Catalytic Efficiency for the Chemoenzymatic Synthesis of Core Human Milk Oligosaccharides and Analogs via Sugar Oxazolines

Biocatalytic production of human milk oligosaccharides (HMOs) depends on efficient synthesis of the sugar backbone core. Previous studies engineered a D746E variant of a GH20 β-N-acetylhexosaminidase (BbhI) that appears promising for industrial synthesis of the trisaccharide HMO core precursor lacto-N-triose II (LNTII) using a chemically prepared N-acetylglucosamine oxazoline (NAG-oxa). In this study, a NMR-based quantitative assay was developed to explore the Michaelis–Menten kinetics of N-acetylated sugar oxazoline consumption by D746E BbhI. These results show that chemical conversion of N-acetylhexosamine to an oxazoline-activated sugar dramatically increases turnover relative to p-nitrophenylated sugar (kcat = 277 ± 9 s–1 for NAG-oxa consumption). Enzyme-catalyzed consumption was also observed for N-acetylgalactosamine and N-acetylmannosamine oxazolines. These results represent the first reported Michaelis–Menten kinetics of a glycosynthase with respect to sugar oxazoline consumption and suggest that utilization of substrates beyond that of standard p-nitrophenylated sugars may be useful in carbohydrate-active enzyme (CAZyme) engineering and broaden product oligosaccharide diversity.