Glycan synthesis with SpyCatcher-SpyTag immobilized Leloir-glycosyltransferases

Human milk oligosaccharides (HMOs) have been proven to play key roles in human development and health benefits. Further research requires high-purity HMOs in large quantities. While enzymatic glycan synthesis has been proven to be highly selective and efficient, it is often associated with costly enzyme production and loss of enzymes during synthesis. Immobilization of enzymes can counteract this disadvantage, making synthesis more cost-effective. In this study, we report a versatile immobilization technique for Leloir-glycosyltransferases (GTs) onto maleimide-activated agarose by employing the SpyCatcher-SpyTag technology. The five GTs: human β1,4-galactosyltransferase 1 (β4GalT), Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase (LgtA), Escherichia coli O55:H7 β1,3-galactosyltransferase (WbgO), Helicobacter pylori α1,2-fucosyltransferase (FutC), and the human α1,3-N-acetylgalactosaminyltransferase GTA/R176G were equipped with SpyCatcher (SpyC), while SpyTag (SpyT) was covalently coupled to the agarose. SpyC-GTs were extensively characterized regarding their preferred buffer system and pH optimum, divalent cation, and substrate kinetics. We successfully immobilized these five SpyC-GTs onto SpyT-agarose, reaching yields between 67 and 100% while retaining their enzymatic activity. Long-term stability over 1 month was enhanced, and reusability for six reactions on three consecutive days was shown. One-pot syntheses employing immobilized SpyC-LgtA, SpyC-WbgO, and SpyC-β4GalT resulted in the primary formation of the tetrasaccharides Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). In sequential syntheses, we were able to produce the HMOs Lacto-N-triose II (LNT II), LNT, LNnT, Lacto-N-fucopentaose type I (LNFP I), Lacto-N-neofucopentaose type I (LNnFP I), and the blood group A antigen hexaoses type I and II (BGA I and II).