Assay of the enzymatic activity.

β-Mannanase activity was assayed by measuring the amount of reducing sugars released from locust bean gum using the 3,5-dinitrosalicylic acid (DNS) method (9). The standard reaction system contained 100 μl of appropriately diluted enzyme sample and 900 μl of 0.5% (wt/vol) locust bean gum in 100 mM Na₂HPO₄-citric acid (pH 6.0). The reactions were carried out at 50°C for 10 min and terminated by the addition of 1.5 ml of DNS reagent. After 5 min of boiling in water, the mixtures were cooled to room temperature and the absorbance at 540 nm was measured. For the control sample, the recombinant enzyme was added after DNS reagent. One unit of β-mannanase activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per min under standard assay conditions. Each experiment was performed in triplicate.