新型抑菌蛋白APn5抑制胡萝卜软腐欧文氏菌

Identification and characterization of novel antimicrobial protein APn5 against Erwinia carotovora

【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。

[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% relative 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 quadrupole-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.