2.6. Cellulase Activity Assay

The bacterial strain was cultured in individual enrichment medium for cellulase fermentation, it contained (g/L): NaCl 5, peptone 5, yeast 10, CMC 5 (for CMCase), or microcrystalline cellulose (for Avicelase), and KH₂PO₄ 1 at 45 °C and 150 rpm for 5 d.

The cellulase assay was performed by extracting the supernatant of the bacterial culture after centrifugation at 10,000 rpm at 4 °C. The reaction mixture contained 0.5 mL of different crude enzyme dilutions and 0.5 mL of 1% CMC as a substrate (in 0.1 M citrate buffer, pH 4.8). The mixture was incubated at 50 °C for 30 min, and the reaction was terminated by adding 3 mL of DNS solution, and the solution was boiled for exactly 5 min for color development. All samples were then cooled rapidly. The reduction in sugar was estimated spectrophotometrically at 540 nm following the method of Miller [41]. One unit of cellulase activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugars (measured as 2 mg of glucose) per milliliter per minute under assay conditions.

Assay of filter paper activity (FPase) was estimated using gravimetric determination. The bacterial culture media was composed of (g/L): KH₂PO₄, 0.5; MgSO₄, 0.25; gelatin 2, Whatman filter paper 50 mg/20 mL/L of distilled water containing the target bacterial inocula. After 3 d of incubation, the cultures were centrifuged at 6000 rpm for 15 min at 4 °C. The collected pellets were used to estimate the constant weight after drying by comparing them with the trial without bacterial inocula. All experiments were performed in triplicates.