Site-specifically wired and oriented glucose dehydrogenase fused to a minimal cytochrome with high glucose sensing sensitivity.

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 kETapp. Electrochemical analysis revealed an efficient electron transfer rate with more than 15 times higher imax 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.