title: Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.
abstract: Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 micromol p-nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p-nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60-70 degrees C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N-terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.
authors.affiliation: Faculty of Health Sciences, Okayama University Medical School, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Mori
authors.forename: M
authors.initials: M
authors.lastname: Okamoto
authors.forename: M
authors.initials: M
authors.lastname: Nishibori
authors.forename: M
authors.initials: M
authors.lastname: Ichimura
authors.forename: J
authors.initials: J
authors.lastname: Sakiyama
authors.forename: H
authors.initials: H
authors.lastname: Endo
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification; D002412:Cations; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D017421:Sequence Analysis; D013696:Temperature
chemical_list: D002412:Cations; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase