Enzyme Assay

BGL activity was assayed in a 0.4 ml of reaction mixture containing 1.25 mM p-nitrophenyl β-D-glucoside (pNPG), 100 mM acetate buffer (pH 5.5), and a suitable amount of enzyme. After incubation for 0–30 min at 30°C, 2 ml of 0.5% Na₂CO₃ was added. The absorbance of the released p-nitrophenol was determined at 420 nm. Activity units (U) were calculated from an initial velocity, and 1 U was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per min. Protein content was measured by the Lowry method [26] using bovine serum albumin as the standard. The transglycosylation ability was also measured under the same conditions as described in the BGL activity using various concentrations of substrate and 50 μg of proteins. After reaction, the products were detected by the thin layer chromatography.

Parameters of glucose inhibition for hydrolysis (K_i) were calculated from Dixon plots [27] using initial velocities. The plots were specially used for calculating in the case of low substrate concentration (0.060–0.34 mM in the absence of glucose and 0.060–2.1 mM for 10 mM glucose addition). Kinetic parameters for hydrolysis were also determined using the Lineweaver–Burk plots after measuring initial reaction velocities at various substrate concentrations (0.060–2.5 mM) [28].