Modified lowry protein assay kit

Produced cellulase activities against filter paper (FP), p-nitrophenyl-d-cellobioside (pNPC), sodium salt of carboxymethyl cellulose (CMC), and 4-nitrophenyl-beta-d-galactopyranoside (pNPG) were measured at pH 5.0 throughout cultivation. One unit of FPase or CMCase activity forms 1 μmol of reducing sugar per minute during the hydrolysis reaction, which was quantified by the 3,5-dinitrosalicylic acid method with glucose as a standard (Miller 1959 (link)). One unit of pNPCase or pNPGase activity was defined as the amount of the enzyme needed to produce 1 μmol of p-nitrophenol per minute during the hydrolysis reaction. Xylanase I and xylanase II activities were measured by xylan degradation at pH 3.7 and 5.0, respectively, as described by Stricker et al. (2008 (link)). One unit of xylanase activity was defined as the amount of the enzyme needed to generate 1 μmol of xylose reducing sugar equivalents per minute under the defined assay conditions. Protein concentration was determined by means of the Modified Lowry Protein Assay Kit (Sangon Biotech, Shanghai, China).

The supernatants collected via centrifugation (10,000×g for 10 min at 4 °C) were used for enzyme and secreted protein concentration assays. The FPase and CMCase activities were measured via the DNS method using glucose as a standard. One unit represents the amount of enzyme that formed 1 µmol of reducing sugar per minute during the hydrolysis reaction. The pNPCase and pNPGase activities were measured against p-nitrophenol-d-cellbioside (pNPC) and p-nitrophenyl β-d-glucopyranoside (pNPG) (Sigma-Aldrich, St. Louis, USA), respectively. One unit of pNPCase and pNPGase activity was defined as 1 μmol of p-nitrophenol released per minute during the hydrolysis reaction. Xylanase I and II activities were determined by xylan degradation at pH values of 3.7 and 5.0 [29 (link)], respectively. One unit of xylanase activity is defined as releasing 1 μmol of xylose reducing sugar equivalents per minute under the defined assay conditions. Protein concentration was determined using the Modified Lowry Protein Assay Kit (Sangon Biotech, Shanghai, China). All experiments were performed in three biological replicates. SDS-PAGE electrophoresis was carried out with 12% polyacrylamide separating gel.

For the fungal growth assay, conidia (final concentration 10⁶/mL) from each T. reesei strain were inoculated into 100 mL of the minimal medium [MM, (NH₄)₂SO₄ 5 g/L; Urea 0.3 g/L; KH₂PO₄ 15 g/L; CaCl₂ 0.6 g/L; MgSO₄ 0.6 g/L; FeSO₄·7H₂O 5 mg/L; ZnSO₄·7H₂O 1.4 mg/L; CoCl₂·6H₂O 2 mg/L; pH 5.5] containing 20 g/L glycerol, lactose, or Avicel in 500 mL Erlenmeyer flasks and were cultivated by shaking (200 rpm) at 28 °C for 72 h. Two milliliters of the culture liquid was collected every 12 h for biomass concentration analysis as described by Bischof et al. (2013 (link)). Intracellular protein contents were measured by means of the Modified Lowry Protein Assay Kit (Sangon Biotech, Shanghai, China). The biomass (in dry weight per liter) was quantified by calculating the intracellular protein content in a glycerol-, Avicel-, or lactose-based medium assuming 0.32 g of intracellular protein per gram of dry biomass (Bischof et al. 2013 (link)).