Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese PeroxidaseIn Vitro

ABSTRACTPyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacteriumKitasatospora aureofaciens(KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substratesin vitro. A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene fromK. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus,KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds betweenKaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity inKaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCEEstablishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase fromKitasatospora aureofaciensis of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively smallK. aureofaciensproteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.

Detection of 1,5-anhydroglucitol as a Biomarker for Diabetes Using an Organic Field-Effect Transistor-Based Biosensor

Sensor devices that can be fabricated on a flexible plastic film produced at a low cost using inkjet-printing technology are suitable for point-of-care applications. An organic field-effect transistor (OFET)-based biosensor can function as a potentiometric electrochemical sensor. To investigate the usefulness of an OFET-based biosensor, we demonstrated the detection of 1,5-anhydroglucitol (1,5-AG) and glucose, which are monosaccharides used as biomarkers of diabetes. An OFET-based biosensor combined with a Prussian blue (PB) electrode, modified with glucose oxidase (GOx) or pyranose oxidase (POx), was utilized for the detection of the monosaccharides. When the GOx- or POx-PB electrode was immersed in glucose solution at the determined concentration, shifts in the low-voltage direction of transfer characteristic curves of the OFET were observed to be dependent on the glucose concentrations in the range of 0–10 mM. For 1,5-AG, the curve shifts were observed only with the POx-PB electrode. Detection of glucose and 1,5-AG was achieved in a substrate-specific manner of the enzymes on the printed OFET-biosensor. Although further improvements are required in the detection concentration range, the plastic-filmOFET-biosensors will enable the measurement of not only diabetes biomarkers but also various other biomarkers.