Polysaccharide Lyase Activity Assay

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).