Title
Purification and characterization of phytase produced from Aspergillus niger using solid state fermentation
Authors
SHAHZAD MAHMOOD, MEMUNA GHAFOOR SHAHID, MUHAMMAD NADEEM, RUBINA NELOFER and MUHAMMAD IRFAN

Received May 12, 2014
Published Volume 58 Issue 9-10 September-December
Keywords Aspergillus niger, phytase, purification, FPLC, SDS-PAGE, characterization

Abstract
The current research work was carried out to purify and characterize phytase. The enzyme was purified through precipitation of ammonium sulfate and gel filtration chromatography. The results revealed that, after all the purification steps, the maximum specific activity of 697 U/mg, 15.2 purification fold with 43% phytase yield was obtained. The result showed the appearance of only one band of purified phytase in SDS-PAGE, which indicates high purification of the enzyme and efficiency of the process used for purification. Phytase, after purification, showed a molecular weight of 45.7 kDa. After that, the characterization of the purified phytase was also conducted. The results exhibited that the enzyme was active optimally at pH 5 and 50 °C temperature for 10 min of the incubation period. Enzyme kinetics revealed that purified phytase exhibited Vmax and Km values of 1207.0 IU/mL and 0.5738 mg/mL, respectively, using Na-phytate as substrate. Phytase showed 98% and 86% thermostability at 50 °C and 60 °C, respectively, for 1 h of pre incubation temperature treatment. The pH stability studies revealed that the enzyme retained 100% and 98% relative activity at pH 5 and 6, respectively. Ca2+ and Mg2+ showed positive effects, whereas Mn2+, Na+, Fe2+, Zn2+, and Cu2+ exhibited negative effects on phytase activity.