黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1产生的脂肽类抗生素的分离和鉴别

Isolation and identification of lipopeptide antibiotic produced by Bacillus amyloliquefaciens B10-6-1 antagonistic to Aspergillus flavus

本文旨在对黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1的脂肽类抗生素相关基因进行克隆,对其所产生的脂肽类抗生素进行组分分离,对有抑菌活性的脂肽类抗生素进行结构鉴别。以解淀粉芽孢杆菌B10-6-1基因组DNA为模板,采用PCR方法对ItuA、ItuB、ItuC、ItuD、FenA、FenB、FenD、Sfp、bmyA、bmyB、bmyC合成基因进行DNA扩增。将该菌发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类抗生素的粗提物,采用高效液相色谱法分离,对收集的洗脱峰组分进行体外抑菌试验,对有抑菌活性的组分进行液质(HPLC-ESI-MS)分析。PCR扩增检测结果显示黄曲霉菌拮抗菌B10-6-1能扩增出ItuA、ItuC、ItuD、FenA、FenD、Sfp、bmyA、bmyB、bmyC合成基因,未能扩增出ItuB、FenB合成基因。高效液相色谱法收集到7种组分,体外抑菌试验证明组分5和7有抑菌活性。经液质测定,组分5和组分7分别为脂肽类抗生素C14Bacillocnycin D和C15Bacillocnycin D。本研究探明了黄曲霉菌拮抗菌B10-6-1所产生的天然抑菌活性成分,为该菌用于黄曲霉菌的生物防治奠定了理论基础。

The lipopeptide antibiotic related genes of Bacillus amyloliquefaciens B10-6-1 antagonistic to Aspergillus flavus were cloned,and the components of the antibiotics were separated and structurally identified.Synthesis genes ItuA,ItuB,ItuC,ItuD,FenA,FenB,FenD,Sfp,bmyA,bmyB,bmyC were amplified by PCR amplification using genomic DNA of Bacillus amyloliquefaciens B10-6-1 as template.The crude extract of lipopeptide antibiotics was obtained from the fermentation broth through centrifugation,acid precipitation and methanol extraction.The extract was separated by high performance liquid chromatography(HPLC),and antifungal activity test in vitro of the elution peak components collected was carried out.The components with antimicrobial activity were analyzed by high performance liquid chromatography combined with multi-stage electrospray tandern mass spectrometry(HPLC-ESI-MS).PCR amplification test results showed that B10-6-1 antagonistic to Aspergillus flavus was able to amplify the ItuA,ItuC,ItuD,FenA,FenD,Sfp,bmyA,bmyB,bmyC synthesis genes,but failed to amplify the ItuB,FenB synthesis genes.Seven components were collected by HPLC and antibiotic test in vitro results showed that components 5 and 7 exhibited antimicrobial activity.HPLC-ESI-MS determination results showed that component 5 is lipopeptide antibiotic C14 bacillomycin D and component 7 is lipopeptide antibiotic C15 bacillomycin D.This study laid a theoretical foundation of biological control of Aspergillus flavus by using natural antimicrobial activity of antagonistic bacteria.