Enzyme activity assays

Polysaccharide-degrading enzyme activities were analyzed using the 3,5-dinitrosalicylic acid (DNS) method by measuring the amount of reducing sugars liberated [14] according to the standard procedure recommended by the Commission on Biotechnology, IUPAC [15] with modifications on the total reaction volume. Reactions of 3.5 mL contained 100 mM sodium acetate phosphate buffers, pH 5.5 with an appropriate dilution of the enzyme using a 1 × 6 cm Whatman no. 1 filter paper as the substrate and incubated at 50 °C for 60 min for determining the Filter paper activity (FPase) as filter paper unit (FPU). The carboxymethyl cellulase (CMCase), xylanase, mannanase, amylase, and pectinase activities were assayed using 1% (w/v) carboxymethyl cellulose, 1% (w/v) birchwood xylan, 0.5% (w/v) locust bean gum, 1% (w/v) soluble starch, and 0.5% (w/v) pectin from citrus peels as the substrates, respectively. The reactions were incubated at 50 °C for 30 min. The amount of reducing sugars was determined at the end of the reaction by measuring the absorbance at 540 nm using a UV-Vis spectrophotometer microplate reader (Multiskan Ascent, Thermo Scientific, Cambridge, MA) and interpolation from a standard curve prepared using dilutions of the corresponding sugar as standards. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugars from a substrate in 1 min under the assay condition. The β-glucosidase and β-xylosidase activities were assayed using 0.1% (w/v) p-nitrophenyl-β-D-glucopyranoside (PNPG) and p-nitrophenyl-β-D-xylopyranoside (PNPX) as the substrates, respectively in 3 mL reactions containing an appropriate amount of enzyme in 100 mM sodium acetate buffer (pH 5.5). The reactions were incubated at 50 °C for 30 min and terminated by the addition of 2 mL of 1 M Na₂CO₃. The quantity of p-nitrophenolate was measured spectrophotometrically at 405 nm at the end of the reaction. One activity unit (U) is defined as amount of enzyme that produces 1 μmol p-nitrophenolate per minute under the experimental conditions. The total protein concentration of the crude enzyme extracts was determined using Bradford’s method with the BioRad’s Protein Assay reagent (BioRad, Hercules, CA) using bovine serum albumin (BSA) as the standard. The experiments were performed in triplicate.