Figure 9.

Time course for release of oxidized products during incubation of 2 mg·ml⁻¹ PASC with 1 μm C4-oxidizing NcLPMO in reactions with H₂O₂ (dashed lines). NcLPMO9A (black squares), NcLPMO9C (red squares), and NcLPMO9D (blue squares) were incubated with 2 mg·ml⁻¹ PASC in 50 mm Bis-Tris (pH 6.5) at 45 °C and 1000 rpm, and ∼45 μm H₂O₂ was added to the reactions every 15 min. Prior to every addition of H₂O₂, ∼12 μm AscA was added to ensure reduction of the LPMO. Control reactions (●, connected with dotted lines) were done in the absence of H₂O₂, meaning that only ∼12 μm AscA was added every 15 min for 4 h. Solubilized oxidized products were enzymatically converted to Glc4gemGlc using 1 μm TrCel7A. The concentrations of Glc4gemGlc, shown on the left y axis, were determined by HPAEC-PAD using a Glc4gemGlc standard prepared as described previously (22). Error bars represent S.D. of triplicate samples. Note that only soluble oxidized products were measured and that the nonsoluble material contains a significant fraction of the oxidized sites. The cumulative amount of H₂O₂ that was added to the reactions is indicated by green asterisks, and the concentration values are shown on the right y axis.