Cellulase assay

The wild-type and T4 strains were fermented in 40 mL of MNN medium inoculated with 1.0 × 10⁶ 12-day-old spores at 42 °C with shaking at 200 rpm. Cellulase activities in supernatants were assayed at various intervals. FPase, CMCase, and xylanase were assayed using the 3,5-dinitrosalicylic acid (DNS) method²¹. Whatman No.1 filter papers (1 × 6 cm in area) were immersed in 2 mL of appropriately diluted supernatants rendered to 100 mM in Na₂HPO₄-citric acid (pH 6.0) at 60 °C for 30 min. Next, 3 mL of DNS were added to terminate the reaction. The mixtures were boiled for 5 min and the absorbances at 540 nm determined. The standard assays for CMCase and xylanase featured the addition of 100 μL of appropriately diluted supernatants to 900 μL of 100 mM Na₂HPO₄-citric acid (pH 6.0) containing 1.0% (w/v) CMC-Na or birchwood xylan, followed by incubation at 60 °C for 10 min. Each reaction was terminated by the addition of 1.5 mL of DNS reagent and boiling for 5 min. The absorbance at 540 nm was determined when the reaction mixture had cooled to room temperature. Glucosidase activity was measured using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate. The reaction featured the addition of 250 μL of 4 mM pNPG in 100 mM Na₂HPO₄-citric acid (pH 6.0) to 250 μL of appropriately diluted solutions, followed by incubation at 60 °C for 10 min. Finally, 1.5 mL of 1.0 M Na₂CO₃ were added to terminate the reaction, and the liberated p-nitrophenyl was detected by measuring the absorbance at 420 nm. Cellobiohydrolase activity was measured in the same way, except that p-nitrophenyl-β-D-cellobioside (pNPC) served as a substrate. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmoL of reduced sugar (for FPase, CMCase, and xylanase) or 1 μmoL of p-nitrophenyl (for glucosidase and cellobiohydrolase) from the substrate, per min, under the conditions described above. Glucose and p-nitrophenyl served as standards.