Glycosynthase Principle Transformed into Biocatalytic Process Technology: Lacto- N-triose II Production with Engineered exo-Hexosaminidase

Glycosynthases are promising enzyme catalysts for glycoside synthesis. Derived from glycoside hydrolases by mechanistic repurposing of their active site, glycosynthases utilize suitably activated glycosyl donors for glycosylation, yet they are unable to hydrolyze the products thus formed. Although primed for synthetic application by their design, glycosynthases have yet to see actual use in carbohydrate production. To challenge limitations on glycosynthase applicability perceived from the process chemistry point of view, here we developed a glycosynthase (D746E variant) from Bifidobacterium bifidum β-N-acetylhexosaminidase that is highly active synthetically (≥100 μmol min^-1 mg^-1) and fully chemo- and regioselective when using N-acetyl-d-glucosamine 1,2-oxazoline for β-1,3-glycosylation of lactose. We thus established a chemoenzymatic process technology for production of lacto-N-triose II, a core structural unit of human milk oligosaccharides. Using equivalent amounts of oxazoline (prepared chemically in 40% yield from N-acetyl-d-glucosamine) and lactose, we obtained lacto-N-triose II (515 mM; 281 mg mL^-1 90% yield; ≤1 h reaction time) immediately recoverable from the reaction in 85% purity. These metrics of process efficiency reveal the prodigious potential of the glycosynthase for trisaccharide production.