P nitrophenyl β d cellobioside

Total extracellular protein content in supernatants was measured using a Bio-Rad DC protein assay kit (Bio-Rad) based on absorbance at 595 nm, with bovine serum albumin used as the standard. FPA was measured by the 3,5-dinitrosalicylic acid method [62 (link)]. Exoglucanase activity was measured at 50°C using 1.0 mg/mL p-nitrophenyl-β-d-cellobioside (Sigma-Aldrich) in 50 mM citrate buffer (pH 4.8) as the substrate. The reaction mixture containing 250 µL of properly diluted enzyme and 250 µL of 1.0 mg/mL substrate in 50 mM citrate buffer (pH 4.8) was incubated for 10 min at 50°C, and the reaction was terminated by the addition of 500 µL of 1 M Na₂CO₃. The release of p-nitrophenol (pNP) was monitored at an absorbance at 420 nm. The control was the inactivated enzyme, which was boiled at 100°C for 10 min. pNP was used to generate a standard curve. In exoglucanase activity analyses, one unit of enzymatic activity was defined as the amount of pNP released from the substrate per minute using 1 mL enzyme under the standard assay conditions. Endoglucanase activity in culture supernatants was determined using an azo-cm-cellulose assay kit (Megazyme, Wicklow, Ireland) as described by the manufacturer. Endo-1,4-β-xylanase activities were assayed using an azo-xylan kit (Megazyme) according to the manufacturer’s instructions.

Wheat bran used for enzyme production was procured from a local market; wheat straw and rice straw were procured from local farmer whereas sugarcane bagasse was procured from sugar industry. All the agro residues were washed thoroughly with water, dried at 80°C and stored at room temperature in airtight plastic bags until use. p-Nitrophenyl-α-L-arabinofuranoside (PNAF), p-nitrophenyl-β-D-xylopyranoside (PNXP), p-nitrophenyl-β-D-glucopyranoside (PNGP), p-nitrophenyl-β-D-cellobioside (PNCB), carboxymethyl-cellulose (CMC), Avicel PH-101, birch wood xylan and cellobiose were purchased from Sigma (St. Louis, MO, USA). Commercial cellulase SIGMA (Cellulases from T. reseei ATCC 26,921, ≥700 U/g) was purchased from Sigma-Aldrich, USA. All the chemicals, reagents and media used in the present study were of analytical grade purchased from Qualigens, Hi-media, Merck, Loba from India.

Orange waste used for laccase production was donated by a local restaurant and subsequently dried and milled. Cellic CTec2 enzymatic cocktail and commercial laccase (Novozymes NS-22127) were kindly donated by Novozymes^® (Bagsvaerd, Denmark). The chromatography columns Hiprep Q FF and Superdex 75 10/300 GL were acquired from GE Healthcare Life Science (Chicago, IL, USA). Aminex HPX-87P column and Precision Plus Protein TM Standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Malt Extract Agar, Vanillin, 3,5-dinitrosalicylic acid (DNS), sodium carbonate, and the substrates for enzymatic activities, 3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxyphenol (DMP), xylan beechwood, carboxymethylcellulose (CMC), locust bean, debranched arabinan, β-glucan, p-nitrophenyl-α-L-arabinofuranoside, p-nitrophenyl-β-d-galactopyranoside, p-nitrophenyl-β-d-glycopyranoside, p-nitrophenyl-β-D-xylanopyranoside, and p-nitrophenyl-β-d-cellobioside, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used for the assays were of analytical grade.