糖苷水解酶通过催化碳水化合物中糖苷键的水解或糖苷转移，在生物体中扮演重要角色，也在发酵和制药等工业领域有巨大应用潜力，一直是酶学基础和应用研究关注的重点。迄今，糖苷水解酶共被划分为129个不同家族，其催化机制可分为保留和反转两种类型，同一家族酶一般只采用其中一种催化类型。特异的是，糖苷水解酶GH97家族同时拥有保留型和反转型成员，且表现出其它家族成员少见的Ca2+依赖性，这暗示其可能在糖苷水解酶进化中拥有特殊地位。不过，GH97家族的两种催化类型是如何分化而来？Ca2+是否在其中扮演重要角色？这些问题都有待解答。本项目拟以课题组前期发现的一个GH97家族新成员Pagl1为研究对象，通过理性设计和定点突变构建系列突变体，并对野生酶和突变酶的催化机制和Ca2+依赖性的生化及结构基础进行研究，为以上问题的解答提供依据，进而帮助揭示GH97家族的进化机制，也为相关糖苷水解酶的改造和应用奠定基础。

Glycoside hydrolases (GHs) catalyse hydrolysis of glycoside or transfer of aglycon in carbohydrate. They play important roles in organisms, and have significant application potential in industries such as fermentation and medicine, thus have been extensively studied. Glycoside hydrolases are classified into 129 families, and their catalytic mechanisms could be divided into two groups: retaining and inverting. Any of the GH families contain only one of the two mechanisms, except for GH97, whose members adopt either of them. Moreover, the activities of GH97 members are Ca2+ dependent, which is rare for other GH families. These indicate GH97 occupies a special position in GH evolution. However, how the two mechanisms developed and what role Ca2+ plays in it are unclear yet. To answer the questions, we plan, by rational design, site-directed mutagenesis, enzymatic and structural characterization of an inverting and Ca2+ independent GH97 member Pagl1, to investigate the biochemical and structural bases of catalytic mechanism divergency and Ca2+ dependencies of GH97 enzymes. Our results will contribute to reveal the evolution mechanism of GH97 family, and provide ground to engineering relevant GHs to improve their application potential.