Purification and Characterization of Phytase from Bacillus subtilis (natto) N-77

Abstract

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 0.5 mM, 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°C. Its activity was greatly inhibited by reagents and metal ions such as EDTA, Zn²⁺, Cd²⁺, Ba²⁺, Cu²⁺, Fe²⁺, and Al³⁺.