纳豆芽孢杆菌Bna05菌株产抗霉脂肽的鉴定

Identification of antifungal lipopeptides produced by Bacillus subtilis natto Bna05

【目的】分析枯草芽孢杆菌纳豆菌亚种Bna05菌株代谢产物中脂肽类物质的存在情况,并探讨它们在抗霉功能中所发挥的作用。【方法】利用特异性引物对Bna05菌株进行脂肽合成酶类基因片段扩增、测序和BLAST比对分析;通过平板抑菌圈区域取样法获得Bna05菌株的高抗霉活性代谢产物,对该产物进行反相高效液相色谱(RP-HPLC)分离;用琼脂微稀释法测定分离物的抗霉活性,并对活性分离物进行质谱鉴定。【结果】Bna05菌株含有sfp和srf AA基因,未检测到itu C、itu D、fen D、fen ACE、bym B、bym C基因;RP-HPLC分离得到3组抗霉活性物质F_2、F_3和F_4,F_2中未检测到脂肽类物质,从F_3和F_4中分别鉴定出两类Surfactin同系物:V_7-surfactin和I/L_7-surfactin。两类Surfactin分别与F_2组合使用时,均表现出抗霉协同作用;此外,与Surfactin单独使用相比,两类Surfactin混合物与F_2组合后的协同抗霉活性得到进一步增强。【结论】Bna05菌株所产脂肽类物质主要是V_7-surfactin和I/L_7-surfactin,Surfactin与Bna05菌株所产其它活性物质之间存在抗霉协同作用,而V_7-surfactin和I/L_7-surfactin的同时存在,对于增强这种协同抗霉作用是有利的。

[Objective]To isolate and identify lipopeptides in metabolites of Bacillus subtilis subsp.natto Bna05 and to assess antifungal activity of the identified lipopeptides.[Methods]Primers specific for lipopeptide synthetases were used to amplify DNA fragments from strain Bna05 by PCR.The amplified DNA fragments were sequenced and analyzed using BLAST against Gen Bank database.The antifungal metabolites were obtained from the inhibition zone formed between Bna05and Aspergillus niger on modified PDA plate and separated by RP-HPLC.Antifungal activity of fractions separated by RP-HPLC was determined by micro-dilution method and the active molecules were identified by mass spectrum analysis.[Results]Genes for srf AA and sfp were identified in strain Bna05.However,genes encoding for itu C,itu D,fen D,fen ACE,bym B and bym C were not detected.Three active fractions F2,F3 and F4 were isolated by RP-HPLC.Lipopeptide was not detected in F2.Two kinds of surfactin variants were identified from F3 and F4：V7-surfactin variants in F3 and I/L7-surfactin variants in F4.Synergistic effect against Aspergillus niger was observed when F2 was used together with either V7-surfactins or I/L7-surfactins.The best antifungal activity was obtained when all three fractions were used together.[Conclusion]V7-surfactins and I/L7-surfactins were the main lipopeptides secreted by B.subtilis natto Bna05.Synergistic antifungal effect was found against Aspergillus niger when surfactins were used together with other active compounds such as F2.The greatest synergistic effect was obtained when both V7-surfactins and I/L7-surfactins were used together with F2.