肠道微生物来源昆布多糖降解酶催化机制及其产物构效关系

昆布多糖因其丰富的功能活性而备受关注，其降解而成的不同聚合度昆布寡糖具有更加显著的营养功能与应用价值。因此开展特定聚合度昆布寡糖的酶法制备及构效关系研究具有重要意义。目前国内外主要集中于昆布多糖降解酶的挖掘及表征研究，对于酶的催化机制及其产物构效关系的研究则鲜有报道。申请人前期在人体肠道微生物中挖掘了多个昆布多糖降解酶基因，并进行了功能验证。在此基础上，申请人拟阐明肠道微生物来源昆布多糖降解酶的催化机制及其产物构效关系。首先，继续表达10个以上肠道微生物来源昆布多糖降解酶，解析其酶学特性与底物降解模式；然后，基于酶的结构分析结合定点突变、分子对接，揭示其生成昆布寡糖的催化机制；最后，实现不同聚合度昆布寡糖的酶法制备，阐明其在调节肠道菌群与降低肠道炎症中的构效关系。研究结果对于突破昆布寡糖的酶法绿色制备技术、阐明肠道微生物在海洋多糖降解中的作用机制、丰富海洋特征寡糖功能信息具有重要意义。

Laminarin attracts much attention owing to its rich functional activities. Laminarin oligosaccharides with specific degree of polymerization (DP) present more significant nutritional function and application value than laminarin. Therefore, it is of great significance to study the enzymatic preparation of laminarin oligosaccharides with specific DP and elucidate the structure-activity relationships of laminarin oligosaccharides in different DP. At present, it mainly focuses on the mining and characterization of laminarinase. However, the catalytic mechanism of the laminarinase and the structural-activity relationship of laminarinase-catalyzed products in different DP remain unclear to date. In our previous study, it was found that there are abundant resources of laminarinases in human gut microbiota and the catalytic function of laminarinase OUC-L1 has been demonstrated. The program aims to illustrate the catalytic mechanism of the laminarinases from human gut microbiota and the structural-activity relationship of laminarin oligosaccharides in different DP. First, based on our previous research on the laminarinases from human gut microbiota, more than 10 genes of laminarinases from human gut microbiota will be further cloned or synthesized, expressed, purified, and characterized. Second, the structures of the laminarinase and the complex of enzyme-substrate will be solved. Meanwhile, the catalytic mechanism of laminarinase will be illustrated by structural analysis, mutational analysis, and molecular docking. Finally, laminarin oligosaccharides with different DP will be prepared by laminarinase, and their effects on gut microbiota composition and inflammation after antibiotic treatment will be explored. This study will be of significant in the establishment of enzymatic technologies for the preparation of marine laminarin oligosaccharides with specific DP as well as the elucidation of the mechanism of human gut microbiota in the degradation of marine polysaccharides. Meanwhile, this study will enrich the library of marine oligosaccharides functionality information.