Purification and characterization of a thermostable extracellular phytase from Bacillus licheniformis PFBL-03.

Extracellular phytase from Bacillus licheniformis PFBL-03 was purified in three steps by using ammonium sulfate precipitation, ion-exchange chromatography on a DEAE Sephadex A-50 column, and gel filtration chromatography on Sephadex G-100. The single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that the enzyme was homogeneous. The molecular mass determined from SDS-PAGE was 36 kD. The enzyme yield was 10% while the purification fold was 39. The purified phytase exhibited optimum activity at 55°C and was able to retain 55% of its original activity after 60 min of incubation at 80°C. The purified enzyme had optimum pH of 6.0 and was stable over a pH range of 4.0 to 7.5. The kinetic parameters K(m) and V(max) of the purified phytase for sodium phytate were 4.7 mM and 49.01 µmol/min. The activity of the enzyme was enhanced in the presence of Ca²⁺ but completely inhibited by Fe²⁺, Mn²⁺, and Cu²⁺. The exhibited characteristics of the purified phytase from Bacillus licheniformis PFBL-03 show that the enzyme has potential for applications in the animal feed industry.