title: Purification and characterization of SepII a new restriction endonuclease from Staphylococcus epidermidis.
abstract: A Type II restriction enzyme SepII has been purified to apparent homogeneity from the gram-positive coccus, Staphylococcus epidermidis. The purification included an ammonium sulfate precipitation followed by Q-sepharose, heparin-sepharose and MonoQ column chromatography on an FPLC system. SDS-PAGE analysis showed a denatured molecular weight of 29 kDa. The effects of temperature, pH, NaCl, Mn(2+), Ca(2+), and Mg(2+) ion concentrations were studied to determine the optimal reaction conditions. The enzyme exhibits near maximal levels of activity between pH 8-10, at 10-20mM MgCl(2), 100-150 mM NaCl and 1mM DTT. The results also show that in NEB Buffer 3 the enzyme is active over a broad temperature range from 0 to 70 °C, and in the absence of DNA, enzyme thermostability is observed up to 50 °C for 20 min, while most of the original activity is conserved in 50% glycerol for weeks at room temperature. Single and double digestion in presence of commercial restriction enzymes of known DNA substrates (lambda, pBR322, pET21, pTrcHisB, pPB67) showed that the purified SepII recognized and cleaved the same site as EcoRV. Genomic DNA modification status was also determined.
authors.affiliation: Laboratoire de Biochimie et de Biologie Moléculaire, Faculté des Sciences, Université Hassan II-Ain Chock, Casablanca, km 8, route d'El Jadida BP. 5366, Casablanca, Morocco. belkebirkarim2003@yahoo.fr
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Belkebir
authors.forename: Houssine
authors.initials: H
authors.lastname: Azeddoug
mesh_terms: D000645:Ammonium Sulfate / Q000378:metabolism; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D003067:Coenzymes / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000201:enzymology*; D013696:Temperature
chemical_list: D003067:Coenzymes; D008670:Metals; D004262:DNA Restriction Enzymes; D000645:Ammonium Sulfate