PURIFICATION, PROPERTIES AND RECOGNITION SEQUENCE OF SITE-SPECIFIC RESTRICTION ENDONUCLEASE FROM "ACETOBACTER LIQUEFACIENS"

Abstract

A type II restriction endonuclease was purified from "Acetobacter liquefaciens" IAM 1834 by consecutive column chromatography on heparin- Sepharose CL-6B, DEAE-Sepharose CL-6B and Sephacryl S-400 superfine. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The enzyme preparation was essentially free from other nuclease activity, as judged by constancy of a lambda DNA-digest electrophoretic pattern after prolonged incubation for 24hr. The enzyme was optimally active at 37° at pH 7.5, and did not require NaCl, which rather inhibited its activity. The recognition sequence for the enzyme was determined to be 5′-G-G-A-T-C-C-3′, and the enzyme was found to cut between G and G in the sequence, being an isoschizomer of the endonuclease from "Bacillus amyloliquefaciens" H (Bam HI).