2.4.3. Cellulase Activity Assay

The cellulase activity was measured using the 3,5-dinitrosalicylic acid (DNS) reducing sugar method [33] with CMC as substrate. The reaction was initiated by adding 1 mL of suitably diluted sample to 1 mL of 1% (w/v) CMC in 50 mM potassium phosphate buffer (pH 7), and incubating the tubes at 37 °C. The reaction was terminated after 30 min by adding 3 mL of DNS reagent. The tubes were incubated for 15 min in a boiling water bath. Then, 1 mL of 40% (w/v) potassium sodium tartrate solution was added immediately and the tubes were cooled at room temperature over 20 min. The absorbance of the reaction solutions was measured at 540 nm. An enzyme blank was prepared by adding the DNS reagent before the enzyme solution.

The amount of glucose released from the substrate was estimated based on a standard curve of glucose generated in the range 1 to 7.5 μmol. One unit of cellulase activity was defined as the amount of enzyme required to release 1 μmol of glucose per minute under the assay conditions. All tests were performed in triplicate.