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作 者:于秀菊[1] 韩小涛 李钰钰 孙铮 董常生[1] Xiuju Yu;Xiaotao Han;Yuyu Li;Zheng Sun;Changsheng Dong(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,Shanxi,China)
机构地区:[1]山西农业大学动物医学学院,山西太谷030801
出 处:《生物工程学报》2021年第7期2453-2462,共10页Chinese Journal of Biotechnology
基 金:山西省青年基金(No.201801D221301);山西农业大学创新团队项目(No.CXTD201201)资助。
摘 要:目前,我国饲料中全面禁止添加抗生素,寻找新型的抗生素替代物成为科学研究的热点之一。为获得新的细菌素,本研究以大肠杆菌、沙门氏菌、金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特氏菌为指示菌,通过牛津杯扩散法从羊驼粪便中筛选到高产抑菌物质的芽孢杆菌。基于菌落特征、革兰氏染色和16S rRNA的鉴定结果,将分离到的产抑菌物质菌株命名为地衣芽孢杆菌SXAU06株。采用硫酸铵沉淀、氯仿抽提、分子截留和SDS-PAGE等技术对所产抑菌物质进行分离纯化,经LC-MS/MS和生物信息学分析发现,抑菌物质为分子量约14 kDa的类细菌素,将其命名为BLISSXAU06。耐受性试验结果显示,BLISSXAU06具有耐高温、耐酸碱和耐蛋白酶K的特性。利用大肠杆菌表达系统对BLISSXAU06进行重组表达,所获得的重组BLISSXAU06具有抑制金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特氏菌生长的活性。综上所述,BLISSXAU06具有优良的特性,可进一步在农业生产、生物医药和食品加工领域开展研究。The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16 S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including(NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance(BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLISSXAU06. BLISSXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLISSXAU06 was heterologously expressed in E. coli and the recombinant BLISSXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.
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