E. coli BL21 CodonPlus (DE3)-RIL strain was transformed with pET23d-BtDex DNA. The transformants were grown in 600 mL Lenox broth containing 50 μg·mL−1 ampicillin and 30 μg·mL−1 chloramphenicol at 37 °C until the culture reached an attenuance of 0.5 at 600 nm. Cultures were agitated (180 rpm) for 30 min at 37 °C, followed by induction of rBtDex expression using 0.2 mm isopropyl β-d-thiogalactopyranoside overnight at 18 °C. Cells were harvested by centrifugation at 8000 g for 20 min at 4 °C, suspended in 50 mL 20 mm potassium phosphate buffer (pH 6.8) containing 0.5 m NaCl and 1.0 mm phenylmethansulfonyl fluoride, and then disrupted by sonication using a Sonifier-250 sonicator (Branson, Danbury, CT, USA). The soluble fraction (764 U, 0.59 U·mg−1) obtained by centrifugation (14 000 g for 20 min at 4 °C), was applied to a DEAE-TOYOPEARL 650M column (2.0 × 15 cm; Tosoh, Tokyo, Japan) equilibrated with 20 mm potassium phosphate buffer (pH 6.5, buffer A), and eluted with a linear gradient of 0–1.0 m NaCl. The pooled active fractions (434 U, 5.40 U·mg−1) were adjusted to 1.5 m ammonium sulfate, and loaded onto a butyl-TOYOPEARL 650M column (1.6 × 12.1 cm; Tosoh) equilibrated with buffer A containing 1.5 m ammonium sulfate, and eluted using a linear gradient of 1.5–0 m ammonium sulfate. The active fractions (214 U, 16.3 U·mg−1) were concentrated and loaded onto a Sephacryl S-100 HR column (3.0 × 80 cm; GE Healthcare, Piscataway, NJ, USA) equilibrated and eluted with buffer A containing 50 mm NaCl. The pool of active fractions (224 U, 20.4 U·mg−1) was dialyzed against buffer A, applied to a DEAE-TOYOPEARL 650M column (2.0 × 15 cm) equilibrated with buffer A, and eluted using a linear gradient of 0–1.0 m NaCl. The pool of active fractions (186 U, 22.7 U·mg−1) was then dialyzed against 20 mm potassium phosphate buffer (pH 6.5) and concentrated using a CentriPrep YM-30 centrifugal filter unit (Millipore, Bedford, MA, USA). All purification steps were performed at 4 °C. Enzyme concentrations were estimated from the amino acid content of 500 pmol protein hydrolysates (6 N HCl for 24 h at 110 °C) using an Amino Tac JLC-500/V amino acid analyzer (JEOL, Tokyo, Japan). One unit (U) of dextranolytic activity was defined as the amount of enzyme that released 1 μmol reducing power per min as determined using the copper bicinchoninate method , with glucose as the standard. The reducing power was measured using 0.4% w/v Dextran T2000 in 20 mm MES/NaOH (pH 6.2) at 35 °C for 10 min.