Enzyme Activity Assays for Lignocellulose Degradation

Equally harvested biomass samples of different treatments were transferred to the medium with rice straw as the sole carbon source and incubated at 37 °C to determine the growth rate. For enzyme activity assays, 1 mL of fresh spore suspension (1.0 × 10⁷ spores·mL⁻¹) of different strains was inoculated for SSF. All samples of different treatments were collected on the 4th day, and three biological replicates were collected at each sampling point. Filter paper activity (FPA) and endoglucanase activity (EG) were measured according to the method described by Xue et al. [27 (link)] with filter paper (Whatman NO.1) and CMC-Na (Sigma, St. Louis, MO, USA) as the substrates. Xylanase activity (XYL) was assayed with oat spelts xylan (Sigma, St. Louis, MO, USA) as the substrate [28 (link)]. The reaction system was executed in 0.1 M acetate buffer (pH 4.8) at 50 °C for 10 min, after which the DNS method was used to measure the released reducing sugars. The cellobiohydrolase activity (CBH) was determined in 0.1 M acetate buffer at 50 °C for 30 min with pNPC (Sigma, St. Louis, MO, USA) as the substrate according to Liu et al. [29 (link)]. One enzyme activity unit was defined as the amount of enzyme required to liberate 1 μmol glucose or pNP per minute under the assayed conditions.