2.6. Cellulase activity assay

Cellulase activity was determined using (3,5-dinitrosalisylic acid) DNS which measures the amount of reducing sugar liberated from CMC or cellulose, according to [19]. The tested bacterial strain was grown in BHM minimal medium or LB broth medium up to three days at 37 °C, and then centrifuged at 13,000 rpm for 5 min. One ml of the supernatant (enzyme solution) was mixed with one ml of CMC solubilized in phosphate buffer (1%) and incubated at 37 °C for 30 min under shaking (120 rpm). One ml dinitrosalisylic (DNS) acid reagent was added and the mixture was boiled for 5 min, then the absorbency was measured at 540 nm. One unit of enzyme activity was defined as µmol substrate consumed or product formed per minute.

Total protein concentration was determined according to [5] and absorbance was measured at 595 nm. Different concentrations of Bovine serum albumin (BSA) were conducted for plotting standard curve according to [15]. Cellulase specific activity was calculated by dividing the end product concentration(μmolreducingsugars/min) expressed as units by the total protein (mg) of the sample.