Engineered Pyranose 2‐Oxidase: Efficiently Turning Sugars into Electrical Energy

Abstract

AbstractDue to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale‐devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi‐rational protein design to improve the catalytic activity of the enzyme pyranose 2‐oxidase (P2Ox) with its alternative soluble electron acceptors 1,4‐benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96‐well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4‐benzoquinone and ferricenium ion compared to the wild‐type enzyme, when either D‐glucose or D‐galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either “wired” to an osmium redox polymer or using soluble 1,4‐benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4‐fold compared to systems employing the wild‐type enzyme.