*4.3. Purification of Dextranase*

First, excess water and other matter were removed from the crude dextranase product by ultrafiltration (Watson Marlow, Cornwall, UK) using a 30,000 NMWC hollow fiber cartridge (GE Healthcare) at room temperature. Deionized water was added three times. The crude enzyme was finally concentrated to one tenth of the original volume of the culture broth. Second, pre-cooled ethanol (−40 ◦C) was added to the crude enzyme solution slowly and agitated gently for 10 min. An ethanol: enzyme ratio of 0.6:1.2 (*v*:*v*) was found to be optimal. After centrifugation for 15 min at 12,000× *g* and 4 ◦C, the precipitate was dissolved in 10 mM Tris-HCl buffer (pH 7.5). Third, ammonium sulfate precipitation was performed using a magnetic stirrer. The supernatant from ethanol precipitation was placed in a beaker within an ice tray. Then 25% saturated ammonium sulfate was added to the solution, which then was allowed to stand at 4 ◦C for 1 h, after which it was centrifuged at 12,000× *g* at 4 ◦C for 20 min. The supernatant was collected, and 60% saturated ammonium sulfate was added. The mixture was allowed to stand for 1 h, and precipitated protein was collected by centrifugation 20 min at 12,000× *g* and 4 ◦C. Then the pellet was dissolved and dialyzed using 10 mM Tris-HCl buffer (pH 7.5). Finally, the enzyme sample was loaded onto a Q-Sepharose column (1.6 cm × 10 cm; GE Healthcare). Chromatographic analysis was conducted by a fast protein liquid chromatography (FPLC;

Bio-Rad, Hercules, CA, USA) at room temperature. Proteins were eluted with several NaCl gradients in 10 mM Tris-HCl (pH 7.5) at a flow rate of 0.8 mL/min. Protein content and enzyme activity for each fraction were monitored. The enzyme-containing fractions were concentrated using ultracel-10k centrifugal filters (Millipore, Burlington, MA, USA).

#### *4.4. SDS-PAGE and Isoelectric Focusing*

SDS-PAGE analysis of dextranase was performed according to Laemmli [48] with minor modifications. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. Isoelectric focusing (IEF) was performed using a Pharmacia PhastGel System (GE Healthcare) on PhastGel IEF 3–9 according to the manufacturer's instructions. An IEF protein marker (mix 3.6–9.3) was used as the pI standard.
