Fig. 2

Effect of pH (a) and H₂O₂ (b) on HMFO stability, and influence of pH on FDCA production from HMF (c) and FFCA (d). a pH stability as shown by residual activities after 72-h incubation of the enzymes in 100 mM B&R buffer (pH 6.5–9) at 25 °C. b H₂O₂ stability as shown by residual activities after 72-h incubation of the enzymes with increasing amounts of H₂O₂ (0–30 mM) in 50 mM Tris/HCl, pH 7.5. The residual activities were measured with vanillyl alcohol as substrate. Percentages of FDCA (referred to initial substrate concentration) in c and d are shown after 24-h reaction of HMF or FFCA, respectively, with the HMFO WT and variants (4-h reaction with 8BxHMFO) at different pH values. The reactions were performed at 28 °C and 180 rpm shaking, using 1.5 mM substrate, and 2.5 µM enzyme, in 50 mM NaPi (pH 6.5) or Tris/HCl (pH 7.5, 8.0 and 9.0). Mean and standard deviation values from triplicate experiments are shown