基于PCR和TOF-MS海洋源产脂肽芽孢杆菌筛选

Screening of Bacillus spp.Producing Lipopeptide from Marine Environment Based on PCR and TOF-MS

【目的】准确高效分离红树林等海洋环境中产脂肽芽孢杆菌。【方法】将前期从红树林环境筛出具有抗菌活性的12株芽孢菌株作为研究菌株,采用溶血活性及排油作用对其中的可能产脂肽菌株进行初步筛选,并基于脂肽合成酶基因PCR扩增法对脂肽合成酶基因携带菌复筛,采用飞行时间质谱(TOF-MS)对所产脂肽进行鉴定。【结果】初步发现6株芽孢杆菌具有溶血活性,其中HY-5、HY-4和HN-9这3株具有排油能力,油层透明圈的直径分别为45、32和3 mm。PCR扩增检测到HY-5含有iturin、surfactin和bacillomycin D合成酶基因,HY-4含有iturin、surfactin合成酶基因,HN-9含有iturin合成酶基因。TOF-MS检测到HY-5和HY-4的发酵液含surfactin、iturin和bacillomycin 3种脂肽,HN-9的发酵液只含iturin。【结论】基于PCR与TOF-MS为核心的级联技术,建立一种海洋源脂肽产生菌快速筛选以及脂肽谱快速鉴定的方法,解决海洋源芽孢杆菌在常规培养基难合成脂肽造成的漏筛情况。

【Objective】To accurately and efficiently isolate lipopeptide-producing bacillus spp.from mangrove and other marine environments.【Methods】The 12 bacillus with antibacterial activity that were identified from the mangrove area in our previously study were used as research strains.Blood plate and oil drainage method were performed out to evaluate the hemolytic activity and surface activity of the fermentation,following PCR was then used to detect the four lipopeptide synthase genes.Finally,TOF-MS method was used to detect the lipopeptide components.【Results】6 bacillus with hemolytic activity were identified,and three of them have the ability to drain oil.The diameter of transparent zone of HY-4,HY-9 and HN-9 were 45,32 and 3 mm,respectively.PCR results showed that HY-5 contained iturin,surfactin and bacillomycin D synthase genes,HY-4 contained iturin and surfactin synthetase genes,and HN-9 contained iturin synthase genes.Surfactin,iturin and bacillomycin were detected by TOF-MS both in the fermentation of HY-5 and HY-4,while only iturin was detected in the fermentation of HN-9.【Conclusion】Based on the combination of PCR and TOF-MS,a cascade method was established to identify and screen marine bacillus and their lipopeptide profile rapidly.The method can solve the problem of false negative screening caused by leakage of lipopeptides produced by marine bacillus in conventional medium.