Cloning, over Expression and Characterization of Alkalin Phytase Enzyme in Escherichia Coli

Phytase (myo-inositol hexakisphosphate) is the hydrolysis enzyme of phytic acid that produces inorganic phosphate. The gene phyC encoding phytase was isolated from Bacillus subtilis ATCC12711 genomic library and sequenced. The nucleotide sequence of the phytase gene contained an open reading frame of 1089 bp, which codes for 90 bp signal peptide and a mature protein with a deduced molecular mass of 42 kDa. Target gene was inserted in pET32a (+) as expression vector and transformed to Escherichia coli BL21 (DE3) as host expression. The fusion phytase phyC-Trx gene was successfully overexpressed in E. coli as an active and cytoplasmic phytase. Recombinant protein production was induced with 1mM IPTG in shaking flasks at 300C in presence of 10 mM calcium. Samples were taken at 0-5 h after induction by 1 hour time intervals, and then protein electrophoresis was done. phyC-Trx protein was expressed in the cytoplasm of E. coli successfully. Molecular weight of recombinant phytas was estimated about 64 kDa. Phytase activity was 7.44 U/ml with using standard phosphates method. Optimum pH for the degradation of phytate was 7. The results of current study showed that thermal stability and cost effective production make this enzyme attractive for using in feed supplements.