Enzymatic Cleavage Assays for LPMOs

All the cleavage assays (300 μl liquid volume) contained 4.4 μM of PaLPMO9s, 1.2 U.ml⁻¹ of PaCDHB or 1 mM of ascorbate, and 0.1 % (w/v) PASC prepared from Avicel as described by [47 (link)] in 50 mM sodium acetate buffer pH 4.8 or 50 μM of cello-oligosaccharides (Megazyme, Wicklow, Ireland) in 10 mM sodium acetate buffer pH 4.8. The enzyme reactions were performed in 2-ml tubes and incubated in a thermomixer (Eppendorf, Montesson, France) at 50 °C and 850 rpm. After 16 h of incubation, all the samples were boiled at 100 °C for 10 min to stop the enzymatic reaction and then centrifuged at 16,000 rpm for 15 min at 4 °C to separate the soluble fraction from the remaining insoluble fraction before carbohydrate determination. For kinetic experiments, reactions were run as described above and stopped after 1, 2, 3, 5, 7, 9, 24, 30, and 48 h of incubation. Assays were performed as triplicate independent experiments. For XG, the reaction mixture (300 μl liquid volume) contained 4.4 μM of PaLPMO9H, 1 mM of ascorbate, and 0.2 % (w/v) tamarind XG (Megazyme) in 50 mM sodium acetate buffer pH 4.8. The enzyme reactions were performed in 2-ml tubes and proceed as described above. Assays were performed as triplicate independent experiments.