4.6. Xylanase Assay

Enzyme activity of xylanase was assayed by determining the liberated reducing end products by standard method (Miller 1959) with some modifications. The reaction solution (2 mL) consisted of 0.5 mL of 1% birchwood xylan, 1.4 mL 100 mM phosphate buffer (pH 6.5) and 0.1 mL enzyme solution. It was incubated for 20 min at 37 °C in a water bath. Three mL of dinitrosalicyclic acid (DNSA) reagent was added, and the volume was brought to 6 mL by the addition of 1 mL of distilled water. The solution was boiled for 10 min in a water bath, cooled, and absorbance was read at 540 nm. A negative control was simultaneously prepared using a thermally denatured enzyme. The concentration of the product (xylose) was determined with the help of a calibration curve. One unit of xylanase activity is defined as the amount of enzyme that liberates 1 μmol of xylose per min per unit volume under the assay conditions.