Oat spelts xylan

The endoglucanase activity and filter paper activity (FPA) were measured using CMC-Na (Sigma, USA) and filter paper (Whatman No.1) as substrates, following the method described by Xue et al. [56 (link)]. Xylanase activity was assayed with oat spelts xylan (Sigma, USA) as the substrates [57 (link)]. The assays for these three enzymes were conducted at 50 °C for 10 min in a 0.1 M acetate buffer (pH 4.8). Subsequently, the released reducing sugars were measured using the DNS method [58 ]. The cellobiohydrolase activity was determined in 0.1 M acetate buffer at 50 °C for 30 min with pNPC (Sigma, USA) as substrates according to Liu et al. [59 (link)]. One enzyme activity unit was defined as the number of enzymes required to liberate one μmol glucose or pNP per minute under the assayed conditions. The respiratory rate of T. guizhouense under SSF was evaluated by tracking the release of CO₂. Briefly, the triangular flasks for SSF were sealed with a parafilm for 2 h before sampling, and then 20 mL of gas was extracted with a syringe. The collected gas was injected into the gas sampling bag (E-SWITCH, China), and the contents of CO₂ were determined by gas chromatography (Agilent 7890A) equipped with a Porapak Q column and a flame ionization detector (FID) according to Zhang et al. [60 (link)].

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.

Endoglucanase, exoglucanase and xylanase activities were measured using carboxymethyl cellulose sodium (CMC-Na) (Sinopharm Chemical Reagent Co., China), p-nitrophenyl-β-d-cellobiose (pNPC) (Sigma, USA), and oat spelts xylan (Sigma, USA) as the substrate, respectively, according to the methods described previously [20 (link), 41 (link)]. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol reducing sugars or pNP from the substrate in 1 min. Protein concentrations were determined using the Bradford protein assay kit (ThermoFisher, USA) based on the product description [42 ]. All the enzyme activities were reported as the means of at least three replicates.