2.3. Xylanase Activity Assay

For xylanase activity determination, 3,5-dinitrosalicylic acid (DNS) method [19] and Bradford assays were performed. Liberation of reducing sugars in the culture filtrate was performed according to [20] with modifications. Reaction mixture containing 0.1 mL of appropriately diluted culture filtrate with 0.1 mL of 1% birchwood xylan (Serva) solution in acetate buffer (0.05 M, pH = 5.0) was incubated for 30 min at 50 °C. The reaction was stopped by the addition of 0.65 mL DNS and heated for 10 min at 100 °C. Then, the mixture was cooled down to room temperature, and the liberated reducing sugars were measured spectrophotometrically at 530 nm and expressed as xylose equivalent. Xylose was taken as standard. For protein determination, Bradford assay was performed according to Jones (2012) with modifications [7]. 0.5 mL of Bradford reagent was mixed with 0.5 mL of appropriately diluted culture filtrates and incubated for 10 min at room temperature. Protein content was measured spectrophotometrically at 595 nm. Protein concentration was calculated using bovine serum albumin (BSA, SERVA) as a standard.

Specific enzyme activity was defined as the amount of enzyme required to produce 1 µmole of xylose per minute under assay conditions per soluble protein in the culture filtrate (U/mg protein).