Fig. 3

Dose–response curves for different components of the rPHP-based LPMO assay determined by the absorbance at 552 nm after 30 min of incubation at 40 °C. Reaction conditions are 300 nM TaAA9A, 200 µM rPHP, 100 µM DHA, and 25 mM citrate–phosphate buffer pH 7.25 with the following modifications: a The relative copper loading of the TaAA9A was varied (0–600 nM CuCl₂). b The concentration of Cu-TaAA9A was varied (0–1 µM) and was found to be linear in the lower range 0–300 nM. Measurements on 16 samples at 0 or 15 nM enzyme concentrations are shown in the insert. The horizontal lines mark the σ = 1.645 thresholds. c The DHA concentration was varied (0–5.0 mM) and a plateau is observed in the range 50–250 µM. d The rPHP concentration was varied (0–1.0 mM). The formation rate of PHP was calculated and plotted together with the best fitting Michaelis–Menten kinetics curve, described by K_M = 244 µM, V_max = 1.6 µM/min, k_cat = 0.09 s⁻¹ with an R² of 0.99