Fig. 4

Effect of temperature and pH on TausLPMO9B. Reaction mixtures containing 1-µM LPMO, 1-mM ascorbic acid and 0.1% w/v PASC were incubated for 24 h at different conditions: a fixed pH 5.0, different temperatures (35 °C, 45 °C, 55 °C, 65 °C, 75 °C and 85 °C); b fixed temperature 45 °C, different pHs (3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0). The amount of total oxidized products was calculated as sum of the peak areas of peaks eluting between 16 and 30 min during HPAEC–PAD (i.e., C1-oxidized cello-oligosaccharides, DP2 – DP6). The stability of TausLPMO9B in the function of pH (c) was evaluated by pre-mixing the enzyme with buffers of varying pH in 50% of the final reaction volume and incubating for 24 h at 45 °C, after which PASC (0.1% w/v) and ascorbic acid (1 mM) were added to initiate the reaction, followed by incubation for 24 h at 45 °C. Control reactions at the same varying pH values but without the pre-incubation step were also set up. The data points represent the percentage of activity retained by the pre-incubated enzyme, relative to the non-pre-incubated enzyme. Activities were calculated as the sum of peak areas corresponding to oxidized products in the HPAEC–PAD analysis. In other words, 100% retained activity (marked by a red line) means that the amount of oxidized products released was identical in the reactions with and without pre-incubation