N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20–70% higher activity (p < 0.05) within pH 6–9, but 15–45% lower activity (p < 0.05) at 45–70 °C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (Td) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower Km vaules, but higher kcat/Km ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.

► We describe an effective enzymatic deglycosylation process of β-D-glucuronidase.
► N-glycosylation increases thermal stability and enzymatic sensitive to metal ions.
► N-glycosylation influences the tertiary structure of β-D-glucuronidase.
► N-glycosylation might be used for functional enhancement of industrial enzymes.