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期 刊 ：植物保护学报 2010年

关键词：解淀粉芽孢杆菌；抗菌蛋白；分离纯化；理化性质；

Keywords:Bacillus amyloliquefaciens, antifungal proteins, purification, physicochemical characteristics,

摘 要 ：解淀粉芽孢杆菌Bacillus amyloliquefaciens LP-5是一株对梨黑斑病菌Alternaria alternata具有抑制作用的内生细菌,其产生的抗菌物质主要是抗菌蛋白。为明确该抗菌蛋白的理化性质,采用盐酸沉淀、DEAE-52层析和Sephadex G-100层析进行纯化,测定了所得抗菌蛋白对热、蛋白酶、紫外辐射及酸、碱的稳定性。结果表明:纯化的抗菌蛋白分子量为20.3kD,等电点为6.33。该蛋白对高温敏感,100℃处理30min,抑菌活性仅为对照的18.4%;对蛋白酶K和胰蛋白酶稳定,酶处理后的抑菌活性分别为对照的92.5%和88.9%;对紫外辐射稳定,照射12h后抑菌活性仍达对照的92.5%;对酸稳定、对碱敏感,pH 13处理后抑菌活性下降为原有活性的74.1%。抑菌活性测定结果显示,该蛋白对梨黑斑病菌的孢子萌发和菌丝生长有明显抑制作用,可造成孢子萌发端泡囊化、菌体破裂、原生质外渗。

Abstract:Endophytic Bacillus amyloliquefaciens strain LP-5 was an antagonistic bacterium to Alternaria alternata. Antifungal protein which produced by LP-5 was the main antifungal substance. To research the physicochemical characteristics of antifungal proteins isolated from strain LP-5,an antifungal protein was purified by using HCl precipitation and column chromatography on DEAE-52 and Sephadex G-100. The effects of heat, proteinase, ultraviolet (UV) and pH on inhibiting activity of antifungal protein were studied. The results showed that the molecular weight (MW) and isoelectric point (pI) value of the antifungal protein were 20.3kD and 6.33 determined by SDS-PAGE and PAGE-IEF, respectively. This protein was sensitive to high temperature and the antifungal activity only retained 18.4% compared to the control after treatment at 100℃ for 30min. This protein was stable after treatment by proteinase K and trypsin with the activity keep at 92.5% and 88.9%, respectively. The activity retained 92.5% after radiating the protein for 12h by UV suggesting its relatively higher stability against the UV radiation. This protein was stable to acid but sensitive to alkali with the activity decreased to 74.1% after treatment at pH 13. This protein showed inhibition activity on conidia germination and hyphal growth of A.alternata. The spore peak was abnormally swollen, with the cell wall disrupted and the cytoplasm leaked.

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