Restriction Endonuclease Aor13HI from Acidiphilium organovorum 13H, a New Isoschizomer of BspMII: Purification and Characterization

A restriction endonuclease, Aor13HI, an isoschizomer of BspMII, was purified to homogeneity from cell extracts of Acidiphilium organovorum strain 13H. The enzyme has a molecular mass of 60,000 daltons and consists of two subunits identical in molecular mass of 30,000 daltons. Aor13HI endonuclease, like BspMII, recognizes the palindromic six-base sequence 5′-TCCGGA-3′, and cleaves between the T and C to produce a four-base 5′ extension. Aor13HI is not inhibited by dam-dependent methylation. The isoelectric point of the enzyme is 5.7. Aor13HI activity was maximum at pH 7.5, 100 mm KCl, 7.5-10 mm MgCl₂, and 55°C. The enzyme was stable up to 60°C. The N-terminal amino acid sequence (30 residues) of Aor13HI did not show any similarity with the sequence of other restriction endonucleases reported.