Green Synthesis of Chitooligosaccharides and Lacto- N -Triose II Catalyzed by a Novel Bifunctional β- N -Acetylhexosaminidase from the GH3 Family

Human milk oligosaccharides (HMOs) and chitin oligosaccharides possess unique nutritional and prebiotic properties, driving interest in their sustainable synthesis. The key enzyme for their production is β-N-acetylhexosaminidase. Herein, we characterized Nag1420 (accession: PQ041313), a novel GH3 family enzyme from Cellulosilyticum sp., which exhibits both hydrolytic and transglycosylating activities. Substrate specificity analysis revealed a higher activity toward the natural substrate chitobiose than the synthetic analog p-nitrophenyl N-acetyl-β-d-glucosaminide (pNP-GlcNAc), enabling complete hydrolysis to N-acetylglucosamine. Notably, Nag1420 synthesized Lacto-N-triose II (LNT II) via transglycosylation when β-lactose was present as an acceptor. Crucially, in a cascade reaction with Chitinase, Nag1420 directly converted chitin waste into the high-value LNT II. This study provides insights into the green and low-cost biosynthesis of high-value-added carbohydrates.