芽孢杆菌中新型非核糖体肽类抗菌活性物质的发掘、分离鉴定及特性研究

doi:10.16431/j.cnki.1671-7236.2020.12.034

摘要/Abstract

摘要： 本研究旨在基于对细菌次级代谢产物的筛选，寻找新型非核糖体肽类抗菌物质。通过对烟台沿海地区的土壤、海水及近海常见海洋生物中的细菌进行培养，分离纯化得到细菌的单克隆，以大肠杆菌（E.coli）ATCC 25922和金黄色葡萄球菌ATCC 29213为指示菌进行初步筛选，以耐多黏菌素和碳青霉烯E.coli B2（bla_NDM-5+mcr-1）和耐甲氧西林金黄色葡萄球菌（MRSA） T144作为指示菌对有活性的细菌进行二次筛选。通过提取基因组进行PCR扩增产物测序比对，确定活性菌种属。采用有机萃取法对细菌的次级代谢产物进行萃取，通过凝胶层析和制备液相色谱进行纯化，利用分析型液相色谱进行纯度检测，进—步利用质谱对纯化后的抗菌活性物质进行结构鉴定。测序结果表明，活性菌属于解淀粉酶芽孢杆菌种，将其命名为解淀粉酶芽孢杆菌9-14（活性菌9-14）。抑菌试验结果表明，活性菌9-14的代谢产物对金黄色葡萄球菌ATCC 29213、MRSA T144、E.coli ATCC 25922和E.coli B2均具有高效抑制作用。活性菌9-14代谢产物是由氨基酸链组成的环状脂肽，属于伊枯草菌素的衍生物。对活性菌9-14代谢产物的生物学特性及其抑菌谱研究发现，活性菌9-14的代谢产物具有良好的热稳定性和酸碱稳定性，该抗菌物质经胰蛋白酶、胃蛋白酶、蛋白酶K和木瓜蛋白酶处理后抗菌活性没有明显减弱，具有较好的稳定性；该抗菌物质对所用金黄色葡萄球菌和大肠杆菌同样具有抑制作用，对绿脓杆菌、肺炎克雷伯杆菌、粪肠球菌和蜡样芽孢杆菌均不表现活性。本研究得到一种新型的抗菌物质，以该抗菌物质为抗菌药物的前体，可为食品安全和疾病控制提供一定的参考依据。

关键词: 非核糖体肽类抗生素; 有机萃取法; 分离纯化; 结构鉴定; 生物学特性

Abstract: The aim of this study was to search for novel non-ribosomal peptide antimicrobial substances based on the screening of bacterial secondary metabolites.The bacteria isolated from soil,sea water and common marine organisms in Yantai coastal area were isolated and purified.E.coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria,and E.coli B2 (bla_NDM-5+mcr-1) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were used as indicator for secondary screening.The genome was extracted and the PCR products were sequenced to determine the active species.The secondary metabolites of bacteria were extracted by organic extraction,purified by gel chromatography and preparative liquid chromatography,and purity was detected by analytical liquid chromatography.The results of sequencing showed that the active strain belonged to Bacillus amyloliquefaciens sp.,and was named as Bacillus amyloliquefaciens 9-14 (active bacterium 9-14).The results of antibacterial test showed that the metabolites of active bacterium 9-14 had high inhibitory effect on Staphylococcus aureus ATCC 29213,MRSA T144,E.coli ATCC 25922 and E.coli B2.The metabolites of active bacterium 9-14 were cyclic lipopeptides composed of amino acid chains,which belonged to the derivatives of ibuprofen.The biological characteristics and antibacterial spectrum of the metabolites of active bacterium 9-14 were studied.The results showed that the metabolites of active bacterium 9-14 had good thermal stability and acid-base stability.And the antibacterial activity of the antibacterial substance treated with trypsin,pepsin,protease K and papain was not significantly weakened and had good stability.The antibacterial substance also had inhibitory effect on Staphylococcus aureus and E.coli,but had no activity against Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterococcus faecalis and Bacillus cereus.In this study,a new antibacterial substance was obtained,which could be used as the precursor of antibacterial drugs,and could provide certain reference for food safety and disease control.

Key words: non-ribosomal peptide antibiotics; organic extraction; separation and purification; structural identification; biological characteristics

中图分类号:

S859.79⁺6

毛馨, 张桂真, 曲劲尧, 张玉豪, 卢国柱, 高永林, 李彦伸. 芽孢杆菌中新型非核糖体肽类抗菌活性物质的发掘、分离鉴定及特性研究[J]. 中国畜牧兽医, 2020, 47(12): 4093-4102.

MAO Xin, ZHANG Guizhen, QU Jinyao, ZHANG Yuhao, LU Guozhu, GAO Yonglin, LI Yanshen. Discovery,Isolation,Identification and Characterization of Novel Non-ribosomal Peptide Antibacterial Active Substances in Bacillus[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4093-4102.

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