Routing the flow of electrons...

The project investigates the function of the cytochrome domain (CYT) which is part of the fungal enzymecellobiose dehydrogenase asanelectrontransferring domain forother oxidoreductases.Thecombinationof CYT andglucose-methanol-choline (GMC) oxidoreductases into chimeric flavocytochromes will reroute the flow of electrons from the catalytic centre to an electrode. The enzymes dependence on cosubstrates like oxygen, quinones or redox mediators shall be replaced by direct electron transfer to an electrode a cheap and truly atom efficient source of reduction equivalents. CYT-modified GMC oxidoreductases with efficient DET are highly interesting for biosensors and bioelectrocatalytic processes. The mechanism and kinetics of CYT interaction with three GMC oxidoreductases (aryl alcohol oxidase, alcohol oxidase and glucose dehydrogenase) will be studied in comparison with CDH. In a second line of the project, the interaction of electrode-immobilized CYT to the unwired GMC oxidoreductases and to lytic polysaccharide monooxygenase will be investigated as a platform to electrically contact oxidoreductases. The project aims to elucidate the domain interaction mechanism, CYT orientation and mobility by fast kinetic and electrochemical methods and to investigate the potential of CYT-GMC chimeric enzymes and CYT-modified electrodes for biosensing and bioelectrocatalytic applications.

A new strategy for the application of enzymes in biosensors was investigated in this project. In biosensors enzymes are used as detection elements and are also called "biorecognition elements". They are the key to specifically and sensitively detect the analyte (the substance of interest) in complex matrices like food or biological samples despite the presence of interfering substances. Of high importance for its correct function is the transfer of electrons between the enzyme and the biosensor electrode and this topic needs to be covered in more detail in fundamental studies. Therefore, the project "electron flow" investigated a possible route to improve the electric connection of GMC-oxidoreductases, which are often used in biosensors for the determination of glucose, e.g. in blood. The electrons obtained by the enzyme from the detection reaction in which glucose is converted into gluconic acid can be transferred to a biosensor electrode via redox mediators. However, this electron transfer can be interfered by electroactive species in the matrix. By establishing a direct electron transfer between the enzyme and the electrode this problem can be circumvented, but a suitable electron transfer protein is required. In nature cytochromes fulfil this purpose. The project's researchers have performed an in silico bioinformatic search to select eight candidates from over 1500 cytochromes, produced and characterized them. Cytochromes from bacteria and fungi with a suitable redox potential and good electron transfer properties were selected and combined with the enzyme glucose dehydrogenase via genetic or biochemical fusion methods. The engineered fusion enzymes showed direct electron transfer, but at a too low rate for commercial applications. Within the project we investigated strategies to increase the electron transfer and thereby the signal of the biosensor by molecular simulation methods. A combination of protein engineering at the interface between both proteins and an improved immobilization of the enzyme on the electrode were identified as the most suitable strategies to improve the electron transfer by future research and development.