2.3. Chitinase assay

Chitinolytic activity was estimated by dinitrosalicylic acid (DNS) method using colloidal chitin as a substrate according to the method described by Sadfi et al. (2001) with some modification. The reaction matrix had 0.5 mL of 1% colloidal chitin suspension in 0.1 M sodium acetate buffer pH5, 0.4 mL of enzyme solution. The mixture then incubated 30 min at 50 °C and then the reaction was terminated by 1mL DNS (NaOH 10 g/L, dinitrosalicylic acid C₇H₄N₂O₇ 10 g/L, phenol C₆H₆O 2 g/L, and adding Na₂SO₃0.05g/100mL and sodium potassium tartrate C₄H₄KNaO₆.₄H₂O 20 g/100mL when using). The color of the mixture was developed by incubating it for 10 min at 100 °C. Centrifugation was performed at 7500 ×g for 10 min, then the supernatant adsorption was measured at 540nm. A standard curve was plotted using N-acetyl glucosamine (NAG, Sigma). One unit (U) of chitinase activity represent the amount of released enzyme of 1μmol N-acetyl glucosamine of colloidal chitin per min under reaction conditions.