*2.4. Determination of Enzyme Activities and Protein Concentration*

For feruloyl esterase activity assay, ethyl ferulate (EFA) was used as substrate. The reaction (1 mL) was carried out in sodium phosphate buffer (50 mM, pH 7.4) that contained 1 mM EFA. The reaction was initiated by the addition of a diluted enzyme solution. After 15 min incubation at 40 ◦C, the enzymatic activity was terminated by incubation at 100 ◦C for 5 min. The released FA from the substrate was quantified using a Shimadzu HPLC system (Shimadzu Corp, Japan) equipped with a diode array detector (DAD), where the chromatographic separation was achieved using a Phenomenex® C18 5 µm (150 × 4.6 mm) LC column (Phenomenex, United States of America). Ambient conditions were used for analysis. The mobile phase A was 0.01 M phosphoric acid and the mobile phase B consisted of HPLC grade acetonitrile. The isocratic mobile phase consisted of A: 70% and B: 30% and ran for 10 min at a flow rate of 0.8 mL/min. The injected volume was 10 µL and the UV absorption of the effluent was monitored at 320 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of ferulic acid per min under standard conditions.

For xylanase activity assay, 10 mg/mL of insoluble wheat arabinoxylan (WAX) was used as a substrate. The reaction mixture consisted of 300 µL of 1.33% (*w*/*v*) substrate dissolved in 50 mM sodium phosphate buffer (pH 7.4) and 100 µL of diluted enzyme. After 15 min incubation at 40 ◦C, the enzymatic activity was terminated by incubation at 100 ◦C for 5 min, followed by centrifugation at 16,000× *g* for 5 min. The concentrations of reducing sugar were measured using 3,5-dinitrosalicylic acid (DNS) method described previously [22]. Briefly, 150 µL of the sample was mixed with 300 µL of DNS followed by boiling for 5 min. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing sugars per min. Reducing sugars were estimated using a xylose standard curve. The yield of reducing sugars was determined using the following equation:

$$\text{\(\text{\(\text{\(\(\%)} = \text{\(\(\%)} = \text{\(\(\%)} \text{released} \times \text{0.88}))} \times 100\text{)}\text{\(\(\text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\(\(\%)} = \text{\(\(\%)} \text{\)} = \text{)} \text{\)} \text{\)} \text{\)}))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))$$

The Bradford method was used to determine the protein concentration of the enzymes [23]. A protein standard curve was constructed using bovine serum albumin as a suitable standard.
