mainly disintegration of the cell wall to make the hyphaeabnormal and the spores germinate abnormally or can not germinate at all.