N-Glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris.

Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.