SUPERVISOR: Doris RIBITSCH

PROJECT ASSIGNED TO: Anna MAGNONE

The ability of oxidoreductases to perform electron-transfer reactions makes them attractive systems in the field of bioelectrocatalysis, which combines the advantages of biocatalysis, such as high selectivity and operation under mild reaction conditions, with the applications of electrocatalysis. Oxidoreductases have been widely employed as catalysts in reactions coupled to electrode surfaces, making them highly versatile tools in this field.

However, electron transfer is often inefficient due to the random orientation of enzymes on the electrode surface. Therefore, improvements in immobilization methods are crucial to enhance electron-transfer efficiency. The goal is to achieve a uniformly oriented enzyme monolayer, thereby increasing catalytic rates and optimizing the overall performance of the system. A promising approach involves the use of target-binding peptides, which have been shown to improve electrode binding and enzyme stability.

In this PhD thesis, cellobiose dehydrogenase, CDH, is used as a model enzyme. Its cellulose-binding module, CBM1, will be substituted with electrode-specific binding modules, with a particular focus on gold and carbon supports. Target-specific sequences will be selected through the screening of carbon- and gold-binding peptides using phage surface display. Subsequently, affinity and binding dynamics on the electrode surfaces will be characterized using surface plasmon resonance and quartz crystal microbalance measurements. The selected binding sequences will then be incorporated into CDH, and the resulting fusion proteins will be produced in Komagataella phaffii.

Electrochemical evaluation will be conducted to assess the performance of the engineered oxidoreductases, with particular emphasis on current density and redox potential. In parallel, protein engineering will be employed to improve enzyme properties, including pH dependence and substrate affinity.

This strategy aims to generate stable, efficient, and reusable biocatalysts for bioelectrocatalysis by combining enzyme engineering with material-specific immobilization, while ensuring compatibility with existing electrode platforms. The resulting systems may contribute to the development of sustainable bioelectrochemical technologies and have potential applications in enzymatic fuel cells, as well as in biosensors for diagnostics and environmental monitoring.