α-新琼二糖水解酶生成奇数琼寡糖的催化机制和产物构效关系研究

项目名称： α-新琼二糖水解酶生成奇数琼寡糖的催化机制和产物构效关系研究

项目编号： No.31471607

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 毛相朝

作者单位： 中国海洋大学

项目金额： 88万元

中文摘要： 琼胶寡糖因具有多种营养功能，在食品领域有广泛应用。琼胶寡糖聚合度对其营养功能和应用范围有显著影响，因此酶法制备特定聚合度琼胶寡糖并阐明其构效关系具有重要意义。目前报道主要集中于β-琼胶酶制备新琼寡糖，较少关于α-琼胶酶制备琼寡糖及催化机制的报道，更未见奇数琼寡糖构效关系的研究。项目组首次从Agarivorans gilvus中克隆并表达了α-新琼二糖水解酶，分析了酶学性质并进行了同源建模。在此基础上，拟对该酶和酶底二元复合物进行蛋白结晶，采用基于分子模拟和理论计算的微观认知手段结合宏观实验学研究揭示其降解不同聚合度偶数新琼寡糖生成奇数琼寡糖的底物结合机制和催化机理；通过理性设计获得针对不同聚合度底物催化效率提高的和底物谱扩增的人工设计酶；进而实现特定聚合度奇数琼寡糖的定向制备，并研究其构效关系。本研究对于建立特定聚合度海洋寡糖的酶法制备技术、丰富海洋特征寡糖库和产物功能信息具有重要意义。

中文关键词： α-新琼二糖水解酶；奇数琼寡糖；催化机制；理性设计；构效关系

英文摘要： Agar oligosaccharides have potential applications in food, pharmaceutical, and cosmetic industries owing to their physiological and biological activities. Degree of polymerization (DP) of agar oligosaccharides has a significant effect on their nutritional function and range of applications. More and more studies have been focused on preparing agar oligosaccharides with specific DP by enzymatic methods and elucidating the structure-activity relationships of the agar oligosaccharides in different DP. In our knowledge, β-agarases are relatively well studied, and much mechanistic and structural insight into the cleavage of the β-bond in agarose has been gained. However, comparatively little is known about the α-agarases and the structure-activity relationship of their products which are odd agaro-oligosaccharides. In this study, a new α-neoagarobiose hydrolase, AgaWH117, was cloned and expressed from Agarivorans gilvus in our group, and the enzymatic character and homology modeling were studied. On these basis obtained, we will get the protein crystallizations of AgaWH117 and the binary complex of enzyme-substrate. Combinding dynamic simuliation, theoretical arithmetic and experimental study in the microcosmic and macroscopic scopes, we will reveal the catalytic mechanism and the binding mechanism of AgaWH117 with different substrates. What's more, through the rational design and mutational analysis, artificial enzymes will be obtained to catalyze the substrates (neoagaro-oligosaccharides) to produce odd agaro-oligosaccharides in different DP with higher catalytic efficiency. Also, the spectrum of the substrates will be amplified in this work. With the novel artificial enzymes obtained in this research, odd agaro-oligosaccharides in different DP would be prepared directionally and efficiently, and their structure-activity relationship would also be studied. Based on this research, we will establish the enzymatic technologies for the preparation of marine oligosaccharides with specific DP, and enrich the marine oligosaccharides library and the product functionality information.

英文关键词： α-Neoagarobiose Hydrolase;Odd Agaro-oligosaccharides;Catalytic Mechanism;Rational Design;Structure-activity Relationship