地衣芽孢杆菌W10抗菌蛋白的分离纯化及其理化性质研究

Isolation, purification and characterization of antifungal protein from Bacillus licheniformis W10 strain

地衣芽孢杆菌W10是一株对多种植物病原菌有拮抗作用的生防细菌,其产生的抗菌物质主要是抗菌蛋白。细菌培养滤液用30%硫酸铵盐析,透析后再经SephadexG-75层析柱纯化得到一种具有抑菌活性的蛋白。该抗菌蛋白的分子量为46049.2Da;等电点为6.71;含有糖基,含糖量为6.83%;含有脯氨酸或羟脯氨酸;不含脂基。这种蛋白在pH6～12范围内均具有抑菌作用,pH6时活性最大;对热稳定,100℃以下水浴20min,抑菌活性与对照相比均在对照的80%以上,121℃蒸汽处理20min,活性虽有所下降,但仍达对照的65.7%;对胰蛋白酶和蛋白酶K不敏感,酶处理后蛋白的抑菌活性分别为对照的98.7%和98.3%;对氯仿和紫外线也不敏感,处理后活性仍达对照的91.0%和98.6%。

Bacillus licheniformis W10 was an antagonistic bacterium to Botrytis cinerea and other important plant pathogens. Antifungal protein which produced by W10 was the main antifungal substance. The crude protein in culture filtrate could be precipitated with 30% amonium sulfate and obtained by dialysis of the precipitate. The antifungal protein was purified by gel filtration on Sephadex G-75 column. Molecular weight of the protein was 46 049.2 Da by DSD-PAGE and PI value was 6.71 by IEF-PAGE. The protein was demon- strated as a glycoprotein that contained 6.83% saccharide, and some proline or hydroxyproline, but no lipid. The inhibitory activity of the protein was observed in the range of pH value from 6 to 12, and the strongest one was at pH 6. The protein was found to be thermostable at 100℃, and not sensitive to trypsin, proteinase K, ultraviolet radiation and chloroform. Key words：