Enzymatic Characterization of HoBGLA Activity

For characterization of the hydrolytic activity of HoBGLA using cellobiose and lactose, the glucose oxidase (GOD) and horseradish peroxidase (POD) assays were used as described by Kunst and coworkers (Kunst et al. 1988 ). The assay solutions were prepared by adding GOD and POD to final concentrations of 2.41 and 1.45 U/mL, respectively, to 200 mL solution of 4 mM KH₂PO₄, 6.4 mM 4-aminoantipyrine, and 11 mM phenol pH 7.0.
When lactose or cellobiose was used as substrate, 20 μL enzyme solutions were added to 480 μL of substrate solution in 20 mM Bis-Tris buffer pH 7. The reaction mixtures were incubated at 50 °C using an Eppendorf heat block. After 5 min, the reaction was stopped by heating the reaction mixture at 99 °C for 3 min and the sample was centrifuged at 13,000 rpm for 1 min to pellet the protein precipitate. The sample was allowed to cool at room temperature, and the release of d-glucose was assessed colorimetrically by adding 60 μL of reaction mixture to 600 μL of the GOD/POD assay solution. The assay mixture (660 μL) was incubated in the dark at room temperature for 40 min, and the absorbance at 546 nm was measured. The amount of glucose produced was calculated from a glucose standard curve obtained by adding 60 μL (0.28–3.89 mM) of standard glucose solutions to 600 μL assay solution and incubated at room temperature in the dark for 40 min. One unit of lactase activity was defined as the amount of enzyme releasing 1 μmol of d-glucose per minute under the given conditions. One unit of cellobiose activity was defined as the amount of enzyme releasing 2 μmol of d-glucose per minute under similar conditions as described for determination of β-galactosidase activity using lactose as the substrate.