Abstract：To evaluate the sufficiency of the N-glycosylation of recombinant phytases from Pichia pastoris systematically， a method for analyzing the amino acid sequence and N-linked glycosylation sites of phytase was established via HPLC-MS/MS. Three strains of pichia pastoris， which have been recombined with different phytase genes， were fermented separately. The phytases were obtained by the sequential purification of ion-exchange and gel filtration. The purified phytases were treated by deglycosylases PNGase F or Endo H. Then the deglycosylated proteins were digested by trypsin. The tryptic mixtures were separated via Nano-HPLC. Peptide sequencing and site identification of N-linked glycosylation were performed on the coupled Tandem MS. The coverage for the detectable protein sequence is above 69% for every protein. Insufficient modifications of glycosylation sites have been observed in all phytases. The glycosylation patterns were different from that reported in the literature. The results demonstrated the efficiency of the detection of N-glycosylation sites in phytase via HPLC-MS/MS， and provide a new basis for the study of glycosylation of phytase.
2018, Vol. 52 
