Purification and characterization of extracellular alkaline phosphatase from an alkalophilic bacterium.

Abstract

From some Japanese soil, we isolated a strain of alkalophilic bacterium, Bacillus sp. OK-1, that excreted alkaline phosphatase into the culture broth. From the cell-free supernatant, the enzyme was successively purified by acetone fractionation, Sephadex column chromatography, and DEAE-cellulose column chromatography. The final preparation thus obtained showed only a single band when analyzed by SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated as 108, 000, and was composed of two subunits having the same molecular weight. The purified enzyme had a pH optimum of 11 and was fairly stable between pH 5-12. This enzyme was inactivated by EDTA. Cobalt ions restored the enzyme activity. The Km was 0.037 mM. On the basis of its properties, this metalloenzyme seems to be a novel extracellular alkaline phosphatase.