Dephosphorylation of the Cel7A enzyme and determination of kinetic parameters

For the dephosphorylation assay of the Cel7A enzyme, the fungi T. reesei and Aspergillus nidulans were cultivated on sugarcane bagasse. Then, 100 µg of proteins extracted from the supernatant were used for Cel7A (T. reesei) and CBHI (A. nidulans) purification (Fig. 5C). Purified proteins were then treated with 10 U of FastAP™ Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, Massachusetts, MA, USA) for 1 h at 37°C. The resultant unphosphorylated samples were used for enzymatic assays to detect Cel7A activity.

Effects of phosphorylation on the holocellulolytic activity of T. reesei. (A) Cellulolytic activities of the crude extract upon incubation with phosphatase (dephosphorylated, DP) and the control (no treatment, phosphorylated, P). (B) FPase activity of Cel7A (T. reesei) and CBHI (A. nidulans) purified and upon treatment with phosphatase (DP) and the control (no treatment, P). (C) SDS-PAGE gel with Cel7A and CBHI enzymes purified from the supernatant of T. reesei and A. nidulans, respectively, in cultures of sugarcane bagasse.

Different concentrations of the substrate (4-Nitrophenyl β-D-cellobioside, Sigma-Aldrich, St. Louis, MO, USA) ranging from 0.2 mM to 2 mM were used to determine the kinetic parameters of Cel7A. The reaction mixture consisted of 10 μL of purified Cel7A (treated or not treated with FastAP™ phosphatase), 50 µL of a 50-mM sodium acetate buffer, and 40 µL of substrate. Reactions underwent incubation at 50°C for 3 h, followed by the addition of 100 μL of 1 M sodium carbonate. The enzymatic activity assays were performed in 96-well microplates, and absorbance was read at 405 nm using the xMark™ Microplate Absorbance Spectrophotometer. One enzyme unit was defined as the amount of enzyme capable of liberating 1 μmol of reducing sugar per minute. Reaction rates were measured, and the Michaelis-Menten constant (K_M) and maximum reaction rate (V_max) were calculated by fitting the initial reaction rates (v) for each substrate concentration (S) to the Michaelis-Menten equation, using the GraphPad Prism software.