新属种来源新型琼胶酶构效关系的研究与应用

Agarases are glycoside hydrolases. Agarases have potential applications in the food, cosmetic and medical industries for the production of neoagarooligosaccharides from agar. It has been proved that oligosaccharides with different degree of polymerization (DP) has different biological activities and applied value. So it was important to obtain specific DP oligosaccharides by agarases. An effectively agar-degrading strain, HQM9, which was isolated from the surfaces of red algae, that represents a novel species in the family Flavobacteriaceae. Based on genomic sequencing of HQM9 and mass spectrometric analysis of extracellular agarase, two novel agarase with different product compositions were chosen as the study subject. The aims of this project are defining the glycoside hydrolases family of two novel agarases by sequence analysis. To study the relationship of function and structure of agarases, main motif of two agarases were expressed effectively in prokaryonic expression system. The three-dimentional strcutural data could be obtained by homology modeling. The key residues were analyzed and exchanged between two genes. The functional difference of mutants and changing patterns of product DP were analyzed. The significance of the project：The catalytic mechanism of two novel agrases （AgaP, AgaW） were approached by comparing analysis of two gene structure. The results made a base for obtaining specific DP oligosaccharides by agarases. It was significant to the theoretical research and practical applications.

琼胶酶降解琼脂生成的琼寡糖在食品、化妆品和医药行业有着广泛的应用，而不同聚合度的琼寡糖生物活性和应用价值差异较大。目前常用的化学水解法很难获得聚合度特定或均一的琼寡糖，大大限制了其深度应用。本项目拟基于前期课题组分离的高效降解琼胶的黄杆菌科新属种HQM9进行的基因组测序和胞外琼胶酶质谱分析，分别挖掘出两条水解产物组成不同的新琼胶酶基因AgaP和AgaW，在此基础上开展两基因序列和蛋白质结构模拟分析，结合酶水解特性确定其各自所属的糖水解酶家族及主要功能模块；通过比较分析各主要功能模块杂合和融合后的水解产物变化的规律，揭示决定琼胶酶水解聚合度的主要结构域；通过理性改造功能结构域阐明新型琼胶酶AgaP和AgaW的作用机制，并开发水解琼胶聚合度可控的琼胶酶设计策略。研究结果为实现特定聚合度琼胶寡糖的酶法制备调控奠定基础，具有重要的理论意义和应用价值。

琼胶酶降解琼脂生成的琼寡糖在食品、化妆品和医药行业有着广泛的应用，而不同聚合度的琼寡糖生物活性和应用价值差异较大。目前常用的化学水解法很难获得聚合度特定或均一的琼寡糖，大大限制了其深度应用。. 本项目以来源于高效降解琼胶的黄杆菌科新属种HQM9的两条水解产物组成不同的新琼胶酶基因AgaP和AgaW为研究对象，开展两条基因序列和蛋白质结构模拟分析，确定了其各自所属的糖水解酶家族分别为GH42和GH16。对两条基因进行基因结构分析，确定了4个主要功能模块，研究了每个功能模块的酶学特性及产物降解模式，明确了与产生新琼二糖有关的功能模块CMP和NMW。构建了琼胶酶基因AgaP和AgaW的同源模型，根据获得的三维结构模型，结合重组功能模块的功能比较分析，构建了用于研究功能模块与产物聚合度相关性的4个置换体，通过比较分析各主要功能模块杂合和融合后的水解产物变化的规律，揭示决定琼胶酶水解聚合度的主要功能模块。结合TLC、MALDI-TOF质谱分析，证实置换体EX1降解琼胶时的终产物为新琼二糖，酶切方式为从琼胶的末端开始裂解的外切琼胶酶。以不同聚合度琼胶寡糖为底物，进行置换体EX1酶解的最小底物分析，结果表明：置换体EX1的最小降解单元为新琼四糖；置换体EX3是以新琼四糖为终产物的内切琼胶酶，能够降解的最小单元为新琼六糖。确定了功能模块P1和W1对于酶降解琼胶生成不同聚合度琼胶寡糖具有重要作用，其中功能模块P1对于产生单一聚合度的新琼二糖具有锚定性。课题组还拓展地研究了HQM9 来源的与琼胶酶基因AgaP、AgaW一级结构相似的两条琼胶酶基因AgaN、Aga9，分别对其进行基因克隆表达、酶学性质及酶解底物特异性研究，为AgaP、AgaW基因建模及比较研究提供理论数据。. 本项目通过理性改造功能结构域，明确决定琼胶酶酶解产物聚合度的功能位点，阐明新型琼胶酶AgaP和AgaW的作用机制，为开发水解琼胶聚合度可控的琼胶酶提供设计策略。研究结果为实现特定聚合度琼胶寡糖的酶法制备与调控奠定基础，具有重要的理论意义和应用价值。