【摘要】 【目的】魔芋软腐病是一种由胡萝卜软腐欧文氏菌引起的病害,严重影响了魔芋产业的发展。枯草芽胞杆菌BSn5所产生的蛋白APn5对胡萝卜欧文氏菌具有良好的抑菌效果,本文将对该蛋白进行鉴定和性质分析。【方法】用饱和度为30%的硫酸铵沉淀和超滤离心的方法从枯草芽胞杆菌BSn5的发酵上清液中分离纯化APn5蛋白。以滤纸片法检测APn5蛋白的抑菌谱。用不同的温度、pH条件和蛋白酶处理APn5蛋白,并检测其抑菌活性变化。测定菌株BSn5的生长周期,同步检测其发酵上清液的抑菌活性。利用基质协助激光解吸附离子化-飞行时间质谱(MALDI-TOF-MS)和电喷雾四极杆飞行时间串联质谱(Q-TOF)分析APn5蛋白。【结果】APn5蛋白对于魔芋软腐病菌具有较好的抑菌活性,抑菌谱较窄,主要对细菌性植物病原菌具有较强的抑制作用;对高温敏感,对蛋白酶E敏感,对蛋白酶K和胰蛋白酶部分敏感;在酸性环境下其抑菌活性稳定,碱性环境下活性不稳定。在菌株BSn5的生长周期中,该蛋白的抑菌活性不能稳定存在,其发酵上清液在对数生长期抑菌活性逐渐升高,进入稳定期后抑菌活性开始下降,直至完全消失。质谱分析未发现其与已知蛋白具有高度相关性。【结论】APn5蛋白具有不同于已报道的枯草芽胞杆菌所产生的抑菌物质的性质,推测为一种新型的抑菌蛋白。更多还原

【Abstract】 [Objective] Amorphophallus soft rot disease,caused by Erwinia carotovora,affects Amorphophallus industry development. We identified and characterized protein APn5 against Erwinia carotovora,isolated from Bacillus subtilis strain BSn5. [Methods] Protein APn5 was purified from BSn5 culture by ammonium sulfate precipitation with 30% rela-tive saturation and ultrafiltration. Inhibition activity was tested by agar well diffusion assay. Protein APn5 was treated at different temperatures,pH conditions and proteinase. The growth curve of BSn5 was examined; meanwhile,the inhibition activity of supernatant of BSn5 culture in different growth phase was tested. Amino acid sequence of protein APn5 was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadru-pole-time-of-flight (Q-TOF). [Results] Protein APn5 showed a narrow inhibition spectrum,mainly strongly inhibiting the growth of a few plant pathogenic bacteria. Protein APn5 was sensitive to high temperature and alkaline pH,and partial sensitive to trypsin,proteinase K and pronase E. During strain BSn5 growth phase,the inhibition activity was unstable,which would gradually lose on stationary phase and disappeared finally. [Conclusion] On the basis of the difference on inhibition spectrum and characteristics,protein APn5 was suggested as a novel antimicrobial protein.更多还原