清酒乳杆菌葡聚糖蔗糖酶的催化机制及功能研究

前期工作表明，清酒乳杆菌L3所产胞外多糖是一种只含有α-1,6糖苷键且存在吡喃型糖基残基的葡聚糖，在食品及医药领域展现出了良好的应用前景。基于前期转录组学结果和文献报道，葡聚糖蔗糖酶在L3葡聚糖合成过程中可能起着关键作用，然而葡聚糖蔗糖酶的催化机制及功能尚不清楚。本课题拟通过全基因组测序分析技术挖掘并确定该菌株合成葡聚糖蔗糖酶基因的数目。在此基础上，构建葡聚糖蔗糖酶基因的缺失及回补菌株，确定调控葡聚糖合成的关键葡聚糖蔗糖酶。随后利用分子模拟技术初步解析L3关键葡聚糖蔗糖酶与底物互作活性位点以及结合模式。利用截短和突变的方法对该酶催化机制进行研究。同时通过定向改造获得能够催化产生更具经济效益的低分子量葡聚糖的高性能酶及功能性葡聚糖产品。本课题不仅对阐明清酒乳杆菌L3葡聚糖蔗糖酶催化机制及功能具有重要意义，而且为定向改造葡聚糖蔗糖酶基因以制备不同类型葡聚糖产品奠定基础。

Previous work demonstrated that the exopolysaccharide produced by Lactobacillus sakei L3 is a dextran containing D-glucose residues with α-1,6 glycosidic linkages, and showed excellent application prospects in food processing and medicine. According to our transcriptome results and other studies, glucansucrase may play key roles in the synthesis of L3 dextran. However, the catalytic mechanisms and functions of the related enzyme(s) are still unknown. This project will utilize whole-genome sequencing to determine the number of glucansucrase in the L3 strain and construct deletion and complementation strains to identify the key glucansucrase responsible for dextran synthesis. Subsequently, the interaction sites and binding modes of the L3 key glucansucrase with the substrate will be preliminarily analyzed by molecular simulation technology. Additionally, truncations and point mutations of the key glucansucrase residues will be constructed to uncover its catalytic mechanism. Based on these mutated enzymes, functional dextran products and high performance enzymes which could yield low molecular weight dextran with excellent economic benefits can be obtained via directional modification. This project is not only of great significance for elucidating the catalytic mechanism and function of Lac. sakei L3 glucansucrase, but also lays a foundation for the targeted modification of the enzyme to yield different types of dextrans.