Expression of the Aspergillus niger var. awamori Phytase Gene in Pichia pastoris, and Comparison of Biological Properties

The PhyA gene, encoding myo-inositol hexakisphosphate phosphohydrolase in Aspergillus niger var. awamori (wild-type), was cloned and sequenced. The cDNA was overexpressed by a multicopy gene expression system in Pichia pastoris KM71. Recombinant, wild-type and commercial phytase from Aspergilus ficuum NRRL 3135 (Natuphos) were purified. The PhyA gene of Aspergillus niger var awamori showed perfect homology to the phytase of Aspergillus ficcum and <TEX>$97\%$</TEX> homology to A. niger var awamori (L02421). Wild-type phytase was highly glycosylated and more thermostable than the other two, while deglycosylated farms of three phytases showed identical molecular weight, 507 kDa. After heating at <TEX>$80^{circ}C$</TEX>, wild-type, commercial, and recombinant phytases retained <TEX>$57\%, 32%,;and;8\%$</TEX> of their original activities, respectively. In conclusion, glycosylation plays a key role in the thermostability of phytase and its enzymatic characterization.