PURIFICATION AND CHARACTERIZATION OF PHYTASE FROM BACILLUS-SUBTILIS (NATTO) N-77

An extracellular phytase from Bacillus subtilis (natto) N-77 was purified 322-fold to homogeneity with the specific activity of 8.7 units per mg protein by ultrafiltration, and a combination of Sephadex G-100 and DEAE-Sepharose CL-6B column chromatographies. The molecular weight of the purified enzyme was estimated to be 36 kDa on gel filtration and 38 kDa on SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a monomeric protein. The enzyme had the isoelectric point of pH 6.25, and Ca2+ requirement for the production and activity, the K(m) value of 0.5mM, and the activation energy of 9.87 kcal/mol for sodium phytate. The enzyme proved to be fairly specific for phytate and was most active at pH 6.0-6.5 and 60-degrees-C. Its activity was greatly inhibited by reagents and metal ions such as EDTA, Zn2+, Cd2+, Ba2+, Cu2+, Fe2+, and Al3+.