Isolation and identification of an extracellular polysaccharide high-producing marine Bacillus and the study on its anti-tumor activity

Abstract

[Objective] The aim of the present study was to determine the biological activities, such as antitumor activity of the extracellular polysaccharide (EPS) from marine bacteria in Beibu Bay, Guangxi, and to provide reference basis for the research, development and application of the biological activity of the EPS secreted by bacteriafrom Beibu Bay..

[Method] The shallow soil samples of Maowei Sea mangrove in Qinzhou, Guangxi were collected. The marine bacteria with high production of EPS were screened by LB-aniline blue culture medium and phenol-sulfuric acid method, and the strains were identified by 16S rRNA sequence. The molecular weight and structure of the EPS were determined by high performance gel permeation chromatography and infrared spectroscopy. Finally, the cytotoxicity of the EPS to Vero cells and the inhibitory effect on the proliferation of HeLa cells were determined by thiazolyl blue (MTT) method.

[Result] The results showed that a strain named QLc-04, with EPS production of 0.2917 mg/mL was screened. The colony of QLc-04 formed on LB-aniline blue solid medium was white, opaque, slightly raised in the center and rough on the surface. QLc-04 was identified to be Gram-positive bacilli. QLc-04 showed 99.45% to 99.82% 16S rRNA sequence similarity to Bacillus, with the highest similarity to B. thuringiensis BT62(CP044978) (99.82%), then 99.81% to B. cereus ATCC 14579 ^T (AE016877) and B. mycoides DSM 2048 ^T (ACMU01000002). In the phylogenetic tree based on 16S rRNA sequence similarity, QLc-04 and Bacillus were clustered into the same branch, so that QLc-04 was identified to be Bacillus. The molecular weight of QLc-04-EPS was about 179.9 kD and the EPS belonged to a polysaccharide with pyranoid structure. The QLc-04-EPS had no significant cytotoxicity to Vero cells. In the concentration between 10-300 μg/mL, the more QLc-04-EPS was used, the stronger proliferation ability of Vero cell was. However, when the concentration of QLc-04-EPS was more than 50 μg/mL, the inhibitory effect on HeLa cell proliferation was significantly enhanced ( P<0.05).

[Conclusion] The production of EPS secreted by marine Bacillus QLc-04 is 0.2917 mg/mL after 36 h fermentation. QLc-04-EPSis a typical pyran polysaccharide with a molecular weight of 179.9 kD, and shows a significantly inhibitory effect on the proliferation of HeLa cells.

Abstract

摘要： 【目的】 明确广西北部湾海洋细菌胞外多糖 (EPS) 独特的抗肿瘤等生物学活性, 为北部湾海域细菌EPS活性 研究及开发利用提供参考依据。 【方法】采集广西钦州茅尾海红树林浅层土壤样品, 使用LB-苯胺蓝培养基及苯酚— 硫酸法筛选EPS高产海洋细菌, 基于16S rRNA序列进行菌株鉴定; 通过高效凝胶渗透色谱和红外光谱测定EPS的分 子量及其结构, 最后使用噻唑蓝 (MTT) 法测定EPS对Vero细胞的毒性及抑制HeLa细胞增殖的效果。 【结果】筛选出1株 EPS产量为0.2917 mg/mL的菌株, 命名为QLc-04, 在LB-苯胺蓝固体培养基上生长形成的菌落呈白色, 不透明, 中央略 微隆起, 表面粗糙, 为革兰氏阳性杆菌。QLc-04与芽孢杆菌属 ( Bacillus) 菌株的16S rRNA序列相似性在99.45%~99.82%, 其中与 B. thuringiensis BT62 (CP044978) 的相似度最高 (99.82%), 与 B. cereus ATCC 14579 ^T (AE016877) 和 B. mycoides DSM 2048 ^T (ACMU01000002) 相似性均为99.81%; 在基于16S rRNA序列相似性构建的系统发育进化树上, QLc-04与芽孢杆菌属聚类为同一分支, 因此确定QLc-04为芽孢杆菌属。QLc-04-EPS分子量约179.9 kD, 属于具有吡 喃型结构的多糖; QLc-04-EPS对Vero细胞无直接毒性, 在10~300 μg/mL浓度范围内Vero细胞随QLc-04-EPS浓度的增 加其增殖能力不断升高, 但QLc-04-EPS浓度超过50 μg/mL时对HeLa细胞增殖的抑制作用显著增强 ( P<0.05)。 【结论】 海洋芽孢杆菌QLc-04经发酵培养36 h其EPS产量为0.2917 mg/mL; QLc-04-EPS为典型吡喃型多糖, 分子量为179.9 kD, 具有显著抑制HeLa细胞增殖的作用。