β-Glucosidase activity assay

After the fourth subculture, cells were harvested by centrifugation (10,000×g, 4 °C, 10 min) and washed twice with 50 mM phosphate buffer pH 6.5. The cells were resuspended in the same buffer and mixed with glass beads (200–300 μm) to perform mechanical disruption of cells by mixing in five cycles, 2 min each with breaks on ice in between [25]. Then, the suspension was centrifuged (17,000×g, 30 min, 4 °C) to remove the cell debris and glass beads. The supernatant was then filtered through 0.45 μm filter (Merck Millipore, Darmstadt, Germany) and used for enzyme activity assays.

β-Glucosidase activity was determined according to the method described previously with minor modifications [25]. Briefly, 150 μL of 5 mM p-NPG in 50 mM phosphate buffer pH 6.5 were mixed with 600 μL of the appropriate dilutions of cell-free extract and incubated at 37 °C. The reaction was stopped on ice by adding 375 μL of cold 0.1 M NaOH. The amount of p-nitrophenol released from the reaction was measured at 410 nm with a Hitachi U-1100 spectrophotometer (Hitachi Ltd. Tokyo, Japan). The enzyme activity was expressed in Units (U), which defined the amount of p-nitrophenol released from the above reaction per ml per min (with a protein concentration of the cell extract of 1 mg/ml), under the reaction conditions. The protein content was determined using Bradford reagent [26] and bovine serum albumin (BSA) as standard. Optical density of the reaction mixture was read at 595 nm.