Characterization of a GH20 β- N -Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto- N -triose II.

β- N -Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto- N -triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family β- N -acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p -nitrophenyl N -acetyl-β-d-glucosaminide ( p NP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward p NP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM p NP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from p NP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM p NP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 β- N -acetylhexosaminidases and promoting the high-value utilization of whey powder.