Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: a biotechnological tool for protein

The elucidation of glycans biological function is essential to
understand their role in biological processes, both normal and
pathological. Immobilized glycoenzymes are excellent tools
for this purpose as they can selectively release glycans from
glycoproteins without altering their backbone. They can be
easily removed from the reaction mixture avoiding their
interference in subsequent experiments. Here, we describe the
immobilization of peptide-N-glycosidase F (PNGase F) onto
silica magnetic nanoparticles with immobilization yields of
86% and activity yields of 12%. Immobilized PNGase F showed
higher thermal stability than its soluble counterpart, and could
be reused for at least seven deglycosylation cycles. It was
efficient in the deglycosylation of several glycoproteins
(ribonuclease B, bovine fetuin, and ovalbumin) and a protein
lysate from the parasite Fasciola hepatica under native
conditions, with similar performance to that of the soluble
enzyme. Successful deglycosylation was evidenced by a
decrease in specific lectin recognition of the glycoproteins
(40%–80%). Moreover, deglycosylated F. hepatica lysate
allowed us to confirm the role of parasite N-glycans in the
inhibition of the lipopolysaccharide-induced maturation of
dendritic cells. Immobilized PNGase F probed to be a robust
biotechnological tool for deglycosylation of glycoproteins and
complex biological samples under native conditions.

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