嗜酸乳杆菌GIM1.208β-葡萄糖苷酶高效释放刺梨槲皮素的底物结合模式及分子作用机制研究

Rosa roxburghii Tratt is the focus in the development of fruit resources with mountainous region characteristic in Guizhou. Our preliminary study found that β-glucosidase produced by L.acidophilusGIM1.208 can effectively induce the release of quercetin--the main aglycones in Rosa roxburghii Tratt，but the mechanism remains unclear. In this program,the recombinase was attained by cloning and heterologous expression of BGL gene from GIM1.208, then its specific substrate was clarified by analyzing enzyme activity and establishing the kinetic model. Meanwhile,the molecular structure combining with different substrates and different features of molecular interaction were revealed by determining the amino acid sequence, the dynamic scattering spectrum, the fluorescence spectrum and the circular dichroism. Finally,through the study of BGL homology model,the docking method for target substrate molecular using three-dimensional structure model and two-dimensional mapping, the relationship between BGL and the structure of active sites, active site, amino-acid residues, and the non-covalent interaction of different complexes were further clarified. Consequently, it will be elucidated that the substrate binding model and Interaction mechanisms of releasing effectively quercetin by BGL from Rosa roxburghii Tratt. This program has great significance for improvement of the flavonoid bioactivity from Rosa roxburghii Tratt and application of the probiotics, and provides guidance for the BGL functional modification and its downstream engineering as well.

刺梨是贵州重点发展的山地特色水果资源。项目前期研究发现，嗜酸乳杆菌GIM1.208产β-葡萄糖苷酶(BGL）对刺梨重要黄酮苷元—槲皮素具有高效释放能力，但相关机制尚不清楚。据此，本项目通过基因克隆外源表达获得GIM1.208BGL重组酶，进而建立BGL催化反应动力学模型，探明BGL对刺梨不同槲皮素糖苷底物识别特异性；采用DLS、FS、CD、MALDI-TOF-MS及分子序列分析，明确BGL催化活性结构、酶与不同底物识别的结构模式及分子间作用差异化表征；同时通过同源建模及分子对接三维结构模型与二维图谱研究，进一步揭示BGL及酶-底物结合的活性中心、活性位点、关键氨基酸残基及非共价作用与催化活性的相关性，最终阐明GIM1.208BGL高效释放刺梨槲皮素的底物结合模式及分子作用机制。该研究对刺梨功能挖掘提升及益生菌功能开发具有重要意义，同时为BGL下游工程研究提供必要的理论依据和指导。

项目根据计划书要求以及研究过程中的实际需要，完成了嗜酸乳杆菌GIM1.208β-葡萄糖苷酶（GIM1.208 BGL）的编码基因序列、理化性质、蛋白分子结构与性质分析；完成了刺梨槲皮素糖苷前体分离纯化鉴定、含量分布检测及生物转化特征分析；探讨了GIM1.208 BGL酶学特性及催化底物反应动力学；开展了GIM1.208 BGL与槲皮素糖苷分子相互作用原子力显微镜、荧光光谱变化及圆二色谱分析，同时，结合进行了GIM1.208 BGL与槲皮素糖苷相互作用的分子对接模拟分析应证。本研究首次阐明了来源于益生菌的嗜酸乳杆菌GIM1.208 BGL的分子结构与酶学特性，阐明了GIM1.208 BGL与槲皮素糖苷底物识别结合关系以及最适底物，揭示了酶蛋白受体与底物识别与结合的行为模式，以及活性中心结构特征与其催化作用关系。. 研究按计划开展相关研究工作，顺利完成了预定目标任务，公开发表相关论文8篇，其中SCI收录4篇，授权技术发明专利2件，培养研究生3名。项目研究结果和发现为后续研究工作提供了重要思路方向，成果为刺梨特色资源功能活性二次开发以及嗜酸乳杆菌BGL催化活性位点修饰和理性改造提供了基础理论依据和技术参考。

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