反相高效液相色谱-质谱联用法分析蝙蝠蛾拟青霉菌发酵液中抑菌肽类物质的氨基酸序列

Analysis of Amino Acid Sequence of Antimicrobial Peptides in the Fermentation Broth of Paecilomyces hepiali by RP-HPLC-MS

测定蝙蝠蛾拟青霉菌发酵液中小分子抑菌肽的氨基酸组成序列对于深入理解其抑菌原理并实际应用有重要意义。本研究通过Amicon^(■)Ultra-15离心分离器(相对分子质量<10^(4))获得对金黄色葡萄球菌、大肠杆菌O157等具有抑菌作用的混合肽,采用液相色谱-质谱联用法(HPLC-MS/MS)对其分离纯化和二级质谱鉴定,并人工合成相应的肽段验证其体外抑菌效果。结果表明鉴定出3个小肽,氨基酸组成序列分别为Phe-Gly-Arg、Cys-Val-Cys-Tyr和Lys-Phe-Val-Pro-Arg。对其进行化学合成并验证有2条高抑菌活性肽段对大肠杆菌O157和金色葡萄球菌均有较好抑菌作用。上述研究为阐明蝙蝠蛾拟青霉发酵液中抗菌肽的分子多样性及进一步合理开发应用提供必要基础实验数据奠定基础。

It is important to determine the amino acid composition sequence of the small molecular antimicrobial peptides in the fermentation broth of Paecilomyces hepipipes for further understanding the antibacterial principle and practical application.In this study,the mixed peptides with antibacterial activity against Staphylococcus aureus and Escherichia coli O157 were obtained by Amicon Ultra-15 centrifugal separator(relative molecular mass less than 10^(4)).They were separated,purified and two-dimensional mass spectrometry identified by liquid chromatography-mass spectrometry(HPLC-MS/MS).The corresponding peptides were synthesized to verify their antibacterial effect in vitro.The results show that three small peptides are identified.The amino acid sequences of the peptides are Phe-Gly-Arg,Cys-Val-Cys-Tyr and Lys-Phe-Val-Pro-Arg.The peptides were synthesized and verified two of them have good antibacterial activity against Escherichia coli O157 and Staphylococcus aureus.The above-mentioned research lays the foundation for clarifying the molecular diversity of antimicrobial peptides in the fermentation broth of Paecilomyces hepialis and providing necessary basic experimental data for further rational development and application.