基于辛烯基琥珀酸淀粉酯的可降解仿生硒酶构筑及构效关系研究

As an important selenium-containing enzyme in human body, glutathione peroxidase plays an important role in removing the excess free radicals and treating the oxidative disease. Based on the similation of the catalytic behavior of native glutathione peroxidase, the construction of novel artificial selenoenzyme is one of the important sources of original innovation in the field of biological chemistry. However, the nondegradable property of the scaffold of traditional macromolecular artificial selenoenzyme has largely limited its application. The construction of a degrable artificial selenoenzyme has become one of the important and urgent scientific problems in the field of artificial selenoenzyme.Therefore, this project is intented to propose the simulation of the biological function of glutathione peroxidase as a guide and the construction of degrable artificial selenoenzyme as a target.To explore the innovative strategy for the construction of starch-based artificial selenoenzyme using octenyl succinic anhydride modified starch (OSA-starch) as raw material. Furthermore, the charactization techniques of FTIR、XRD、NMR、SEM and EDS were empolyed to explore these basic scientific problems, such as the structural features,catalytic properties and structure-activity relationships of the degrable artificial selenoenzyme, etc.And this project is aimed to optimize the preparation technology of starch-based artificial selenoenzyme,to investigate the influence of selenium modification on the structure of OSA-starch. Moreover, this project is aimed to clarify the influence mechanism of the structural changes of starch-based artificial selenoenzyme on its catalytic behavior, and to investigate the formation mechanism of hydrophobic microenvironment with special binding ability for hydrophobic sbstrates in the skeleton of starch-based artificial selenoenzyme. Significantly, the theory system of structure-activity relationship of the degrable artificial selenoenzyme would be established and the general regularity for the consturtion ofdegrable artificial selenoenzyme could be revealed. The successfully research of this project in future may provide the theoretical basis for the industrial applications of degrable artificial selenoenzyme.

谷胱甘肽过氧化物酶是人体内重要的含硒酶，对清除过量自由基、治疗氧化性疾病具有重要作用。模拟天然硒酶的催化行为，构筑新型仿生硒酶是生物化学领域进行原始创新的重要源头之一。传统大分子仿生硒酶骨架的难降解性对其应用具有较大限制，构筑可降解仿生硒酶已成为该领域亟需解决的关键科学问题之一。本课题以模拟谷胱甘肽过氧化物酶为导向，以构筑可降解仿生硒酶为目标，以辛烯基琥珀酸淀粉酯为原料，探索淀粉基仿生硒酶的构建策略；拟采用FTIR、XRD、NMR、SEM和EDS等测试手段，研究淀粉基仿生硒酶的结构特征、酶学性质和构效关系等基础科学问题；优化淀粉基仿生硒酶的制备工艺，研究硒化改性对辛烯基琥珀酸淀粉酯结构的影响规律，阐明淀粉基仿生硒酶结构变化对其催化活力的影响机制，探索仿生硒酶骨架中疏水微环境的形成机制，建立可降解淀粉基仿生硒酶构效关系的理论，揭示构筑可降解淀粉基仿生硒酶的一般性规律，为其产业化应用奠定基础。

谷胱甘肽过氧化物酶（GPx）是人体内重要的含硒抗氧化酶，模拟GPx催化行为，制备仿生GPx对开发抗氧化药物、治疗氧化性疾病具有重要意义。目前，除蛋白骨架以外大分子骨架仿生硒酶均是以不可降解的合成聚合物制备的，导致其在医疗、保健、功能食品等领域的应用受到极大限制。本项目分别以糯玉米淀粉和木薯淀粉酯化改性制备的辛烯基琥珀酸淀粉酯为原料，建立了制备淀粉基仿生GPx 的新策略，成功开发了糯玉米粉仿生硒酶Se-starch30和Se-starch80、基于木薯淀粉为材料开发的木薯基仿生硒酶Se-ca-starch和Se-ca-starch80。利用H1 NMR、EDS、XPS、SEM、XRD、FT-IR等表征证明了淀粉基仿生硒酶的成功制备，研究表明四种淀粉基仿生硒酶呈现出典型的饱和动力学催化行为和酶学催化行为，Se-starch30、Se-starch80、Se-ca-starch和Se-ca-starch80的最高催化活力为分别是经典小分子仿生GPx PhSeSePh的1.04×105、1.53×105、3.11×105和7.63×105倍。研究表明：NaSeH的强碱性和反应体系的碱性条件导致淀粉表面呈现更多带有褶皱和凹槽的风化状糊化作用结构，有利于形成疏水微环境和底物识别位点，进而使四种淀粉基仿生硒酶表现出较高的底物选择性和催化活力。对淀粉基仿生硒酶的催化机制研究表明，疏水微环境和底物识别位点对提升底物结合能力的贡献均比较重要，但是过强的底物识别位点结合作用和淀粉链段对催化中心的包埋作用会抑制催化活力的提高；GPx催化中心与疏水微环境、底物识别位点的有效匹配是维持淀粉基仿生硒酶的高催化活力重要因素。本工作实现了基于可降解天然高分子骨架的仿生GPx的高效制备，为解决传统大分子仿生硒酶骨架不可降解的问题提供了新思路。