Analytical methods Cellulase activity The
Commercial dry yeast was used in the SSF test (Dry yeast ethanol Red™, S. cerevisiae, Fermentis, Co., Ltd., France).
2.2.1. Non-enzymatic protein pretreatment
Biomass substrates (filter paper, 2 g dry weight/100 mL; xylan, 1 g dry weight/100 mL; pretreated rice straw, 2 g dry weight/100 mL) were added into AMD3100 shaking flask containing 50 mM citrate buffer (pH 4.8) and were then autoclaved.
Non-enzymatic protein was added into the flasks at a room temperature to adjust the initial protein concentration to 1.0 mg/mL. These flasks were then placed in a shaking incubator at 150 rpm and 50 °C for 12 h. The substrate at the solid liquid ratio 2 g dry weigh/100 mL substrate could be fully stirred by the shaking incubator.
2.2.2. Enzymatic hydrolysis
Enzyme experiments were carried out using 50 mM citrate buffer (pH 4.8). Enzymatic hydrolysis of substrates was conducted at 7.5 and 15 FPU/g substrate cellulase, with and without a 12 h BSA pretreatment. Incubation conditions were identical as followed during BSA pretreatment (i.e. 150 rpm shaking at 50 °C). Aliquots of 0.5 mL were collected at 0, 3, 6, 24, 48, and 72 h and immediately chilled on ice followed by centrifugation at 10,000 rpm for 5 min. Supernatants were filtered using a 0.22 μm filter paper. Glucose concentration and free cellulase activity was estimated in the clarified supernatants as described below. Unless specified otherwise, all experimental results are cortex an average of two experiments.