Catalytic Rate Constant (kcat)


Spectrophotometric/Colorimetric Assay

0.2 M sodium phosphate, 0.1 M citric acid

50 °C



540 nm


The activity of the endo-1,4-β-xylanases was determined by using the 3, 5-dinitrosalicylic acid (DNS) colorimetric method. In brief, 40 μL of enzyme diluted with McIlvaine buffer (0.2 M sodium phosphate, 0.1 M citric acid, pH 6.0) was added to 360 μL of 1% (w/v) beech wood xylan (Sigma) that was suspended with the same buffer and incubated at 50 °C for 10 min. The reducing sugars hydrolysed by xylanase were quantitated by adding 600 μL of DNS and boiling at 100 °C for 10 min. The absorbance of the solution was then measured at 540 nm by using a visible-spectrophotometer. One unit (IU) of xylanase activity was defined as the amount of enzyme that liberated 1 μmol of reducing sugar equivalent per minute under standard conditions (at pH 6.0 and 50°C for 10 min).