title: Determination of oxidoreductase activity using a high-throughput microplate respiratory measurement.
abstract: High-throughput multiparallel activity profiling for oxygen consuming cell layers has been recently developed for extracellular flux analysis. This technology has great potential for determining the enzymatic activity of oxidoreductases (i.e., laccase) both in vivo and in vitro, which is usually measured using photometrical tests monitoring the colored oxidation products. Improvements in terms of sample throughput, comparability, and gain of information (i.e., stoichiometry, electron transfer rate) can be achieved by means of a multiwell plate-based fluorimetric oxygen sensor. In the present study, various laccases have been applied to develop protocols that allow the multiparallel measurement of O(2)-consumption by enzymatic reactions. The developed and validated method enables the comparative quantitation of laccase characteristics (i.e., profiles of activity at various pH values) and minimizes the time it usually takes to collect respiratory data of oxygen-consuming enzymes. Furthermore, the possibility to assess differences between single and multisubstrate kinetics of laccases has been demonstrated.
authors.affiliation: Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland.
authors.forename: Gregor
authors.initials: G
authors.lastname: Hommes
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Gasser
authors.forename: Erik M
authors.initials: EM
authors.lastname: Ammann
authors.forename: Philippe F-X
authors.initials: PF
authors.lastname: Corvini
mesh_terms: D055162:Biocatalysis; D004396:Coloring Agents / Q000737:chemistry; D004579:Electron Transport; D005470:Fluorometry; D057166:High-Throughput Screening Assays; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000032:analysis*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D006861:Hydrogen Peroxide; D042845:Laccase; D010100:Oxygen