2.3. Enzyme activity assayPolysacch

2.3. Enzyme activity assay
Polysaccharide degrading activities were analyzed based on the amount of liberated reducing sugars using the 3,5-dinitrosalisylic acid (DNS) method [19]. One-hundred microliter reaction mixtures contained the appropriate dilution of enzyme in 50 mM sodium acetate buffer, pH 5.0 and 1% of the corresponding substrate: carboxymethyl cellulose for CMCase activity, birchwoodxylan for hemicellulase activity, pectin from citrus for pectinaseactivity, -glucan for -glucanase activity, and locust bean gumfor mannanase activity. The reaction was incubated at 45◦C for30 min. Cellulase activity, expressed as filter paper unit (FPU),was analyzed in a 1 mL reaction using Whatman number 1 filterpaper (size 1 cm × 6 cm) as the substrate and incubated at 45◦Cfor 60 min. The amount of reducing sugars was determined fromthe absorbance measurement at 540 nm and interpolated froma standard curve of the corresponding sugar. -Glucosidase and-xylosidase activities were determined using p-nitrophenyl--d-glucopyranoside (PNPG) and p-nitrohenyl--d-xylopyranoside(PNPX) as the substrate [20]. One international unit (IU) wasdefined as the amount of enzyme which produced 1 mol of reducing sugar or p-nitrophenolate in 1 min under the assay conditions.