Extracellular Protein and Enzyme Assays

Total extracellular protein contents in the culture supernatants were measured using a Bio-Rad DC protein assay kit (Bio-Rad) based on absorbance at 595 nm, with bovine serum albumin used as the standard. For protein gel electrophoresis, 30-µL aliquots of concentrated culture supernatants were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis on Novex NuPAGE pre-cast protein gels (Thermo Fisher Scientific). Endoglucanase activity in the culture supernatants was determined using an azo-cm-cellulose assay kit (Megazyme, Wicklow, Ireland) according to the manufacturer’s protocol. Endo-1,4-β-xylanase activities were assayed with an azo-xylan kit (Megazyme) following the method specified by the manufacturer. FPA activities were assayed with Whatman No.1 filter paper as the substrate. The enzyme reactions were performed in 50 mM citrate buffer (pH 4.8) at 50 °C for 60 min, using the DNS method to quantify the released reducing sugar. Exoglucanase activity was assayed according to the method described by Zou et al. [60 (link)] and measured at 50 °C using 1.0 mg mL⁻¹p-nitrophenyl-β-D-cellobioside (Sigma-Aldrich) as the substrate in 50 mM citrate buffer (pH 4.8) containing 1 mg mL⁻¹d-glucono-1,5-σ-lactone. Each reaction mixture containing 250 µL of properly diluted enzyme and 250 µL of 1.0 mg mL⁻¹ substrate in 50 mM citrate buffer (pH 4.8) was incubated for 10 min at 50 °C, and the reaction was terminated by adding 500 µL of 1 M Na₂CO₃. Released p-nitrophenol (pNP) was measured at an absorbance of 420 nm. Inactive enzyme, which was boiled at 100 °C for 10 min, was used as a control. pNP was used for the standard curve. In the exoglucanase activity analyses, one unit (U) of enzymatic activity was defined as the amount of 1 μmol glucose or pNP released by 1 mL of enzyme from the substrate per minute under the standard assay conditions. All estimates were performed in three repeated assays. The statistical significance of differences among WT and mutant strains was assessed by one-way analysis of variance.