桑氏链霉菌KJ07抗菌蛋白的分离纯化及部分特性

Isolation, purification and partial characterization of an antifungal protein from Streptomyces sampsonii KJ07

【目的】桑氏链霉菌(Streptomyces sampsonii)KJ07对无性阶段的杨树紫纹羽病菌(Rhizoctonia violacea)有较强的拮抗作用。为研究其抗菌物质,对其发酵液中主要抗菌物质进行分离纯化并明确其部分性质。【方法】采用硫酸铵分级沉淀、DEAE Sepharose Fast Flow离子交换层析、Sephadex G-50分子筛层析等方法进行分离纯化。【结果】获得单一抗菌活性蛋白,分子量约为28.4 k D。该抗菌蛋白抑菌谱较广,能使R.violacea菌丝畸形,菌丝隔膜不明显,细胞壁及胞内原生质开始降解,并产生黑色物质。稳定性试验显示抗菌蛋白最适温度为25°C,最佳p H为6.0,当温度≥60°C时,抑菌活性下降大于20%,当p H<4.0或≥8.0时,抑菌活性下降大于12%。其活性还受金属阳离子影响,但对蛋白酶K不敏感。利用自动Edman降解法测得抗菌蛋白N端10个氨基酸序列,但通过NCBI BLAST程序未检索到与其相似性较高的已知抗菌蛋白。【结论】推测该抗菌蛋白可能是一种新的蛋白质。

[Objective] Streptomyces sampsonii KJ07 showed a strongly antifungal activity against Rhizoctonia violacea which caused poplar purple root rot. To investigate its antifungal substances, the main antifungal substance was isolated and purified, and also the partial characteristics were studied. [Methods] Methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Sephadex G-50 were used. [Results] A purified antifungal protein was obtained with a molecular mass of 28.4 k D by SDS-PAGE. It had a wide antifungal spectrum, which made the hyphae deformed, as well as the septa, cell wall and intracellular protoplasm of R. violacea abnormal along with the black substances produced. Stability tests showed that the optimum temperature and p H were 25 °C and 6.0 respectively. The antifungal activity of the protein decreased more than 20% at the higher temperature（≥60 °C）, and it declined more than 12% when the p H4.0 or ≥8.0. Meanwhile, it was affected by metal cation, but was tolerant of proteinase K. 10 Amino acids of the N-terminal sequence were measured by Edman degradation, however no one which had the high homology with the antifungal protein was retrieved through NCBI BLAST. [Conclusion] We speculated that the antifungal protein we obtained maybe a novel protein.