Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris

Phytases catalyze the cleavage of phosphate groups from phytic acid. Here, we have studied the effects of glycosylation on the properties of Aspergillus japonicus C03 phytase expressed in Pichia pastoris. The enzyme ORF of 1338 nucleotides was cloned from genomic DNA, and encoded a secreted mature protein of 446 amino acids, which included the sequence motif RHGXRX and dipeptide HD, classifying the phytase as a histidine acid phosphate. After transformation and 72 h of induction, P. pastoris GS115 expressed a 75 kDa protein showing 526 U/mg phytase activity and 143 mg/L of protein. The amino acid sequence showed 8 and 3 potential N- and O-glycosylation sites, respectively. Analysis by ESMS showed two glycoform masses of 75,467 and 72,793, which after deglycosylation decreased to 54,327 and 54,128, respectively, indicating a carbohydrate content of 27-30%. A single GlcNAc was assigned at Asn6, Asn38, Asn84, Asn99, Asn209, Asn218, Asn355 and Asn367. The recombinant phytase showed maximum activity at 50 °C, a half-life of 40 min, and farUVCD spectroscopy indicated a secondary structure rich in α-helix. Thermal denaturation analyses reveal the melting temperature varied from 50 °C at pH 6 to a maximum of 66 °C at pH 3 and pH 4.