2.4. β-Xylosidase and β-glucosidase activity analysis of LXYL-P1−1 mutants

The β-xylosidase and β-glucosidase activities of the purified LXYL-P1−1mutants were measured by detecting the amount of p-nitrophenol released from the substrate p-nitrophenyl-β-d-xylopyranoside (PNP-Xyl) or p-nitrophenyl-β-d-glucopyranoside (PNP-Glc) under the optimum reaction conditions12. Concretely, the 60 μL reaction volume contained 50 μL of 5 mmol/L PNP-Xyl or PNP-Glc and 10 μL of 0.1 mg/mL enzyme in 50 mmol/L sodium acetate buffer with pH 5.0. The reaction was performed under 50 °C for 20 min. Reactions were terminated by adding 1 mL saturated Na₂B₄O₇ solution. The enzymatic activity was assayed using spectrophotometry based on the absorbance at 405 nm12. One unit of activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol/L p-nitrophenol per minute.