4.3. 3,5-Dinitrosalicylic Acid (DNSA) Assay for the Determination of Enzyme Activity

The determination of enzyme activity was done according to a DNSA colorimetric method as described in Ghose [42] and Miller [43] with modifications. Enzymes (cellulase, pectinase, and xylanase) were added to MiliQ water to a final concentration of 0.02 mg/mL. Next, 10 mg of the appropriate substrate (cellulose, pectin, and xylan, respectively) was added to 1 mL of enzyme solution and the reaction was incubated at 40 °C for 60 min in a thermoshaker (Biosan, TS-100) at 900 rpm. The sample was then cleared by centrifugation (5 min, 1568× g). Next, sample aliquots (600 μL final volume) were mixed with 600 μL of DNSA reagent containing 10.0 g/L DNSA, 0.5 g/L sodium sulfite, 10 g/L sodium hydroxide, and 2 mL/L phenol. The mixtures were incubated for 15 min at 95 °C before adding 200 μL of 40 g/L potassium sodium tartrate solution (1.4 mL final volume). The samples were chilled on ice for 5 min, and then the absorbance at 575 nm was measured (Cary Series UV-Vis Spectrophotometer, Agilent Technologies). Product concentrations were calculated from calibration curves generated with the corresponding reducing sugars (glucose, galacturonic acid, xylose, respectively). If needed, the original samples were appropriately diluted to produce the absorption values within the range of the calibration curves, and dilutions were taken into account in the calculation of the enzymatic activity. One unit of enzymatic activity was defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under the specified assay conditions.