Colorimetric β-glucosidase Activity Assay

The β-glucosidase activity was measured by determining the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma) using the initial rate of accumulation of coloured reaction product as described by^{9 (link)}. One hundred eighty microliter of 5 mM pNPG substrate was diluted in 50 mM Tris HCl buffer (pH 8.5) and mixed with a 20 μl aliquot of the acquired enzyme. Then, the mixture was incubated at 50 °C for 10 min, and the reaction was terminated by adding 100 μl of ice-cold 0.5 M Na₂CO₃. The release of p-nitrophenol (pNP) via enzymatic hydrolysis was indicated by the appearance of a yellow colour and the absorbance was measured with a UV/Vis microplate spectrophotometer (Multiskan GO, Thermo Scientific) at 405 nm. One unit (U) of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of pNP per minute from the substrate. All experiments were performed in three technical replicates. Statistical significance between treatments and a control group was assessed using post-hoc Tukey's Honestly Significant Difference (HSD) test. Differences were considered statistically significant at a p-value of < 0.05.
Relative activity is as follows: $$\mathrm{Relative}\mathrm{Activity}(\%)=(\mathrm{Activity}\mathrm{of}\mathrm{sample}(\text{U})/\mathrm{Maximum}\mathrm{enzyme}\mathrm{activity}(\text{U}))\ast 100$$