J Agric Food Chem. 2026 May 27. doi: 10.1021/acs.jafc.5c12942. Online ahead of print.

ABSTRACT

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 (k_cat = 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.

PMID:42200609 | DOI:10.1021/acs.jafc.5c12942