Engineering of pyranose 2-oxidase for modified oxygen reactivity

Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity

“…Contrary to the previous biochemical study on this variant, 15 the electrochemical method used in this study is sufficiently sensitive to demonstrate in a volume of only 50 μL that the dehydrogenase function of POx remains unaltered by this amino acid exchange, and proves an unmodified reactivity of the variant towards glucose. To our knowledge, this is the first study presenting and demonstrating the transformation of an oxidase into a robust and catalytically efficient dehydrogenase.…”

“…Electrochemical experiments demonstrate the complete switch-off of the oxidase function of pyranose 2-oxidase (POx) by only one single amino acid exchange at position 593, replacing asparagine (N) with cysteine (C). Contrary to the previous biochemical study on this variant, 15 the electrochemical method used in this study is sufficiently sensitive to demonstrate in a volume of only 50 mL that Table 1 Michaelis-Menten constants (K m ) for glucose and maximum I DH current at +400 mV (I max ) recorded at saturation concentration of substrates. Apparent substrate efficiency (I max /K m ) and their relative values (%).…”

“…15,16 After concentration and washing steps, both POx enzymes (wt-POx and POx-C) were stored at 8 °C. The activity assay, applied to wild-type POx (wt-POx) and the mutant N593C (POx-C) was previously described.…”

“…The generation of mutant N593C (herein denoted POx-C) and its counter-partners N593S and N593K, as well as the purification of the enzyme variants were carried out repetitive and separate by two co-authors (DB and LS) according to previous publications. 15,16 After concentration and washing steps, both POx enzymes (wt-POx and POx-C) were stored at 8 1C. The activity assay, applied to wild-type POx (wt-POx) and the mutant N593C (POx-C) was previously described.…”