The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, β-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including β-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.

中文翻译：

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从 Aureobasidium pullulans 中纯化一种杀手毒素，用于植物病原体的生物防治

本研究的目的是净化和评估 Aureobasidium pullulans 在绿色霉菌 (Penicillum digitatum) 和酸腐病 (Geotrichum citri-aurantii) 的散剂下产生的杀伤毒素作用。最初，测试了不同的蛋白质沉淀方法。测定蛋白水解活性和作用于细胞壁受体的蛋白质、β-1,3-葡聚糖酶和几丁质酶的存在，并通过Sephadex G-75凝胶排阻色谱和纤维素色谱（中纤维）进行毒素纯化。随后，通过聚丙烯酰胺凝胶电泳确认纯化，并在用柠檬酸盐-磷酸盐（0.1 M，pH 4.6）缓冲的固体 YEPD-亚甲基蓝中进行杀伤活性的检测。毒素鉴定采用液相色谱-质谱法。结果表明，最好的蛋白质沉淀方法是 2:1 乙醇（vol/vol 乙醇/上清液）。可以观察到具有蛋白水解活性的酶的存在，包括 β-1,3-葡聚糖酶和几丁质酶。在纯化过程中，验证了酵母产生的杀伤毒素具有属于泛素家族的低分子量蛋白质，该蛋白质对P. digitatum和G. citri-aurantii具有杀伤活性。