The Development of Interface Molecules for Enzyme Electrochemical Reaction

在大多数氧化还原酶中，假基深埋在蛋白质壳内。因此，很少遇到在电极表面对酶的假基进行直接的电化学再循环，从而产生相应的电流信号。为了提高酶传感器的信号性能，我们重点研究了利用电子转移蛋白、细胞色素作为氧化还原酶氧化还原中心与电极表面之间的分子界面。在自然界中，含有PQQ或FAD的各种脱氢酶通过细胞色素与呼吸链偶联。基于这种电子转移特性，我们考虑利用细胞色素作为界面分子促进酶与人工电子介质或电极之间的电子转移。本课题主要研究细胞色素b_<562>作为界面分子在新型生物电子器件和工艺中的应用和工程化。研究了细胞色素b_<562>的存在对各种氧化还原酶的影响，并通过观察这些组合对传感器电化学响应的改善，证实了其作为界面分子的性质。然后尝试了细胞色素b_<562>的工程制备。通过修饰PQQ葡萄糖脱氢酶的表面电荷或引入新的生物亲和配体来提高对PQQ葡萄糖脱氢酶的亲和性。利用工程界面分子与PQQGDH发生生物电化学反应，从而提高了传感器的性能。最后，尝试利用界面分子构建PQQ葡萄糖脱氢酶与细胞色素_<562>共表达的新工艺。PQQ葡萄糖脱氢酶和细胞色素_<562>的共表达，在没有人工合成电子介质的情况下，构建了能直接与电极传递电子的工程大肠杆菌细胞。结果表明，利用在体内表达的界面分子，构建了一种新的生物电化学过程。少

In most of oxidoreductases, the prosthetic group is buried deeply and within the protein shell. Thus a direct electrochemical recycling of the enzyme's prosthetic group at the electrode surface leading to a corresponding current signal is rarely encountered. In order to improve enzyme sensor signal property, we focused on the application of electron transfer proteins, cytochromes as the molecular interface between oxidorcductase redox center and electrode surface. In nature, various dehydrogenases harboring either PQQ or FAD are coupled with the respiratory chain via cytochromes. Based on this electron transfer property, we considered the utilization of cytochrome as the interface molecule to facilitate the electron transfer between enzyme and the artificial electron mediator or the electrode in the enzyme based biosensor. In this project, we focused on the application and engineering of cytochrome b_<562> as the interface molecule for novel bio-electronic devices and processes employi … More ng various oxidoreductases. The impact of the presence of cytochrome b_<562> on the various oxidoreductases was investigated, and its properties as the interface molecule were confirmed, by observing improved electrochemical response in the sensors employing these combinations. The engineering of cytochrome b_<562> was then attempted. The improvement of affinity toward PQQ glucose dehydrogenase was achieved by either modifying its surface charge or by introducing novel bio-affinity ligands. The employment of engineered interface molecules resulted in the bioelectrochemical reaction with PQQGDH, consequently improved the sensor properties. Finally, the co-expression of PQQ glucose dehydrogenase and cytochromeb_<562> was attempted to construct novel process employing interface molecules. The co-expression of PQQ glucose dehydrogenase and cytochromeb_<562> resulted in the construction of engineered E.coli cell which was able to transfer electron directly with electrode in the absence of artificial synthetic electron mediators. The results suggested the construction of novel bio-electrochemical process employing in vivo expressing interface molecules. Less