抗UV-B辐射菌Pantoea agglomerans KFS-9的选育及其抗辐射机制的研究
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摘要
采用双定向筛选方法,筛选到一株胞外代谢产物具有紫外保护作用的抗UV-B辐射的菌株。经过多次富集和反复分离纯化,从深圳红树林海域中获得263株纯培养的菌株,从中筛选出5株具有较高抗UV-B辐射的菌株。通过考察胞外代谢产物对菌体细胞的辐射保护作用,最终确定KFS-9为出发菌株。采用16S rRNA基因测序与传统的表型特征结合的方法进行了菌种鉴定。最后确定菌株KFS-9为Pantoea agglomerans,提交到Genbank中的注册号为DQ350072。诱导获得了一株类胡萝卜素缺失的突变菌株KFS-98,确定野生菌株与突变菌株的抗UV-B辐射性能没有明显的差别。提示菌体的类胡萝卜素含量对其抗UV-B辐射性没有影响。
为考察胞外小分子代谢产物对菌体抗辐射性的作用,对成团泛菌胞外具有紫外吸收作用的代谢产物进行了组分及性质研究。代谢产物通过一系列的硅胶柱层析、Sephadex LH-20凝胶柱层析、RP-18反相柱层析等方法,提取分离了具有紫外吸收作用的胞外小分子代谢产物,并利用LC-TOP-MS、GC-MS、1H NMR、13C NMR等技术分析组成和结构。Pantoea agglomerans KFS-9胞外代谢产物中具有高光稳定性的紫外吸收物质为一系列溶于2,3-丁二醇的高不饱和芳香族色素类化合物(BAPs),该类化合物在290~340 nm的波长范围内能有效的吸收紫外光辐射。
从Pantoea agglomerans KFS-9发酵液中分离提取胞外多糖,并对其基本性质进行了初步的研究。确定泛菌胞外多糖的最佳提取条件为在70℃下旋转蒸发浓缩7倍体积后,加入3倍乙醇沉淀多糖。根据确定的工艺从Pantoea agglomerans KFS-9菌株发酵液中提取得到水溶性胞外多糖。采用5%三氯乙酸(TCA)除蛋白, Q-Sepharose Fast Flow阴离子交换柱层析及Sephacryl S-300HR凝胶柱层析进一步分离纯化,经Sephacryl S-300HR凝胶柱层析证实,获得均一较好的成团
One UV-B tolerance strain that its extro cellular metabolites can protect it from UV-B irradiation is isolated using the two-step isolation method. 263 Strains are isolated from the mangrove forest marine environment in Shenzhen after repeating enrichment and selection. 5 Strains with high UV-B tolerance are selected from the 263 strains. After selected according to the protection of metabolites against UV-B irradiation, strain KFS-9 is selected as the aim strain. The isolate is identified as Pantoea agglomerans by 16S rRNA gene sequence analysis and conventional identification method, and its accession number in Genbank is DQ350072. A mutant strain KFS-98 that lack of carotenoids gene is obtained, and its tolerance to UV-B irradiation is no obvious change vs. wild-type strain. That indicates that carotenoids have no active contribution to the tolerance of cells against UV-B.
In order to study the contribution of small molecular metabolites on the anti-ultraviolet ability, the component and property of UV-B absorbing metabolites from Pantoea agglomerans are studied. From the cell free supernatant of cultures, the extro cellular metabolites with UV absorbing ability are extracted and separated. Their components and structures are analyzed by LC-TOP-MS, GC-MS, 1H NMR, 13C NMR. A series of high unsaturated aromatic pigments dissolved in 2,3-butanediol (BAPs) are obtained from the extro cellular metabolites of strain Pantoea agglomerans KFS-9, which effectively absorb UV irradiation range from 290~340 nm, and have high light stability.
The exopolysaccharides from Pantoea agglomerans (PAEPS) is obtained and its basis characteristics are studied elementarily. The suitable procedure for extraction
为考察胞外小分子代谢产物对菌体抗辐射性的作用,对成团泛菌胞外具有紫外吸收作用的代谢产物进行了组分及性质研究。代谢产物通过一系列的硅胶柱层析、Sephadex LH-20凝胶柱层析、RP-18反相柱层析等方法,提取分离了具有紫外吸收作用的胞外小分子代谢产物,并利用LC-TOP-MS、GC-MS、1H NMR、13C NMR等技术分析组成和结构。Pantoea agglomerans KFS-9胞外代谢产物中具有高光稳定性的紫外吸收物质为一系列溶于2,3-丁二醇的高不饱和芳香族色素类化合物(BAPs),该类化合物在290~340 nm的波长范围内能有效的吸收紫外光辐射。
从Pantoea agglomerans KFS-9发酵液中分离提取胞外多糖,并对其基本性质进行了初步的研究。确定泛菌胞外多糖的最佳提取条件为在70℃下旋转蒸发浓缩7倍体积后,加入3倍乙醇沉淀多糖。根据确定的工艺从Pantoea agglomerans KFS-9菌株发酵液中提取得到水溶性胞外多糖。采用5%三氯乙酸(TCA)除蛋白, Q-Sepharose Fast Flow阴离子交换柱层析及Sephacryl S-300HR凝胶柱层析进一步分离纯化,经Sephacryl S-300HR凝胶柱层析证实,获得均一较好的成团
One UV-B tolerance strain that its extro cellular metabolites can protect it from UV-B irradiation is isolated using the two-step isolation method. 263 Strains are isolated from the mangrove forest marine environment in Shenzhen after repeating enrichment and selection. 5 Strains with high UV-B tolerance are selected from the 263 strains. After selected according to the protection of metabolites against UV-B irradiation, strain KFS-9 is selected as the aim strain. The isolate is identified as Pantoea agglomerans by 16S rRNA gene sequence analysis and conventional identification method, and its accession number in Genbank is DQ350072. A mutant strain KFS-98 that lack of carotenoids gene is obtained, and its tolerance to UV-B irradiation is no obvious change vs. wild-type strain. That indicates that carotenoids have no active contribution to the tolerance of cells against UV-B.
In order to study the contribution of small molecular metabolites on the anti-ultraviolet ability, the component and property of UV-B absorbing metabolites from Pantoea agglomerans are studied. From the cell free supernatant of cultures, the extro cellular metabolites with UV absorbing ability are extracted and separated. Their components and structures are analyzed by LC-TOP-MS, GC-MS, 1H NMR, 13C NMR. A series of high unsaturated aromatic pigments dissolved in 2,3-butanediol (BAPs) are obtained from the extro cellular metabolites of strain Pantoea agglomerans KFS-9, which effectively absorb UV irradiation range from 290~340 nm, and have high light stability.
The exopolysaccharides from Pantoea agglomerans (PAEPS) is obtained and its basis characteristics are studied elementarily. The suitable procedure for extraction
引文
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