2.5. Enzyme Activity Assay

The xylanase and β-xylosidase activities of the complete enzyme mixture and immobilized enzymes were also compared over the course of 24 h to determine any changes in their respective stability and hydrolytic performance. Briefly, β-xylosidase activities were determined by using p-nitrophenyl-β-D-xylobioside (p-NPX) as substrates according to [16,17]. For xylanase activities, birchwood glucoronoxylan was dissolved in 50 mM sodium acetate buffer (pH 4.3) by stirring overnight at room temperature, then 70 μL dissolved xylan substrates were added in microplates with 30 μL of the appropriately diluted enzyme samples and mixed in an incubator at 400 rpm (PHMP Thermoshaker for Microplates, Thomas Scientific Swedesboro, NJ, USA) for various incubation times at 50 °C. The enzymatic reaction was stopped by adding 200 μL of 3,5-dinitrosalicylic acid (DNS) reagent after exactly 5 min, 10 min, 20 min and 30 min incubation. Afterwards, the microplates were placed in an oven at 105 °C and boiled for 30 min, and the reducing sugar content of the samples were analyzed by measuring the absorbance at 540 nm. Xylose standards were used for calibration. The reducing sugar released (μmol) at different hydrolysis times was plotted, and the enzyme activity (μmol/min) was determined by the slope of the linear phase of the hyperbolic curve. The relative xylanase activity was calculated according to Formula (2).