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Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus
Author(s) -
Mori Shuji,
Okamoto Motoi,
Nishibori Masahiro,
Ichimura Mitsuko,
Sakiyama Junko,
Endo Hiroshi
Publication year - 1999
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1999.tb00555.x
Subject(s) - alkaline phosphatase , edman degradation , thermophile , bacillales , enzyme , bacillaceae , thermostability , mesophile , bacillus (shape) , chromatography , specific activity , chemistry , phosphatase , biochemistry , molecular mass , thermal stability , bacteria , biology , peptide sequence , bacillus subtilis , microbiology and biotechnology , organic chemistry , genetics , gene
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 μmol 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 K m 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 °C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg 2+ . In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N‐terminal sequence was T 1 FSIVAFDPATGELGIAVQ 19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.