The Development of Interface Molecules for Enzyme Electrochemical Reaction

在大多数氧化还原酶中，辅基深埋在蛋白质壳内。因此，在电极表面的酶的辅基的直接电化学再循环导致相应的电流信号很少遇到。为了改善酶传感器的信号特性，我们重点研究了电子传递蛋白、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的应用。在自然界中，携带PQQ或FAD的各种酶通过细胞色素与呼吸链偶联。基于这种电子传递特性，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传递，在酶基生物传感器。在本项目中，我们重点研究了细胞色素B_作为新型生物电子器件和工艺的界面分子的应用和工程。<562> 关于我们 ng各种氧化还原酶。细胞色素B_的存在下对各种氧化还原酶的影响进行了研究，并确认其作为界面分子的性质，通过观察改善电化学响应的传感器采用这些组合。<562>然后尝试了细胞色素B_的工程化。<562>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，实现了对PQQ葡萄糖脱氢酶的亲和力的改善。工程化界面分子的应用导致了与PQQGDH的生物电化学反应，从而改善了传感器的性能。最后，我们尝试将PQQ葡萄糖脱氢酶与细胞色素b_2共表达，构建界面分子的新工艺。<562>将PQQ葡萄糖脱氢酶和细胞色素b_2共表达，构建了能够在不需要人工合成电子介质的情况下直接用电极传递电子的工程大肠杆菌细胞。<562>结果表明，构建新的生物电化学过程中使用在体内表达的界面分子。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