2.4. Extracellular Cellulase/CMCase enzyme activity assay

Cellulase activity was measured following standardized procedure of Miller by estimating reducing sugar content (Miller, 1959). In brief, 0.5 mL of cell free supernatant was taken as crude enzyme to form a reaction mixture with 0.5 mL of 0.5% carboxymethyl cellulose (CMC) in 50 mM citrate buffer (pH 4.8) and incubated at 37 °C in a shaking water bath for 30 min. The reaction was terminated by adding 3 mL of DNS reagent and the colour was developed by boiling the mixture for 5 min. The reaction mixture was cooled down and diluted in 20 mL of distilled water. Absorbance of samples was measured at 540 nm against a blank containing all the reagents except crude enzyme.

To check intracellular enzyme activity, 1 mL culture broth was taken and centrifuged to collect the cell pellet. The pellet was resuspended in 300 µl Bugbuster (Novagen) for inducing cell lysis at 37 °C for 1 h and after centrifugation supernatant was used as crude enzyme for performing enzyme activity assay as per standard protocol (Miller, 1959).