Enzyme activity assays

The substrates were purchased from Sigma–Aldrich (USA). The substrates and enzyme preparations were dissolved in 0.05 M sodium citrate buffer (pH 4.8).

FPA, which represented the overall cellulase activity of the samples, was measured using Whatman No. 1 filter paper as the substrate. The reaction mixtures contained 50 mg of filter paper, 1.5 mL of citrate buffer, and 500 µL of the suitably diluted enzyme fraction (protein concentration was 200 µg/mL). These mixtures were then incubated at 50 °C for 60 min. EG and xylanase activities were assayed with CMC-Na and beechwood xylan as the substrates, respectively. The enzyme reactions (protein concentration was 5 µg/mL) were performed in 1 mL of 1 % substrate-containing citrate buffer (pH 4.8) at 50 °C for 30 min. The amount of reducing sugar released was determined via the dinitrosalicylic acid (DNS) method using glucose and xylose as the standard, respectively [41].

CBH activity was assayed with 1.0 mg/mL p-nitrophenyl-beta-D-cellobioside (pNPC) as a substrate, which contained 1.0 mg/mL D-glucono-1,5-lactone to inhibit the hydrolysis of the substrate by BG. BG activity was assayed with 1.0 mg/mL p-nitrophenyl-beta-D-glucoside (pNPG) as a substrate. Beta-xylosidase activity was assayed with 1.0 mg/mL p-nitrophenylxyloside (pNPX) as a substrate. The reactions were carried out using 50 µL of the substrate solution and 100 µL of the enzyme fraction. After incubation was performed at 50 °C for 30 min, the reaction was terminated by adding 150 µL of 10 % (w/v) Na₂CO₃ and the released p-nitrophenol (pNP) was quantified by measuring the absorbance at 420 nm. The inactive enzyme boiled at 100 °C for 10 min was used as the control [42].

One enzyme activity unit (U) was defined as the amount of enzyme that liberates 1 μmol glucose, xylose, or pNP per minute under the assay conditions.