Tailoring of pyranose oxidase for biofull cells

Biofuel cells have attracted recent interest as possible alternative to conventional fuel cells for specific applications. Biocatalysts or enzymes have evolved to function in complex physiological environments, efficiently and selectively catalyzing reactions at physiological temperature and pH, and involving fuels and oxidants present in such environments. This makes enzymatic biofuel cells a promising future application as an implantable power source, and a number of implantable medical devices might benefit from these power supplies. Another possible application, the exploitation of ambient fuels, is attractive in situations where power needs for small electronic devices are distributed, disconnected, and long term, e.g., for electronic sensors monitoring air quality, weather, presence of biohazards, etc. Since most of these applications aim at long-term use of the biofuel cell, stability is a key aspect. In addition, a wide substrate specificity for the enzyme used in these cells, both pertaining to the sugar substrate and the redox mediator, is desirable. The fungal enzyme pyranose oxidase (P2Ox) has a number of advantages over glucose oxidase, which is commonly studied for this use in biofuel cells. These advantages include the much wider substrate specificity of P2Ox, favourable catalytic properties, and any lack of anomeric preference. Yet, to make biofuel cells commercially viable pyranose oxidase has to be further improved, e.g. with respect to stability and reactivity. In this project both rational design and directed evolution (random mutagenesis combined with DNA shuffling) are used to tailor P2Ox from the fungus Trametes multicolor for this proposed use in biofuel cells. This combination of rationale and random methods of protein modification is likely to be the most productive approach to enzyme optimisation and we expect significant improvements of the properties of the enzyme will be achieved. Screening will be performed by conventional methods (formation of colour in microtitre plates) but also by using cell display in which enzyme variants are displayed on the cell surface of the micro- organism overexpressing the enzyme. Selected variants are characterised in detail including structural studies which give important results about the structure/function relationship of these commercially attractive enzymes. Expected results include information of the interaction of the substrate with the active site, or the dimer interaction and the function of the substrate channel. Finally, selected improved biocatalysts will be used in biosensors and their performance is evaluated.