Characterization of restriction endonuclease BdiSI, an isoschizomer of SfeI, from Bacteroides distasonis strain S-7.

weight), cultivated anaerobically at 37°C for 24hr in a medium containing 0.4% casamino acids, 0.3% yeast extract, 0.3% peptone, 1.0% glucose, 0.15% sodium chloride, 0.1% Tween 80 and 0.04% L-cysteine, pH 7.2, were disrupted by passing through a French pressure cell (Ohtake Works Co.) at 1500-1700kg/cm2. Nucleic acids were removed from the cell extracts by 1%streptomycin treatment, and then the solution was adjusted to 60% (NH4)2SO4 (w/v) by addition of solid (NH4)2SO4. Precipitates were collected and dissolved in buffer A (50 him Tris-HC1, 7mM2-mercaptoethanol and 7 mMMgCl2, pH 7.5). The solution was dialyzed overnight against buffer A. The dialyzed solution (240ml) was used for HPLC on a DEAE-Toyopearlpak 650M column (2.2 x 20cm). The enzyme was eluted with a linear gradient of buffer A containing 0-400raM NaCl (90ml in total). The enzyme was recovered at 60-250 mMNaCl. To the enzyme solution, glycerol was added at a final concentration of 10%(v/v). The solution (420 ml) was put onto a TSKgel Heparin-5PW Glass column (0.8 x 7.0cm). A fraction containing the enzyme was obtained by elution with buffer B (30ml in total, 50mM Tris-HCl, 7mM 2-mercaptoethanol, 7him