齐整小核菌内源小核菌胶水解酶的挖掘及其功能验证

Discovery and functional verification of endogenous glucanases for scleroglucan hydrolysis in Sclerotium rolfsii

摘要:

小核菌胶是一种高分子水溶性微生物胞外多糖，主要用于石油开采、食品加工、医药制造和化妆品等领域。由微生物发酵法生产的小核菌胶分子量较高，存在溶解性低、粘度高和分散性差等问题，对提取、保存和应用带来了一系列困难。研究用于改性小核菌胶分子量的降解方式对扩大小核菌胶的工业应用具有重要意义。文中以齐整小核菌Sclerotium rolfsii WSH-G01为对象，通过基因组测序从全基因组范围内对菌株葡聚糖降解酶基因进行功能注释，设计可能引起β-葡聚糖酶差异表达的培养体系，结合转录组学分析，挖掘菌株内源β-葡聚糖酶，对可能的多糖水解酶进行功能验证。将获得的14个可能的内源小核菌胶水解酶基因进行扩增，在毕赤酵母Pichia pastoris中进行异源表达，经SDS-PAGE验证和酶活测定，得到10个可溶性蛋白，其中5个具有昆布多糖水解活性。这5个内源水解酶中，GME9860具有对小核菌胶的酶解效果。研究结果为应用酶水解法获取中低分子量小核菌胶以及代谢工程改造调控齐整小核菌发酵生产不同分子量小核菌胶提供了参考。

关键词:齐整小核菌，硬葡聚糖，葡聚糖酶，分子量，多糖水解

Abstract:

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.

Key words:Sclerotium rolfsii, scleroglucan, glucanase, molecular weight, polysaccharide hydrolysis