Biochemical characterization

The xylanase activity was assayed by determining the release of reducing sugars from beechwood xylan using the dinitrosalicylic acid (DNS) method [54]. One unit of endo-xylanase activity (1 U) correlates with the release of 1 μmol of xylose equivalent for each minute under the valuation states of pH 4.5, 70 °C, and 10 min.

The determination of the pH activity of the wild-type XylE along with the hybrid mutants was conducted at the optimal temperature for each enzyme for 10 min in 100 mM McIlvaine buffer (pH 3–8). The pH stability of each enzyme was determined by measuring the residual activities under standard conditions (pH 5, 70 °C, and 10 min) after preincubation at 37 °C and pH 1–10 for 1 h. The buffers used were 50 mM glycine–HCl (pH 1–2.5), 100 mM McIlvaine buffer (pH 3–8.5), and 50 mM glycine–NaOH (pH 9–10).

The temperature for the maximal activity of the enzyme was assessed at the optimal pH (100 mM McIlvaine buffer) and at temperatures between 60 and 90 °C for 10 min. The determination of half-lives (t_1/2) of the enzymes was quantified by the remaining activities under standard conditions after different periods of incubation at 70 and 75 °C in the absence of substrate, which was followed by assaying the enzyme activity under standard conditions. To determine the T₅₀ (the temperature at which 50% of the maximal activity of an enzyme is retained), the enzymes (50 μg/mL) were incubated inside a temperature scope of 30 °C to 80 °C for 30 min in the absence of the substrate. Enzymes were put on ice immediately after heating for 10 min, and the remaining activities were then estimated under the standard conditions. All reactions were performed in triplicate.

The K_m, V_max, and k_cat values of the purified wild-type XylE and the hybrid mutants were carried out under standard conditions (pH 4.5, 70 °C, and 5 min) in 100 mM McIlvaine buffer containing 0.5–10 mg/mL beechwood xylan as the substrate. The records were plotted in accordance with the Lineweaver–Burk method. All reactions were conducted in triplicate.