Extracellular β-Glucosidase Activity Assay

Cell concentration was monitored by measuring OD at 600 nm using a UV-visible spectrophotometer (Biomate5, Thermo, USA). Glucose, cellobiose, cellodextrin and ethanol concentrations were determined by high performance liquid chromatography (HPLC; Agilent Technologies 1200 Series, Agilent, USA) equipped with a refractive index (RI) detector using a Rezex ROA-Organic Acid H⁺ (8%) column (Phenomenex Inc., USA). The column was eluted with 0.005 N of H₂SO₄ at a flow rate of 0.6 ml/min at 50°C.
Extracellular β-glucosidase activity was measured according to the methods reported previously [20 (link)-22 (link, link)]. The culture broth was harvested and centrifuged at 15,000 ×g for 20 min at 4°C. After filtration of the supernatant with a 0.22-μm spin filter, the filtrate (500 μl) was mixed with the same volume of 50 mM sodium citrate buffer (pH 4.8). After addition of 6.7 mM p-nitrophenyl-β-D-glucopyranoside (p-NPG), release of p-nitrophenol (p-NP) was monitored by a spectrophotometer at 400 nm. One unit (U) of β-glucosidase was defined as the amount of enzyme that catalyzes the release of 1 μmol of p-NP per min at 30°C. Volumetric extracellular β-glucosidase activity was expressed as units of β-glucosidase per liter. Specific extracellular β-glucosidase activity was expressed as units of β-glucosidase per gram of dry cell.