Azoxylan kit

Protein concentration in the supernatants was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Absorbance was measured at 595 nm, and bovine serum albumin was used as the standard. For protein gel electrophoresis, 20 μL of unconcentrated culture supernatant was loaded onto a polyacrylamide gel (Novex NuPAGE Precast Protein Gels, Thermo Fisher Scientific) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Endoglucanase and endo-1,4-β-xylanase activity in the culture supernatants was determined using an Azo-CM-cellulose assay kit (Megazyme) and an Azoxylan kit (Megazyme), in accordance with the manufacturer’s instructions. All estimates were performed in triplicate assays. The statistical significance of differences between two conditions was analyzed using two-tailed Student’s t test. For all tests, significance was set at a P value of <0.01 (*).

Total extracellular protein content in supernatants was measured using a Bio-Rad DC protein assay kit (Bio-Rad) based on absorbance at 595 nm, with bovine serum albumin used as the standard. FPA was measured by the 3,5-dinitrosalicylic acid method [62 (link)]. Exoglucanase activity was measured at 50°C using 1.0 mg/mL p-nitrophenyl-β-d-cellobioside (Sigma-Aldrich) in 50 mM citrate buffer (pH 4.8) as the substrate. The reaction mixture containing 250 µL of properly diluted enzyme and 250 µL of 1.0 mg/mL substrate in 50 mM citrate buffer (pH 4.8) was incubated for 10 min at 50°C, and the reaction was terminated by the addition of 500 µL of 1 M Na₂CO₃. The release of p-nitrophenol (pNP) was monitored at an absorbance at 420 nm. The control was the inactivated enzyme, which was boiled at 100°C for 10 min. pNP was used to generate a standard curve. In exoglucanase activity analyses, one unit of enzymatic activity was defined as the amount of pNP released from the substrate per minute using 1 mL enzyme under the standard assay conditions. Endoglucanase activity in culture supernatants was determined using an azo-cm-cellulose assay kit (Megazyme, Wicklow, Ireland) as described by the manufacturer. Endo-1,4-β-xylanase activities were assayed using an azo-xylan kit (Megazyme) according to the manufacturer’s instructions.

The mature conidia of M. thermophila strain were inoculated into 100 mL of 1 × VMM with 2% carbon source (arabinan, xylan, or Avicel) at a concentration of 2.5 × 10⁵ conidia/mL in 250-mL Erlenmeyer flasks and incubated at 45 °C with rotary shaking at 150 rpm. Samples were taken at the indicated time for the assays of secreted protein and enzyme activity. The total extracellular protein in culture supernatants was measured using a Bio-Rad protein assay kit. Endo-1,5-l-arabinanase, endo-1,4-xylanase, and endo-glucanase activities in the supernatants of culture of M. thermophila strains were measured using an Azo-Xylan kit (Megazyme), an endo-1,5-l-arabinanase assay kit (Megazyme), and an Azo-CM-Cellulose assay kit (Megazyme), respectively.