Xylanase Activity Assay Protocol

Xylanase activity was measured by mixing the cell-free supernatant with an equal volume of beech-wood xylan (1% w/v; Sigma Aldrich, U.S.A.) dissolved in 0.1 M phosphate buffer (pH 6) and incubating at 50 °C for 10 min in a water bath. The test tubes were then placed in boiling water for another 10 min, after which the reducing sugar content was estimated via the DNS method [16 (link)]. The amount of enzyme that released one µmol of D-xylose per minute during the enzyme-substrate reaction was defined as one unit of xylanase activity (IU/ml/min). D-xylose (Himedia, Mumbai, India) was used as a standard reference [17 (link)].