鞘糖脂糖苷合成酶可以催化氟化寡糖与鞘氨醇合成溶血鞘糖脂，在鞘糖脂类药物的酶法合成中具有重要应用潜力。但由于目前对于糖苷合成酶的催化和活性调节机制了解不充分，构建新的高效糖苷合成酶或对现有酶进行分子改造难度较大。在前期研究中，我们克隆了具有高活性和宽泛底物选择性的鞘糖脂内切糖苷酶、解析了其晶体结构、将其突变为鞘糖脂糖苷合成酶，并建立了基于单细胞微反应器的酶活性超高通量筛选方法。在此基础上，本项目拟构建该酶所有氨基酸位点的全局饱和突变库，结合高通量筛选和二代测序技术对酶进行“序列-结构-功能”关系的全面分析；进而依据结构信息提取对酶功能有正向作用的突变位点进行合理重组，快速构建具有高活性、宽底物谱的新型糖苷合成酶。本项目将有助于深入理解该酶的催化机制，并为建立新的高效酶分子改造技术体系奠定基础。

Endoglycoceramide glycosynthases is an artificial enzyme derived from the catalytic nucleophilic residue mutant of endoglycoceramide glycosidase (EGC) in GH5 family. It shows enormous potential in the enzymatic synthesis of glycosphingolipid medicines. However, the application of EGC glycosynthases is subject to several limitations, such as low catalytic activity and stringent substrate specificity. The design and optimization of more efficient EGC glycosynthase is hampered by the limited understanding of the catalytic mechanism of this class of enzymes. Recently, we cloned a new EGC endoglycosidase with high activity and broad substrates specificity, solved its crystal structure, and converted it into an EGC glycosynthases. Also we established an ultra-high throughput screening technology (>10^7 clones per day) for both the endoglycosidase and glycosynthase activities. In this project, we aim to reveal the “sequence-structure-function” relationships of EGC glycosynthases by combineing the ultra-high throughput screening technology and next generation sequencing technology through the analysis of overall single site-saturated libraries of the enzyme. Optimize mutants with high activity and broad substrate spectrum will be rapidly obtained by recombination of positive mutations guided by the structure-function relationship information. This project will be helpful to reveal the catalytic mechanism of glycosynthases, and can also lay the foundation for the establishment of novel, high-efficiency enzyme engineering systems.