P nitrophenyl β d glucopyranoside

The cactus cultivar Opuntia Milpa Alta was purchased from Suqian, Jiangsu Province and identified by Professor Chen Huaguo from Guizhou Normal University. Lactase, o-nitrophenyl-β-D-galactoside, α-glucosidase, p-nitrophenyl-β-D-glucopyranoside, PBS, and acarbose were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Petroleum ether (60–90 °C) and 95% ethanol were obtained from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Hyaluronidase, sodium hyaluronate, and cetyltrimethylammonium bromide (CTAB) were obtained from Soleibao. Sodium acetate, potassium dihydrogen phosphate, dipotassium phosphate, magnesium sulfate, and ethylenediaminetetraacetic acid disodium salt were purchased from Zhiyuan Biotechnology Co., Ltd. (Tianjin, China). All other reagents were analytically pure.

For this study, the following instruments were purchased: an LGJ-12 vacuum freeze-dryer from Songyuan Huaxing Technology Develop Co., Ltd. (Beijing, China), an SP-756P spectrophotometer from Shanghai Spectrum Instrument Co., Ltd. (Shanghai, China) and a LightCycler 480 II Real-Time PCR System from Roche Group (Basel, Switzerland). In addition, an AWL-1002-M Aquapro ultrapure water machine (Aquapro, United States) was also used to produce the ultrapure water used in the experiments.
Glucose (cas#: 50–99-7), carboxymethylcellulose (cas#: 9000-11-7), avicel (cas#: 9004-34-6), xylan (cas#: 9014-63-5), dinitrosalicylic acid (DNS, cas#: 609–99-4), p-nitrophenyl-β-D-glucopyranoside (cas#: 2492-87-7), 2,6-dimethoxyphenol (DMP, cas#: 91–10-1), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, cas#: 30931–67-0) and sodium acetate (cas#: 127–09-3) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China).

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).