Molecular modification of a GH84 β-N-acetylglucosaminidase from Streptomyces violascens for synthesis of lacto-N-triose II using whey powder and chitin-derived N-acetyl chitobiose

β-N-acetylhexosaminidases garnered attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as the backbone precursor of human milk oligosaccharides (HMOs). In this study, β-N-acetylglucosaminidases Hex(Sv)-2557 from Streptomyces violascens ATCC 27968 was engineered based on a stabilizing intermediate strategy to improve its transglycosylation activity for LNT2 synthesis. A mutant Hex(Sv)-2557(D297K) with a transglycosylation activity of 38.4 U/mg with pNP-GlcNAc −1.9-fold higher than that of Hex(Sv)-2557– was obtained and characterized. Instead, the hydrolase activity of the mutant was 73 % lower compared to the wild-type enzyme. Importantly, the mutant can use N-acetyl chitobiose (GlcNAc2) as the donor for LNT2 synthesis. The LNT2 yield of 14.85 % was obtained when the synthetic reaction, catalyzed by the mutant Hex(Sv)-2557(D297K), started from whey powder and GlcNAc2-prepared from chitin by chitinase ChiA and ChiB. This study has altered the donor for the action by directed modification and promoting the high-value utilization of whey powder and chitin.