Algov Itay, Feiertag Aviv, Alfonta Lital
Departments of Life Sciences, Chemistry and Ilse Katz institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PoBox 653, Beer-Sheva, 8410501, Israel.
Departments of Life Sciences, Chemistry and Ilse Katz institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PoBox 653, Beer-Sheva, 8410501, Israel.
Biosens Bioelectron. 2021 May 15;180:113117. doi: 10.1016/j.bios.2021.113117. Epub 2021 Feb 27.
Direct electron transfer based enzymatic biosensors are highly efficient systems where electrons are transferred directly from the enzyme's electroactive site to the electrode. One way of achieving it is by 'wiring' the enzyme to the electrode surface. The wiring of enzymes to electrode surfaces can be reached in many different ways but controlling its orientation towards the electrode surface is still a challenge. In this study we have designed a Flavin-adenine dinucleotide dependent glucose dehydrogenase that is fused to a minimal cytochrome with a site-specifically incorporated unnatural amino acid to control its orientation towards the electrode. Several site-specifically wired mutant enzymes were compared to each other and to a non-specifically wired enzyme using atomic force microscopy and electrochemical techniques. The surface and activity analyses suggest that the site-specific wiring through different sites maintains the correct folding of the enzyme and have a positive effect on the apparent electrochemical electron transfer rate constant k. Electrochemical analysis revealed an efficient electron transfer rate with more than 15 times higher i and 10-fold higher sensitivity of the site-specifically wired enzyme variants compared to the non-specifically wired ones. This approach can be utilized to control the orientation of other redox enzymes on electrodes to allow a significant improvement of their electron transfer communication with electrodes.
基于直接电子转移的酶生物传感器是高效的系统，其中电子直接从酶的电活性位点转移到电极。实现这一点的一种方法是将酶“连接”到电极表面。酶与电极表面的连接可以通过多种不同方式实现，但控制其相对于电极表面的取向仍然是一个挑战。在本研究中，我们设计了一种黄素腺嘌呤二核苷酸依赖性葡萄糖脱氢酶，它与一种最小化细胞色素融合，并带有一个位点特异性掺入的非天然氨基酸，以控制其相对于电极的取向。使用原子力显微镜和电化学技术，将几种位点特异性连接的突变酶相互比较，并与非特异性连接的酶进行比较。表面和活性分析表明，通过不同位点的位点特异性连接保持了酶的正确折叠，并对表观电化学电子转移速率常数k有积极影响。电化学分析显示，与非特异性连接的酶变体相比，位点特异性连接的酶变体具有超过15倍的更高电流和10倍的更高灵敏度的高效电子转移速率。这种方法可用于控制电极上其他氧化还原酶的取向，以显著改善它们与电极的电子转移通讯。
