Preparative Isolation of N‐Glycans from Natural Sources Mediated by a Deglycosylating Heterogeneous Biocatalyst in Flow

Abstract Efficient methods for isolating N‐glycans are essential to understanding the functions and characteristics of the entire N‐glycome. Enzymatic release using PNGaseF is the most effective approach for releasing mammalian N‐glycans for analytical purposes. However, the use of PNGaseF for preparative N‐glycan isolation is precluded due to the enzyme′s cost and limited stability. In this work, we develop a PNGaseF heterogeneous biocatalyst for the preparative isolation of N‐glycans from natural sources. By controlling the immobilization conditions, 100–51 % of offered PNGaseF is immobilized on aldehyde‐functionalized agarose porous microbeads through distinct protein orientations, achieving different performances. The enzyme orientation through the N‐terminus provides the best activity/operational stability balance, being 20 % more efficient than that randomly oriented. This active and stable heterogeneous biocatalyst eases its application in a packed bed reactor (PBR) for continuous release of free N‐glycans from a model glycoprotein. This PBR processes 1 g of ovalbumin from chicken egg white to isolate 95 % of its N‐glycans upon operating the PBR for 7 days. Finally, by tuning the flow rate, we can control the profile of N‐glycans isolated due to different enzyme kinetics for the deglycosylation reactions. In‐line methodologies to isolate N‐glycans open new paths for more sustainable protocols to prepare relevant glycans.