Purification and Properties of Restriction Endonuclease from Deleya marina IAM 14114, a Marine Bacterium (DmaI)(Microbiology & Fermentation Industry)

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A restriction endonuclease, designated as Dmal, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37℃ in a reaction mixture (50μl) containing 1.0μg λDNA, 10mM Tris-HCl, 7mM 2-mercaptoethanol, 7 mivi MgCl_2 and 100mM NaCl (pH 7.5). The enzyme was stable up to 55℃ and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5'-CAGCTG-3', cuts between G and C and produces a flush end (isoschizomer of Pvull).
社団法人日本農芸化学会の論文
1990-11-23