2.7. 7-glucosidase mediated hydrolysis of cellobiosan

Each reaction was set up in 1.5 mL microcentrifuge tubes using cellobiosan at a concentration of 2 mg/mL in the manufacturers recommended buffers: 50 mM sodium maleate, pH 6.5 (T. maritima and Agrobacterium sp.), and 100 mM sodium acetate, pH 5.0 (P. chrysosporium and A. niger). In each reaction, 2.5–20 µg of enzyme was loaded and total reaction volume was 400 µL. A no-enzyme reaction was run simultaneously to ensure cellobiosan cleavage was enzyme-dependent. Reactions were run at 40 °C using a dry-block for 90 min. In a separate experiment evaluating the substrate concentration, 0.125 μM of purified A. niger β-glucosidase was incubated at 40 °C in a 96-well microtiter plate with 0, 0.5, 1, 2, 4, 8, 16 or 32 mM cellobiose or cellobiosan. Reactions were initiated by the addition of enzyme and quenched by boiling in a thin walled PCR tube at 0.5, 1, 5 and 10 min. Following the reaction periods, the tubes were placed on ice, filtered through a 0.2 µm filter and analyzed via HPLC using a carbohydrate column. Standard curves were generated for the substrate (cellobiosan) and products (glucose and levoglucosan) to quantify results. Additionally, the enzymes were analyzed via HPLC in buffer alone (without the addition of cellobiosan) to ensure no carry-over products inherent to the enzyme preparations.