β-半乳糖苷酶转糖苷活性的功能进化与调控机制研究

项目名称： β-半乳糖苷酶转糖苷活性的功能进化与调控机制研究

项目编号： No.31301523

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 董艺凝

作者单位： 滁州学院

项目金额： 23万元

中文摘要： 低聚半乳糖以其特有的功能性，成为新型食品添加剂。但其生产量却一直受到生物催化剂β-半乳糖苷酶合成能力的制约。本项研究针对β-半乳糖苷酶转糖苷活性低、存在逆向水解催化，导致低聚半乳糖合效率受限这一共性瓶颈问题，拟通过针对功能差异位点的分子改造优化催化剂性能，并对其双向反应的分子调控机制进行探讨。研究拟依据低聚半乳糖合成能力差异，对现有糖苷酶数据资源进行分类汇总。以具有共性催化特性的家族为单位，通过进化历程分析探索其功能分歧产生的氨基酸位点。针对糖苷酶TIM保守催化结构域内的功能分歧位点进行体外体外改造，结合"转糖苷-水解"双向催化活性筛选，以期达到优化低聚糖合成能力的目标。在功能分歧位点改造与双向筛选结果基础上，对反应过渡态进行分析，探索β-半乳糖苷酶低聚半乳糖合成与水解反应的分子调控机制，为开发新型低聚半乳糖生物催化剂提供理论指导。

中文关键词： β-半乳糖苷酶；转糖苷活性；低聚半乳糖；功能进化；调控机制

英文摘要： Galactooligosaccharide as the new food additive, owns unique functionality. However, the yield in galactooligosaccharide is limited by the synthetic capability of biocatalyst β-galactosdase. The low transgalactosylation activity and the reverse-hydrolysis reaction associated with transgalactosylation are the common disadvantage for production galactooligosaccharide，which limited by the synthesis efficiency. The objects of this study are the function optimization of transgalactosylation activity, based on the evolution divergences modification and the research on the regulation mechanism of bidirectional catalytic reaction. According to the differences of the capability on galactooligosaccharide production, the glycosidase will be classified in this study. The amino acid sites, where the evolution divergences occur, will be research using the family as the unit for evolution analysis. The amino acids in the TIM domain are the targets for molecular evolution. The optimization mutants could gain through bidirectional screening. The regulation mechanism of bidirectional catalytic reaction could be studied based on the result of molecular modification and transient analysis. The research result of this study could direct the development of new biocatalyst using for galactooligosaccharide production.

英文关键词： β-galactosidase；transgalactosylation activity；galactooligosaccahride；function evolution；regulation mechanism