2.8. Production of enzymes and zymographic detection of isoforms
2.8.1. Endocellulase production
Carboxymethyl cellulose (CMC) was used as substrate to determine endocellulase activity. Samples were incubated with 2.0% (w/V) CMC in 50 mM acetate buffer pH 5.0 at 50 C for 60 min. Reducing sugars released during the reaction were determined as described in Section 2.7.2. Glucose was used as standard. One unit (U mL1) of endocellulase activity was defined as the amount of enzyme required to produce 1 mmol of glucose in 1 min at 50 C. Specific endocellulase activity (U mg1) was expressed as 1 U per 1 mg of protein present in the FE.