摘要
目的旨在从盐碱土壤中分离获得一株耐盐细菌B-11,对其进行分类鉴定及抗菌活性优化及抗菌特性研究。方法基于形态学分析和16S rRNA基因序列对菌株进行分类鉴定。以金黄色葡萄球菌为指示菌,采用双层平板法对菌株B-11发酵液特性进行研究,并通过发酵培养基的正交优化试验,获得菌株产抗菌活性物质最佳的发酵培养条件。通过对菌株B-11基因组次级代谢产物合成基因的分析,研究其基因组上的功能基因。结果结合形态学和分子鉴定表明该菌为Bacillus属成员,在0-4%的Na Cl浓度范围内生长良好,为耐盐细菌。该细菌发酵上清液对蛋白酶K和链霉蛋白酶敏感,对pH、温度和紫外光具有良好的耐受性。通过正交优化得到菌株B-11最适发酵条件为:可溶性淀粉15g/L,黄豆粉15g/L,NaCl 15g/L,p H为自然pH,接种量为1.5%,发酵温度为34℃或37℃,优化后发酵液对金黄色葡萄球菌的抑菌圈直径较优化前提高25.5%。利用基因组测序及次级代谢产物合成相关基因分析,发现Bacillus sp.B-11基因组中含有非核糖体肽合成酶(NRPS)基因,并结合该菌次级代谢产物对蛋白酶的敏感性,表明该菌具有产多肽类化合物的潜力。结论盐碱土壤中的极端微生物资源可作为抗菌活性物质的潜在新来源。
Objective To isolate a halotolerant bacterium B-11 from saline-alkali soil, which has the antimicrobial activity against Staphylococcus aureus, and to study its identification, optimization of antimicrobial activity and antimicrobial properties. Methods The strain B-11 was identified by morphological analysis and 16S rRNA gene sequence analysis. Using Staphylococcus aureus as the indicator, antimicrobial activity of the fermentation broth of strain B-11 was detected by cylinder plate method and we got the best fermentation medium and culture conditions via orthogonal optimization experiment. The functional genes in the B-11 genome were studied by the analysis of its secondary metabolite biosynthetic gene cluster. Results The strain is a halotolerant bacterium which can grow well over a range of NaC1 concentration of 0-4%, and it is characterized as the genus of Bacillus and named as Bacillus sp. B-11. Fermentation supernatant of the strain B-11 was found to be sensitive to protease K and pronase, but showed excellent stability towards pH, temperature and ultraviolet. Orthogonal optimization experiment was used to determine the optimal medium composition and fermentation conditions as 15g/L amylogen, 15g/L soybean meal,15 g/L NaC1, natural pH, 1 percent inoculum amount and fermentation temperature of 34℃/37℃. Under the optimal medium and culture conditions, the antimicrobial activity against S. aureus was increased by 25.5%, compared with that before optimization. Through genomic sequencing and secondary metabolite biosynthetic gene cluster, we found that Bacillus sp. B-11 genome contains nonribosomal peptide synthetase （NRPS） gene, and combined with the sensitivity of its secondary metabolite to protease, which indicated that the strain possess potential of producing nonribosomal peptides. Conclusion Results from the present study showed that extremophilic exmicroorganisms from saline-alkali soil may be developed as a potential new source of antimicrobial substances.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2016年第7期531-537,540,共8页
Chinese Journal of Antibiotics
基金
中央高校基本科研业务费专项资金资助(No.SCU2015D008)
四川应用基础研究计划项目(No.2014JY0199)
关键词
耐盐芽孢杆菌
金黄色葡萄球菌
抗菌活性
正交优化
功能基因
Salt-tolerant bacillus
Staphylococcus aureus
Antimicrobial activity
Orthogonal optimization
Functional gene
