糖基转移酶CgtB底物特异性改造及其机理研究

Glycosyltransferases (GTs) play a critical role in chemoenzymatic synthesis of oligosaccharides and glycoconjugates. However, the successful applications of biocatalysts have been largely constrained by their inherent poor activity, specificity or stability. The CgtB,a bacterial β-1,3-galactosyltransferase, was used for galactosylation of GM1 ganglioside and synthesis of the O-linked T-antigen glycan. Previously, we have focused our research efforts on improvement of enzyme activity. By using directed evolution with fluorescence-activated cell sortor (FACS) high through screening, we successfully obtained a CgtB variant with a 300 fold higher catalytic efficiency. But the specificity of the CgtB variant to its natural substate (GalNAc) is poor, yielding several unexpected by-products, which limited the CgtB application in biocatalysts. Here, we plan to apply a modified a FACS method in directed evolution of CgtB to enhance its specificity and activity. Futhuremore, we can get an insight of the catalysis and substrate specificity mechanism by strucural biology study on CgtB.

糖基转移酶是酶法合成生物活性寡糖及糖基化合物的关键酶。但实际应用中，糖基转移酶往往受到其活性较低，底物特异性和稳定性较差的局限，对这类酶的分子改造一直受到广泛关注。CgtB是来自细菌的beta-1,3-半乳糖糖基转移酶，在合成GM1类神经节苷脂和肿瘤相关T抗原上具有重要的应用前景。先前，我们使用超高通量的FACS筛选方法，成功将该酶催化活性提高了近300倍。然而由于CgtB对受体底物GalNAc特异性较差，生成大量副产物，限制了其广泛应用。因此，本项目拟通过应用FACS筛选体系对不同荧光分子标记的底物分别创造正向及负向选择压力，实现大规模随机突变库的高通量筛选，从而获得底物特异性改善的突变酶。在此基础上，我们拟解析野生型和突变体CgtB的晶体结构，结合动力学数据揭示突变体性质变化的结构基础，以促进对糖基转移酶家族的催化机理和底物特异性识别机制深入理解，为酶分子改造提供科学依据。

糖基转移酶是酶法合成生物活性寡糖及糖基化合物的关键酶。实际应用中，糖基转移酶往往受到其活性较低，底物特异性和稳定性较差的局限。目前，针对来自细菌的 beta-1,3-半乳糖糖基转移酶CgtB ，我们通过酶学方法合成了适用于糖基转移酶的双荧光底物，并利用该类底物创造正向及负向选择压力，使用FACS 筛选方法实现3.6×107的CgtB随机突变库的高通量筛选，将CgtB 针对受体底物 GalNAc 特异性相对野生型提高了约21倍，从而获得底物GalNac特异性改善的突变酶。在此基础上，我们利用特异性活性改善的CgtB酶突变体合成了神经节苷脂GM1一系列衍生物，开展了GM在哺乳动物们mTORC1信号通路和激活免疫系统参与抑制格兰仕阳性细菌感染研究。研究中对酶分子稳定性，底物选择性等催化机制进行结构研究，力图为该家族酶分子改造和应用提供科学依据。