Peroxidase-like Activity Assay for LPMOs

To test peroxidase-like activity of the LPMOs, we used an assay adapted from Breslmayr et al. (66 (link)). The reaction mixtures (100 μL) contained 3 μM LPMO, 50 μM 2,6-DMP, 100 μM H₂O₂ in 50 mM BisTris-HCl buffer, pH 6.5. The LPMOs were preincubated with a 0.5 molar equivalent of CuSO₄ for a minimum of 30 min before initiating the reactions by adding the LPMO to the buffer solution containing 2,6-DMP and H₂O₂. The absorbance at 469 nm was monitored using a Varioscan LUX plate reader (Thermo Fisher Scientific) at 30°C for 3,600 s. The absorbance in each well was measured every 30 s.

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Tõlgo M., Hegnar O.A., Østby H., Várnai A., Vilaplana F., Eijsink V.G, & Olsson L. (2022). Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi. Applied and Environmental Microbiology, 88(6), e00096-22.

Publication 2022

Varioscan LUX plate reader

Assay Bistris Buffer H2o2 Molar Peroxidase

Corresponding Organization : Norwegian University of Life Sciences

Other organizations : Chalmers University of Technology, Wallenberg Wood Science Center, KTH Royal Institute of Technology

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Variable analysis

independent variables

LPMO concentration (3 μM)

dependent variables

Absorbance at 469 nm

control variables

2,6-DMP concentration (50 μM)
H2O2 concentration (100 μM)
Buffer (50 mM BisTris-HCl, pH 6.5)
Incubation time (minimum 30 min) with CuSO4 (0.5 molar equivalent)
Temperature (30°C)
Measurement interval (30 s)

controls

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

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