Sodium carboxymethylcellulose

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.

Polysaccharide lyase could catalyze depolymerization reaction through β-elimination, forming Δ-4,5-unsaturated ends, resulting in the increase of absorbance at 235 nm [20 (link)–22 (link)]. The lyase activity of Cip1 was determined by measuring the absorbance of supernatant at 235 nm after hydrolysis. Supernatants from enzymatic hydrolysis process with and without Cip1 were taken at 24 h, 48 h, 72 h, respectively, and absorbance at 235 nm was measured. Meanwhile, in order to avoid the interference of products with cellulase on absorbance measurement, the absorbance at 235 nm after hydrolysis in buffer with Cip1 alone was also measured (no cellulase addition), and same procedure without Cip1 was as control.
Different p-nitrophenyl derivatives (Sigma, St. Louis, USA) such as p-Nitrophenyl-β-d-glucopyranoside (pNPG), p-Nitrophenyl-d-cellobioside (pNPC), p-Nitrophenyl-β-d-xyloside (pNPX) and p-Nitrophenyl-l-arabinofuranoside (pNPAf), as well as sodium carboxymethylcellulose (Yuanye Bio-Technology Co., Ltd., Shanghai, China), beechwood xylan (Sigma, St. Louis, USA) and glucan (Yuanye Bio-Technology Co., Ltd., Shanghai, China) were used as substrates for measuring potential activities of purified Cip1 according to the methods described in the literatures [23 (link)–25 (link)]. All the activities measurements were performed in 0.05 M sodium acetate buffer (pH 4.8).