2.4.1. Enzyme Activity Assays

The filter paper activity was determined using a standard method with 1 cm × 5 cm (50 mg) Whatman No. 1 filter paper strips in 0.05 sodium citrate buffer pH 4.8 at 50 ◦C for 1 h and DNS reagent for measuring reducing sugars [26]. The CMCase and xylanase activities were determined by a reducing sugar release at pH 5.0 and 50 ◦C after 10 min using a substrate concentration of 5 mg/mL in the reaction mixture [27]. Avicelase activity was determined by the reducing sugar release at pH 5.0 and 40 ◦C after 60 min of enzyme reaction with Avicel PH105 (5 mg/mL) [28]. Reducing sugars were assayed by the Nelson-Somogyi spectrophotometric method based on molybdenum blue formation in the reaction of molybdic acid reduction by cuprous oxide. A total of 0.2 mL of reducing sugars containing sample solution with 0.2 mL of Somogyi reagent were mixed and incubated at 100 ◦C for 1 h, then sequentially 0.2 mL of Nelson, 0.4 mL of acetone and 1 mL of distilled water were added. Adsorption was measured at 610 nm [29]. One unit of activity corresponded to the quantity of enzyme releasing 1 µmol of reducing sugars (in glucose equivalents) for one minute. The activity against p-NP-β-glucospiranoside was determined at pH 5.0 and 40 ◦C by measuring the p-nitrophenol released, as described elsewhere [28]. One β-glucosidase unit of activity is the quantity of enzyme which liberates 1 micromole of *p*-nitrophenol in one minute. Cellobiase was determined by the incubation of 2.5 mM of cellobiose solution with the enzymes at 40 ◦C and pH 5.0 and measuring the released glucose with the glucose oxidase/peroxidase method [30]. One unit of cellobiase activity is the quantity of enzyme which liberates 1 micromole of glucose in one minute. The activities of the enzyme preparations are

given in the Table 1. Protein concentration was determined according to Lowry protein assay [31] using bovine serum albumin as a standard.


**Table 1.** Properties of *P. verruculosum* enzyme preparations (EP).
