Purification and properties of restriction endonuclease from Deleya marina IAM 14114, a marine bacterium (DmaI).
A restriction endonuclease, designated as DmaI, 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 degrees C in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 100 mM NaCl (pH 7.5). The enzyme was stable up to 55 degrees C 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 PvuII).
Department of Agricultural Chemistry, Shizuoka University, Japan.
H
H
Mizuno
T
T
Suzuki
Y
Y
Yamada
M
M
Akagawa
K
K
Yamasato
D000645:Ammonium Sulfate; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002474:Cell-Free System; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism
D002412:Cations; D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D000645:Ammonium Sulfate