Pyranose dehydrogenase (PDH), a fungal flavin-dependent oxidoreductase, catalyzes the oxidation of a broad variety of sugars substrates. The enzyme is unable to utilize dioxygen as an electron acceptor. Benzoquinones and complexed metal ions naturally present during lignocellulose degradation, the supposed biological function of PDH, are preferred. PDH represents a promising biocatalyst which can be applied in sugar conversions, organic synthesis or electrochemistry. For this purpose, PDHs from Agaricus meleagris, A. xanthoderma and A. campestris were recombinantly expressed in the methylotrophic yeast Pichia pastoris, purified and characterized biochemically. Steady-state kinetic parameters and molecular properties were investigated using UV-Vis spectroscopy, SDS-PAGE and TCA precipitation. Batch lactose conversion experiments and HPLC analysis confirmed the suitability of the enzymes from A. xanthoderma and A. campestris for the production of lactobionic acid or 2-dehydrolactose, a key intermediate for the production of lactulose. The physiological electron acceptor of PDH is undesirable for an application in food technology, therefore site-saturation mutagenesis libraries of twelve amino acids in the active site of A. meleagris PDH were expressed in Saccharomyces cerevisiae. High-throughput screening resulted in one position altering oxygen reactivity. Mutant H103Y, produced in P. pastoris, showed a five-fold increase in oxygen reactivity. Although carrying a non-covalently linked FAD-cofactor in contrast to the wild-type, mutant H103Y was still catalytically active but to a lower degree. Stopped-flow experiments revealed that only the reductive half-reaction was negatively affected by the mutation. Thermal and chemical denaturation experiments were performed and confirmed the lower stability caused by the non-covalent FAD linkage. This semi-rational approach provides a scaffold for further engineering of PDH towards a highly competent biocatalyst.