Fig. 5

Binding of NcLPMO9C to PASC and substrate degradation during H₂O₂-mediated PASC degradation. a SDS-PAGE analysis of supernatants obtained from titration of reduced NcLPMO9C with aliquots of 40 µM H₂O₂ (Fig. 4a, red line). Three µM LPMO was incubated for 10 min in buffer solution (“-PASC”) or together with 0.8 mg mL⁻¹ PASC (“+PASC”). To the latter reaction mix, ascorbate was added to a final concentration of 2 mM (“+PASC/AscA”), and titration with H₂O₂ in aliquots of 40 µM was performed. The intensity (in %) denotes the intensities of the bands relative to those of NcLPMO9C incubated in buffer solution. Intensities were calculated with the Image Lab Software Suite (BioRad). All reactions were carried out in 50 mM potassium phosphate buffer, pH 6.0. The SDS-PAGE analysis was repeated twice with the error being within 5%. b Weight determination of PASC in different control experiments. The total concentration of H₂O₂ added to the samples was 400 µM, which was added in aliquots of 40 µM every 90 s (as shown in Fig. 4a, red line). All assays were performed in 3 mL quartz cuvettes at 30 °C and under constant stirring. For each data point shown, 2 reactions containing 2.5 mL of sample solution were pooled, centrifuged and washed twice with deionized water. The dry weight of PASC in each reaction mix was determined after drying the sample at 55 °C to a constant weight (ca. 16 h of incubation)