Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity.
This study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold-nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active equivalent dehydrogenase via a single residue exchange.
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de and BioToP - The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190 Vienna, Austria.
Dagmar
D
Brugger
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de and BioToP - The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190 Vienna, Austria.
Leander
L
Sützl
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de.
Kawah
K
Zahma
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de and BioToP - The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190 Vienna, Austria.
Dietmar
D
Haltrich
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de and BioToP - The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190 Vienna, Austria.
Clemens K
CK
Peterbauer
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria. leonard.stoica@rub.de.
Leonard
L
Stoica
D016227:Benzoquinones / Q000737:chemistry; D015374:Biosensing Techniques; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*
D016227:Benzoquinones; D004800:Enzymes, Immobilized; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose; D010100:Oxygen