鞘氨醇胶Ss酸沉分子机理的研究

Sphingan Ss, a novel biopolymer with excellent properties of thickening, gelling and emulsification, is produced by a new species of the genus Sphingomonas. Sphingan Ss can be precipitated after the fermentation broth is adjusted to pH 3.0. In contrast to the extraction with organic solvent, extraction with acid is distinct character of sphingan Ss, which significantly decreases the cost of production. It was found in the previous studies that Ss can not be precipitated with acid after protease digestion. Meanwhile, a 14 kDa protein fragment was found on SDS-PAGE. Taking this protein fragment as the starting point, this project intends to illustrate the molecular mechanism of Ss precipitated with acid. By the method of amino acid sequence analysis and gene knockout, protein-coding gene and glycoprotein ligase-coding gene can be found. The sites of carbohydrate chain bonding with protein will also be studied. Moreover, the research can provide theoretical and experimental foundation for changing the current situation of other sphingans must be precipitated with organic solvent.

鞘氨醇胶Ss是由鞘氨醇单胞菌属新种合成的一种新型生物聚合物，具有优良的增稠、胶凝和乳化性能，是一个具有原始创新的微生物胶。调节发酵液pH至3.0, 鞘氨醇胶Ss即可从发酵液中沉淀出来,与其它鞘氨醇胶所采用的有机溶剂沉淀法提取相比，酸沉提取能够显著地降低生产成本。本课题旨在解析其酸沉提取的分子机理：前期研究发现，经蛋白酶处理后鞘氨醇胶Ss的酸沉提取特性消失，同时得到一个约14kDa的蛋白片段；本研究以此蛋白片段为出发点，通过氨基酸序列分析和基因敲除等方法，获得鞘氨醇胶Ss的蛋白链编码基因和糖蛋白连接酶编码基因，结合鞘氨醇胶Ss糖蛋白连接位点解析，阐明这种新型鞘氨醇胶能够酸沉提取的分子机理，为改变其它鞘氨醇胶有机溶剂提取现状提供理论和实验依据。

鞘氨醇胶Ss是由鞘氨醇单胞菌属新种合成的一种新型生物聚合物，具有优良的增稠、胶凝和乳化性能，是一个具有原始创新的微生物胶。调节发酵液pH至3.0, 鞘氨醇胶Ss即可从发酵液中沉淀出来，与其它鞘氨醇胶所采用的有机溶剂沉淀法提取相比，酸沉提取能够显著地降低生产成本，本项目从聚合物结构和基因角度解析了鞘氨醇胶Ss酸沉的分子机理。研究结果表明，先对鞘氨醇胶Ss样品进行碱解和纤维素酶处理，进而用蛋白酶处理，可分离得到样品的蛋白组分，根据目的蛋白氨基酸序列信息，结合鞘氨醇胶Ss合成菌的基因组比较分析，克隆得到1704 bp的鞘氨醇胶Ss蛋白链编码基因，其编码蛋白的等电点为4.7，与该基因相邻的orf0915显示糖基转移酶活性，与鞘氨醇胶Ss糖蛋白连接有关。经氢谱、碳谱和HMBC等二维核磁谱分析，确定了鞘氨醇胶Ss糖链连接顺序为-4-Man-1-4-GlcA-1-3-Rha-1-3-Glc-1-，蛋白组分通过Asn与多糖组分的Rha形成N-连接。鞘氨醇胶Ss酸沉分子机理的解析，将为其他微生物多糖的菌种改良和提取方法改进提供理论依据。