To separately determine the activities of the secreted CBH1 and EG2, we used the cellulase specific substrates resorufin-labeled cellobiose and azurine-crosslinked hydroxyethylcellulose (AZCL-HE-cellulose; Megazyme International Ireland Ltd., Wicklow, IRL), respectively. First, we assayed the activity of CBH1 secreted into the supernatant by means of SDS-PAGE analysis with resorufin-labeled cellobiose using a MarkerGene− Fluorescent Cellulase Assay Kit (Marker Gene Technologies, Inc., OR; [31 (link)]). Briefly, 0.25 mM substrate was added to 10-times diluted supernatant and the mixture was incubated for 30 min at room temperature. After the addition of stop buffer, fluorescence intensity was measured (excitation 535/25 nm and emission 590/20 nm) by using an Infinite F500 microplate reader (Tecan Group Ltd., Mannedorf, CHE).
Next, we assayed the activity of EG2 secreted into the supernatant by using the substrate AZCL-HE-cellulose. Diluted supernatants were added to 50 mM citric acid buffer (pH 5.0) containing 200 mg/l AZCL-HE-cellulose and the mixture was incubated for 30 min at 40°C. After the addition of 2% w/v Trizma base solution (Sigma-Aldrich, St. Louis, MO, USA) to stop the reaction, the absorbance of the filtrate at 590 nm was measured against a blank with a SpectraMax Plus 384 microplate reader (Molecular Devices LLC., CA).
Next, we assayed the activity of EG2 secreted into the supernatant by using the substrate AZCL-HE-cellulose. Diluted supernatants were added to 50 mM citric acid buffer (pH 5.0) containing 200 mg/l AZCL-HE-cellulose and the mixture was incubated for 30 min at 40°C. After the addition of 2% w/v Trizma base solution (Sigma-Aldrich, St. Louis, MO, USA) to stop the reaction, the absorbance of the filtrate at 590 nm was measured against a blank with a SpectraMax Plus 384 microplate reader (Molecular Devices LLC., CA).