P nitrophenyl d cellobioside pnpc

Six recovered GH5 genes (Cel1–Cel6) with full-length sequence were expressed in E. coli (S2 and S3 Tables) and the purified enzymes were subjected to testing for lignocellulase activity on the substrates carboxymethyl-cellulose (CMC, Sigma, St. Louis, MO, USA), locust bean gum (Sigma, St. Louis, MO, USA), xylan from beechwood (Sigma, St. Louis, MO, USA), and p-nitrophenyl-D-cellobioside (pNPC, Sigma, St. Louis, MO, USA). The six GH5 genes were assigned to different GH5 subfamilies according to phylogenetic analysis based on their closest GH5 gene relatives. Gene sequences, gene expression, enzymatic analyses, phylogenetic analysis, and signal peptide prediction details for the GH5 genes are described in S1 File.

Cellobiohydrolase and β-glucosidase activities were determined by measuring the amount of released p-nitrophenol using p-nitrophenyl-D-cellobioside (pNPC; Sigma, St. Louis, MO, USA) and p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma, St. Louis, MO, USA) as the substrates respectively. The assays were performed in 200 μl of reaction mixtures containing 50 μl of culture supernatant and 50 μl of the respective substrate plus 100 μl of 50 mM sodium acetate buffer (pH 4.8), and were then incubated at 45°C for 30 min. The reaction was stopped by addition of 50 μl of 10% Na₂CO₃ solution. One unit (U) of pNPCase or pNPGase activity is defined as the amount of enzyme releasing 1 μmol of pNP per minute [49 (link)]. SDS-PAGE and western blotting were performed according to standard protocols and CEL7A (CBH1) was immunoblotted using a polyclonal antibody raised against amino acids 426–446 of the protein, as previously described [55 (link)].

p-Nitrophenyl-D-cellobioside (pNPC; Sigma, United States), sodium carboxymethylcellulose (Yuanye Bio-Technology Co., Ltd., Shanghai, China), and p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma, United States) were selected to measure the enzymatic activities of cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BG), respectively. In brief, for determining the enzymatic activities of CBH and BG, 100 μL of diluted enzyme was incubated with 50 μL pNPC (gluconolactone was added as an inhibitor) or pNPG solution (1 mg/mL) for 30 min at 50°C, and then 150 μL of Na₂CO₃ solution (10%, w/w) was added to the reaction system, and the absorbance of mixture at 420 nm was determined. The EG activity was determined by mixing 60 μL of sodium carboxymethylcellulose solution (1%, w/v) and 20 μL of diluted enzyme and then incubating at 50°C for 30 min. DNS method was used to determine the reducing sugar yield in the reaction system (Ghose, 1987 (link)). A unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of product per minute.