海洋源枯草芽孢杆菌2713抗白色念珠菌活性物质的鉴定  

Identification of bioactive substances against Candida albicans by marine Bacillus subtilis 2713

作　　者：鞠晓月 薛梅 乔晓妮 王淑婷 孙晓雯 唐涛 何增国 JU Xiaoyue;XUE Mei;QIAO Xiaoni;WANG Shuting;SUN Xiaowen;TANG Tao;HE Zengguo(School of Medicine and Pharmacy,Ocean Univeristy of China,Qingdao 266003,China;Marine Microbiological Engineering&Research Center,Qingdao Marine Biomedical Research Institute,Qingdao 266071,China;Qingdao Bioantai Biological Technology Co.,Ltd,Qingdao 266000,China)

机构地区：[1]中国海洋大学医药学院,山东青岛266003 [2]青岛海洋生物医药研究院海洋微生物工程研发中心,山东青岛266071 [3]青岛百奥安泰生物科技有限公司,山东青岛266000

出　　处：《中国海洋药物》2024年第6期1-10,共10页Chinese Journal of Marine Drugs

基　　金：泰山产业领军人才工程项目(tscy202006017)资助。

摘　　要：目的探究海洋源枯草芽孢杆菌(Bacillus subtilis)2713拮抗白色念珠菌(Candida albicans)的潜力,并分析其天然抗菌活性物质。方法通过平板对峙、Spot-on-lawn及扫描电镜分析拮抗白色念珠菌潜力;通过基因测序及anti-SMASH在线分析有关抗菌活性物质基因簇;通过中试规模液体深层通气发酵验证该菌株合成抗菌活性物质的能力,借助大孔树脂吸附法分离抗菌活性物质,高效液相色谱法(HPLC)进一步纯化活性组分,然后对其稳定性进行表征;最后通过液相色谱质谱联用(LC-MC)确定抗菌活性物质的化学本质。结果菌株2713能显著拮抗白色念珠菌,扫描电镜显示其会导致白色念珠菌细胞皱缩,甚至破坏其完整性。生物信息学分析表明菌株2713基因组中含有fengycin、surfactin、bacilibactin、bacillaene、macrolactin、difficidin、bacilysin等多种次生代谢产物合成簇。中试发酵证实菌株2713能合成拮抗白色念珠菌的生物活性物质。通过大孔树脂一步法分离提取发酵液中活性物质,回收率达到75%。粗提物经HPLC纯化得到活性组分。该纯化物能耐受30 min的100℃高温处理,并在pH 2~12条件下稳定;纯化物对胰蛋白酶和胃蛋白酶不敏感,但能被木瓜蛋白酶处理后失活。通过LC-MS分析证实拮抗白色念珠菌的纯化物质是由1条16个碳的脂肪酸链和1个10个氨基酸的环肽组成的C16-Fengycin A,其分子式为C_(72)H_(110)N_(12)O_(20)。Objective To investigate the antagonistic potential against Candida albicans by marine Bacillus subtilis 2713 and to analyze the natural antimicrobial active substances produced.Methods Evaluation of antagonistic activity against Candida albicans by the combination of plate inhibition zone observation,Spoton-lawn test,and scanning electron microscopy.The relevant antimicrobial genes were analyzed by genome sequencing and anti-SMASH online analysis.Submerged fermentation was applied to verify the ability of antimicrobials production at a spot scale.The antimicrobials were separated by macroporous resin adsorption and then further purified by HPLC.The active fractions were subjected to stability tests.Finally,the chemical nature of the antimicrobial was elucidated by LC-MC.Results It was found that strain 2713 could inhibate the growth of Candida albicans.Scanning electron microscopy revealed that it caused the crumbling of Candida albicans cells and even disrupted their integrity of their cell membrane.Bioinformatics analysis showed that it harbored a variety of gene clusters encoding secondary metabolites such as fengycin,surfactin,bacilibactin,bacillaene,macrolactin,difficidin,and bacilysin.Pilot fermentation convinced that strain 2713 could produce the anti-Candida albicans substance by submerged fermentation.The substances were recovered from the fermentation by one-step macroporous resin absorption,with a recovery rate of up to 75%.The crude extract was purified by HPLC and the activity was identified in one fraction corresponding to a single peak.The purified substance was stable at pH 2−12 and remained its activity when treated at 100℃for 30 minutes.It was not sensitive to trypsin and pepsin treatment but could be inactivated by papain.Lastly,by using LC-MS,the purified anti-Candida albicans substance was confirmed to be C16-Fengycin A,composed of a 16-carbon fatty acid chain and a 10-amino acid cyclic peptide,with the molecular formula as C_(72)H_(110)N_(12)O_(20)。

关 键 词：枯草芽孢杆菌 海洋源 脂肽 fengycin A 

分 类 号：R931.77[医药卫生—生药学]