2.2.2 Determination of enzyme activity
A total of 10 mmol/L p -nitrophenyl–β-d-glucopyranoside was used as the substrate to analyze the activity of β-d-glucosidase. We then mixed 50 μL of suitably diluted enzyme solution and 100 μL substrate with 200 μL 0.05 mol/L acetic acid buffer (pH 5.0), then added 650 μL deionized water to preheat at 45 ℃ for 5 min. Then, 500 μL 1 mol/L sodium carbonate was added to the mixture to stop the reaction at 45 ℃ for 10 min. The absorbance value was measured at 420 nm. The amount of enzyme required to produce 1 μmol/Lp -nitrophenol per unit time per unit volume defines the β-d-glucosidase activity. The standard curve was plotted by dissolving different concentrations of p -nitrophenol. The calculation formula of enzyme activity is as follows:
Enzyme activity assay =\(\frac{\mathbf{\text{A\ }}\mathbf{\times}\mathbf{\ }\mathbf{\text{N\ }}\mathbf{\times}\mathbf{\text{\ Coefficient}}}{\mathbf{\text{V\ }}\mathbf{\times}\mathbf{\text{\ t}}}\),
A : Absorbance, N : Diluted multiples, V : Sample volume, t : Time, Coefficient : 5.7615.