Highly Efficient Flavin–Adenine Dinucleotide Glucose Dehydrogenase Fused to a Minimal Cytochrome C Domain

As the incidence of Type 1 and Type 2 Diabetes rise around the world, there is a constant improvement of blood-glucose monitoring tools. Some glucose biosensing instruments utilize Flavin-adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) as a glucose oxidizing enzyme. FAD-GDH does not use oxygen as its final electron acceptor, and is thermostable, which renders it a good candidate for enzyme-based glucose biosensors. Electrons from the redox active catalytic site of the enzyme are entrapped within the insulating protein matrix and cannot be transferred directly to an electrode. In this study, natural minimal C-type cytochrome domain was fused to the c-terminus of FAD-GDH, in order to achieve direct electron transfer abilities. Our new fusion enzyme introduced highly efficient direct electron transfer compared to the native enzyme, with ca. seven times higher catalytic current, three times higher k_cat and lower onset potential. The onset potential of the new enzyme is about (-)0.15 V vs. Ag/AgCl reference electrode. This low potential is beneficial since it diminishes the electrode sensitivity to known interfering molecules such as Vitamin C that is being oxidized at the electrode at higher redox potentials. To conclude, the fusion enzyme can simplify glucose biosensing, it is highly sensitive and selective towards glucose and thus can be utilized in blood-glucose monitoring.