Isolation and characterization of a novel phytase from Penicillium simplicissimum

摘要

Eighty-three isolates from different soil samples exhibited the potential for producing active extracellular phytase. The most active fungal isolate with phytase activity was identified as Penicillium simplicissimum. In shaking culture with enrichment medium, the highest extracellular phytase activity of the producing strain was 3.8 U/mL. The crude enzyme filtrate was purified to homogeneity using uitrafiitration, IEC and gel filtration chromatography. The molar mass of the purified enzyme was estimated to be 65 kDa on SDS-PAGE. The saccharide identification with periodic acid-Schiff reagent (PAS) and activity recognition by l-naphthyl phosphate was all positive. The isoelectric point of the enzyme, as deduced by isoelectric focusing, was pH 5.8, the optimum pH and temperature being pH 4.0 and 55 deg C, respectively. The purified enzyme revealed broad substrate specificity and was strongly inhibited by Fe~(2+), Fe~(3+) and Zn~(2+); however, no inhibition was found by EDTA and PMSF. Phytase activity was inhibited when 2 mmol/L of dodecasodium phytate was added and the K_m for it was determined to be 813 mmol/L.