一种新的葡萄糖脱氢酶FAD-GDH的晶体结构与催化机制研究

植入式医疗设备在临床诊断和治疗中的应用越来越普遍，但其电池的局限严重影响了它的进一步发展。酶燃料电池能利用体内的葡萄糖氧化供能，理论上可以实现医疗设备的永久性植入，是最有发展前途的一种电池。这种电池的主要元件之一是各种氧化还原酶。其中，FAD型葡萄糖脱氢酶（FAD-GDH）稳定性好、底物专一性高、催化效率高，综合性能优于葡萄糖氧化酶或其它的各种葡萄糖脱氢酶；但FAD-GDH很难实现重组表达，特别是在大肠杆菌中。到目前为止，FAD-GDH的结构信息和催化机制都尚未阐明。本课题拟从一个土曲霉来源的FAD-GDH出发，通过结合多种表达和纯化策略，建立一个简单有效的表达纯化方案，来大量制备高纯度的FAD-GDH进行酶学性质测定和蛋白质结晶。通过测定FAD-GDH的母体和复合物晶体结构，结合相关突变体的酶学性质数据，来阐明FAD-GDH的催化机制，为深入了解和合理开发利用FAD-GDH奠定结构基础。

Implantable medical devices are applied with increasing frequency in field of clinical diagnosis and treatment, but the limits of their batteries impair the further application of them. Enzymatic fuel cells (EFCs) can offer the power by oxidizing glucose in human body, which can realize permanent implantation of medical devices in theory and they are thought to be the most promising batteries. All kinds of REDOX enzymes make up one of the main components in EFCs..In all common used REDOX enzymes, FAD-dependent glucose dehydrogenases (FAD-GDHs) are endowed with good thermostability, high substrate specificity and high catalytic efficiency, of which are superior to those of glucose oxidases and other glucose dehydrogenases. However, FAD-GDHs are hard to recombinant expression, especially in Escherichia coli. There is no structural information of FAD-GDHs available and the catalytic mechanism of which is unclear either till now. Here the program focuses on the FAD-GDH from Aspergillus terreus. By combining several methods of expression and purification, a simple and effective protocol will be set up to produce large amounts of FAD-GDH with high purity. The purified FAD-GDH will be used to enzymatically mensuration and crystallization. After getting the structural information of FAD-GDH and its compound, mutation in FAD-GDH will be carried out to make sure the special functions of some amino acids. Combined the structural information and enzymatic data from mutant FAD-GDHs, the catalytic mechanism of FAD-GDH will be clarified. The result of the program will pays the way for further understanding, reasonable development and utilization of FAD-GDH.