Enzymatic bio-fuel cells transform chemical into electrical energy, with the help of enzyme modified electrodes. Due their membraneless design, they have been suggested for powering transportable micro devices and implantable sensors. One of the major remaining challenges is the achievable maximum power output of these systems. Here, rational enzyme engineering of an oxidoreductase for anode modification, pyranose dehydrogenase, was employed to increase achievable maximum current densities. The fungal enzyme pyranose dehydrogenase is of interest due to its broad substrate specificity, capability of dioxidation, and inability of reducing O2 to H2O2. Pyranose dehydrogenase from Agaricus bisporus was heterologously expressed in this work and compared to the three other reported recombinant pyranose dehydrogenases, to select the most promising target for enzyme engineering. Agaricus meleagris pyranose dehydrogenase I was chosen due to its generally moderately better catalytic efficiency and enzyme stability. The rational engineering strategy was based on reducing N-glycosylation, which was previously shown to be disadvantageous for bio-fuel cell performance. A systematic set of partially N-glycosylated Agaricus meleagris pyranose dehydrogenase I variants was tested on Os-polymer modified graphite electrodes. Knocking out the only apparently overglycosylated site, N252, did not create a variant with improved performance on Os-polymer modified electrodes. The site N319, on the other side, could not be knocked out without preventing functional recombinant expression in Pichia pastoris. However, electrodes based on the variant N75G/N175Q yielded an approximately 10-fold increased maximum current density (290 A cm-2) compared to the equivalent electrodes with recombinant wild type enzyme. It is therefore a promising candidate for application to future enzymatic bio-fuel cell anodes.