Purification and Properties of an Extracellular Acid Phytase from Pseudomonas fragi Y9451

Abstract

An extracellular acid phytase from Pseudomonas fragi Y9451 was purified to homogeneity from the culture supernatant by salting-out, DEAE-Sepharose column chromatography, CM-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 74 kDa on gel filtration and 54 kDa and 25 kDa on SDS-PAGE, suggesting that the native enzyme was a heterodimeric protein. The purified enzyme was most active at pH 4.5 and 70°C and fairly stable from pH 4.0- 6.0. It was specific for phytate and exhibited a K m value of 27 mM (sodium phytate, pH 4.5, 50°C). The. phytase activity was strongly inhibited (at maximum by 87%) by Fe 3+ , Cu 2+ , Fe 2+ , and Zn 2+ at 5 mM concentration, and greatly inhibited by Ca 2+ at 10 mM concentration. However, EDTA notably stimulated the phytase activity at 10 mM concentration. With optimum pH and stability, Pseudomonas fragi phytase could be a potential candidate for animal feed applications.