Measuring AA9-X282s Oxidase Activity

Oxidase activity of the six AA9-X282s was measured according to the protocol described by Kittl et al.^{26 (link)}. The reaction mixture contained Amplex Red reagent (200 µM; Thermo Fisher Scientific), Horseradish peroxidase (HRP; 0.1 mg.mL^-1 final) and AA9-X282s (1 µM). The reactions were performed in 96-well microplates in sodium phosphate buffer (50 mM, pH 7.0) and started by adding 50 μM of ascorbic acid. The H₂O₂ production rate is reflected by the production rate of resorufin, i.e. the product of Amplex Red oxidation by HRP and H₂O₂, which was monitored at 575 nm over a 30 min period. The initial slope of the curve, normalized with the enzyme concentration, was used to calculate the H₂O₂ production rate.

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Reyre J.L., Grisel S., Haon M., Xiang R., Gaillard J.C., Armengaud J., Guallar V., Margeot A., Arragain S., Berrin J.G, & Bissaro B. (2023). Insights into peculiar fungal LPMO family members holding a short C-terminal sequence reminiscent of phosphate binding motifs. Scientific Reports, 13, 11586.

Publication 2023

Amplex Red reagent

Ascorbic acid Buffer Enzyme H2o2 Oxidase Resorufin Sodium phosphate

Corresponding Organization :

Other organizations : IFP Énergies nouvelles, Biodiversité et Biotechnologie Fongiques, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Barcelona Supercomputing Center, Universitat Politècnica de Catalunya, Institut de Biologie et Technologies, Laboratoire Innovations Technologiques pour la Détection et le Diagnostic, Technologies pour la Santé, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, CEA Paris-Saclay, Institució Catalana de Recerca i Estudis Avançats

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Variable analysis

independent variables

Concentration of Amplex Red reagent (200 µM)
Concentration of Horseradish peroxidase (HRP; 0.1 mg.mL^-1 final)
Concentration of AA9-X282s (1 µM)
Concentration of ascorbic acid (50 μM)

dependent variables

H2O2 production rate, reflected by the production rate of resorufin (the product of Amplex Red oxidation by HRP and H2O2), monitored at 575 nm over a 30 min period

control variables

Sodium phosphate buffer (50 mM, pH 7.0)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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