Buffer optimization for improvement of SpNOXDH thermostability and activity.
(A) Thermal unfolding curves of SpNOXDH (0.2 mg.mL-1) in 50mM B bis TRIS-propane buffer pH 6.5, 300 mM NaCl with various glycerol percentages: 5% (–), 10% (–), 15% (–), 20% (–). (B) Thermal unfolding curves of SpNOXDH (0.2 mg.mL-1) in bis TRIS-propane buffer pH 6.5, 5% glycerol with various NaCl concentrations: 100 mM (–), 200 mM (–), 300 mM (–), 400 mM (–), 500 mM (–), 750 mM (–), 1 M (–). (C) Cytochrome c reduction in presence of SpNOXDH (1 µg) NADPH (200 µM) FAD (10 µM) in the standard 50mM bis TRIS-propane buffer at pH 6.5, 300mM NaCl in presence of various glycerol percentages: 0% (–), 5% (–), 10% (–). (D) Cytochrome c reductase activity with SpNOXDH (1 µg), NADPH (200 µM), FAD (10 µM), glycerol (5%), in presence of various NaCl concentrations: 0 mM (–), 100 mM (–), 200 mM (–), 400 mM (–), 1 M (–). (E) 3D surface representation of Tm dependence with NaCl concentrations and glycerol percentages. (○) depicts the optimum used for further experiments of this study.