A type II restriction endonuclease was purified from "Acetobacter xylinus" IFO 3288 by consecutive column chromatographies on heparin-Sepharose CL-6B, DEAE-Sepharose CL-6B, DNA-cellulose and Sephacryl S-400 super fine. The purified enzyme was homogeneous on gel disc electrophoresis and free of other endonuclease, exonuclease and phosphatase activities. The enzyme was optimally active at 37°C at pH7.5 and required 50-150mM NaCl for the enzyme reaction. The enzyme cleaved lambda and M13 mp7 RF DNAs at two and one site, respectively, but did not cleave pBR322, SV40 and φX174 RF DNAs. The recognition sequence for the enzyme was determined to be 5'-C-C-T-N-A-G-G-3', and the enzyme was found to cut between C and T in the sequence, being an isoschizomer of endonuclease from a blue-green alga, Microcoleus species UTEX LB2220 (MstII).
Japan Society for Bioscience, Biotechnology, and Agrochemistry