An aliquot of the substrate stock solution (0.16 mL, 6.25 mg/mL in 0.1 M Na-acetate buffer) was preliminary heated at 50°C for 5 min. Then the enzyme reaction was initiated by adding 0.04 mL of the enzyme solution (also preheated at 50°C for 5 min). The mixture was incubated at 50°C for 10 min (5 min in the case of CMCase and β-glucanase activities); the reaction was stopped by addition of 0.2 mL of the Somogyi copper reagent. The tightly stoppered test tube was incubated in a boiling water bath for 40 min; then it was cooled to room temperature and 0.2 mL of the Nelson arsenomolybdate reagent was added. The solution was carefully mixed and incubated for 10 min at room temperature and then 1.4 mL of water was added (0.4 mL of acetone to dissolve the precipitated CMC or β-glucan and then 1 mL of water were added in the case of CMCase and β-glucanase activity measurements). After centrifugation at 13,000 rpm for 1 min, the absorbance of the supernatant at 610 nm (A610) was measured. The A610 values for the substrate and enzyme blanks were subtracted from the A610 value for the analyzed sample. The substrate and enzyme blanks were prepared in the same way as the analyzed sample except that the necessary amount of the acetate buffer was added to the substrate (enzyme) solution instead of the enzyme (substrate) solution.
An aliquot of the substrate stock solution (0.3 mL, 10 mg/mL in 0.1 M Na-acetate buffer) was mixed with 0.3 mL of the enzyme solution (both solutions were preheated at 50°C for 5 min). After 10 min of incubation at 50°C, 0.9 mL of the DNS reagent was added to the test tube and the mixture was incubated in a boiling water bath for 5 min. After cooling to room temperature, the absorbance of the supernatant at 540 nm was measured. The A540 values for the substrate and enzyme blanks were subtracted from the A540 value for the analyzed sample. The substrate and enzyme blanks were prepared in the same way as the analyzed sample except that 0.3 mL of the acetate buffer was added to the substrate (enzyme) solution instead of the enzyme (substrate) solution.