The reactionswere incubated in glass tubes at 20◦C for 10 min with agitationin a water bath and then terminated by adding 1.95 mL of 0.2 MNa2CO3.
The released 4MU was measured fluorometrically usinga microplate spectrofluorometer (SpectraMax Gemini EM, Molec-ular Devices, Sunnyvale, CA, USA).
All assays were conducted intriplicate. To quantify chitinolytic activity, one unit was defined asthe amount of enzyme required for the release of 1 mol of 4MUper minute.
The protein concentrations were determined with aprotein quantitative analysis kit (Shenergy Biocolor, China).