Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis.

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining β4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of β4GalT and β3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.