Glucose oxidase assay kit

To determine the substrate specificity of AaBGL1 and AaBGL2, cellobiose, beechwood xylan, CMC, Avicel, and different p-nitrophenyl derivatives, such as p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-α-D-glucopyranoside (pNP-α-G), p-nitrophenyl β-D-xylopyranoside (pNPX), and p-nitrophenyl-β-D-cellobioside (pNPC), were used as substrates to measure enzymatic activity. All these substrates were purchased from Sigma, United States. The release of pNP was determined by measuring the absorbance at 405 nm of the mixture using pNP as the standard. One unit (U) of activity was defined as the amount of enzyme released from 1 μmol pNP/min. β-glucosidase activity for cellobiose was tested using the Glucose Oxidase Assay Kit (Abnova, China). One unit (U) of β-glucosidase activity was defined as the amount of enzyme required to release 2 μmol glucose/min from cellobiose. Beechwood xylan, glucan, CMC, and Avicel were measured using the 3,5-dinitrosalicylic acid assay (Miller, 1959 (link)). One unit (U) of enzymatic activity was defined as the amount of enzyme required to release 1 μmol glucose or xylose-equivalent reducing sugars per minute. All the substrates were purchased from Sigma (St. Louis, MO, United States).

β-glucosidase activity was assayed using cellobiose and p-nitrophenyl-β-D-glucopyranoside (pNPG). Activity against cellobiose was determined by adding 10 μg of purified protein to a 200-μl reaction mixture containing 2% (w/v) cellobiose. After 10 min, the activity was tested using a Glucose Oxidase Assay Kit (Abnova, China). One unit (U) of β-glucosidase activity was defined as the amount of enzyme required to release 2 μmol glucose from cellobiose per minute. When using pNPG as the substrate, 10 μg protein was added to a 200-μl reaction mixture containing 2.5 mM pNPG (Sigma, St. Louis, MO, United States). After 5 min of incubation at the optimal temperature, the reaction was stopped by adding 0.6 ml of 1 M Na₂CO₃ (Yang et al., 2015 (link)). The pNP was measured by monitoring the absorbance at 405 nm (Harnpicharnchai et al., 2009 (link)). One unit of β-glucosidase activity was equivalent to 1 μmol of pNP released from the pNPG in 1 min.

Enzymatic hydrolysis of different lignocellulosic materials, such as CMC, Avicel, birch sawdust, and corncob, was performed by adding the appropriate cellulases (Sangon Biotech, China) and/or AaBGL1 and AaBGL2 to a 0.5 ml reaction system containing 50 mM Na₂HPO₄-citric acid buffer (pH 6.0) and 0.5 ml of 1% (w/v) substrate at 40 and 50°C for 24 h. The dosages of cellulases and β-glucosidases (AaBGL1 and AaBGL2) were 0.2 and 0.02 U/mg, respectively, based on the dry weight of substrate (Ye et al., 2017 (link)). Pretreatment of birch sawdust and corncob with ionic liquid (1-ethyl-3-methylimidazolium acetate) was carried out according to the method described in previous studies (Cheng et al., 2012 (link)). After enzymatic hydrolysis, the reactions were terminated by boiling for 10 min. The supernatants were collected by centrifugation at 4°C at 10000 × g for 10 min. The glucose contents of the hydrolysis liquors were measured using the Glucose Oxidase Assay Kit (Abnova, China).