PL7家族褐藻胶裂解酶底物偏好性关键氨基酸的作用机制及定向改造

褐藻胶寡糖及其衍生物在免疫调节、降血脂等方面表现出显著的生物活性，而单糖组成对其生物活性和营养功能具有决定性作用。因此，利用不同底物偏好性的褐藻胶裂解酶制备不同组分的褐藻胶寡糖具有重要的应用意义，而明确酶底物偏好性关键氨基酸是科学指导获得不同底物偏好性褐藻胶裂解酶的理论基础。申请者前期从海洋细菌Microbulbifer sp. Q7中获得了一种热稳定性PL7家族褐藻胶裂解酶，该酶可作用于聚甘露糖醛酸（polyM）和聚古洛糖醛酸（polyG）且具有polyG偏好性。本项目拟对该酶和不同酶底二元复合物进行结晶，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，挖掘影响酶底物偏好性的关键氨基酸、阐明其作用机制，并通过定向改造获得可定向制备特定单糖组成褐藻胶寡糖的褐藻胶裂解酶，丰富海洋酶资源库，对褐藻胶裂解酶催化机制的完善和酶法定向制备褐藻胶寡糖产业的发展具有重要意义。

Alginate oligosaccharides and their derivatives show significant bioactivity in immunomodulatory and hypolipidemic. However, the monosaccharide compositions are essential for their biological activity and nutritional function. Therefore, the use of alginate lyases with different substrate preferences to prepare specific alginate oligosaccharides has important application significance, and the discovery of key amino acids for enzyme substrate preferences is the scientific guidance to obtain alginate lyase with different substrate preferences. The applicant previously obtained a thermostable PL7 family alginate lyase from the marine bacteria Microbulbifer sp. Q7, which is a bifunctional enzyme with a preference for guluronic acid. In this project, we will acquire the protein crystallizations of cAlyM and the binary complex of enzyme-substrate, explore the key amino acids for enzyme substrate preference and clarify its action mechanism by molecular simulation, theoretical arithmetic and experimental study. Furthermore, alginate lyase that can directionally prepare a specific monosaccharide composition alginate oligosaccharide will be obtained through directed evolution. The study is of great significance to the elucidation on the catalytic mechanism of alginate lyase and the development of the enzymatic directional preparation of alginate oligosaccharide.