Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus

Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stear othermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 mu mol 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-ni trophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of app rox. 9.0 and exhibited its highest activity at 60-70 degrees C, It also sho wed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was obser ved in comparison with counterparts from mesophiles. Its partial N-terminal sequence was T(1)FSIVAFDPATGELGIAVQ(19) as estimated by automated Edman de gradation method. A search on the SwissProt database did not reveal any sim ilar protein sequences from other sources.