一株紫罗兰蓝色素产生菌的分离、鉴定及其活性产物的特性分析

Isolation, identification and bioactive metabolites property of the strain secreting violet-blue pigment

摘要:

【目的】调查秦巴山地区微生物物种的多样性, 建立用于活性产物筛选的微生物代谢产物库和菌种资源库, 通过数据库平台进行资源共享。【方法】采用不同的分离方法, 分离存在于不同生境的微生物类群, 通过形态、生理生化及分子生物手段鉴定菌种, 发酵并通过甲醇提取代谢产物。【结果】从本地区土壤样本中分离获得一株产水溶性紫罗兰蓝色素的放线菌Str-4331, 在高氏培养基上其气生菌丝为白色, 产生水溶性紫罗兰色素, 镜检观察孢子丝螺旋形, 孢子椭圆形至长圆形, 通过形态、生理生化特征初步确定为链霉菌。利用ClustalX对其16S rRNA进行序列比对, 用Neighbor-Joining (NJ)构建系统发育树, 并用Bootstraping法对其评价。实验结果表明, 该菌基因序列与砖红链霉菌(Streptomyces lateritius) LMG 19372基因序列有99%的最高相似性。抑菌试验表明, 活性产物具有较强的抗革兰氏阳性细菌活性。色素在水溶液中颜色随着pH的变化而变化。质谱分析显示3个主要化合物, 分子量分别为557.68、588.43、485.18 kD, 其中分子量为557.68 kD化合物与榴菌素B分子量一致。【结论】根据菌株的16S rRNA序列分析结果, 结合菌株的培养特性和生理生化特性, 将菌株Str-4331归属为砖红链霉菌(S. lateritius)。该菌种首次在本地区分离到, 具有进一步研究开发的价值。

Abstract:

[Objective] In order to investigate microbial diversity of the Qinling-Daba region, collect microbial culture and metabolites aimed to screen for active compounds, and establish the collection database as a platform for sharing inform. [Methods] The microbial strains from different habitats were isolated by the different methods and identified by physiological, biochemical and morphological characteristics and 16S rRNA gene sequence analysation, while the microbial metabolites were prepared for screening for active compound by fermentation in shaker flasks, extraction with methanol. [Results] An actinomycete, named strain Str-4331, was isolated from a soil sample in the Qinling-Daba region. It produced grayish-white aerial mycelium and solvable-violet-blue pigment on Gause’s synthetic agar, microscopic observation revealed that it produced helically spore hypha, the spores were elliptic to oblong. It was primarily identified as genus Streptomyces according to its morphological, physiological and biochemical characteristics. The 16S rRNA sequence of strain Str-4331 was analyzed by ClustalX, the phylogenetic tree was derived with NJ and analyzed with bootstrapping. The results showed that the 16S rRNA gene sequence of the strain Str-4331 shared the highest identity (99%) with that of S. lateritius strain: LMG 19372. The antibacterial activity of the extracted metabolites was determined and the results showed that the metabolites had the activity against the Gram-positive bacteria. The aqueous solution containing the metabolites showed different colors under different pH conditions. Liquid chromatography-mass spectroscopy analysation showed that the metabolites produced by the strain Str-4331 composed of three main compounds with the molecular weight separately 557.68 kD, 588.43?kD, 485.18 kD, while the molecular weight of granaticin B was 558 kD according to the documents. [Conclusion] The strain Str-4331 was identified as S. lateritiu from its morphological and cultural characteristics and the 16S rRNA gene sequence. This is the first report that S. lateritius strain was isolated in the Qinling-Daba region, and this study provided basis for the further exploration of the strain.