Biochemical Characterization of a Novel β- N -Acetylhexosaminidase for Efficient Production of N -Acetyl Chitotriose

In this study, a novel β-N-acetylhexosaminidase (AuHex92) was expressed in Escherichia coli. AuHex92 could utilize both p-nitrophenyl-N-acetylglucosamine (pNP-GlcNAc) and N-acetyl chitobiose [(GlcNAc)2] as donors. pNP-GlcNAc as the donor afforded an exceptional N-acetyl chitotriose [(GlcNAc)3] yield (74.5%), whereas (GlcNAc)2 gave a yield of 12.6%. AuHex92 featured a broad entrance, large internal volume, and good flexibility in its catalytic groove, which enabled utilization of both donors, while the hydrophobicity and π-π stacking interactions in the groove improved its transglycosylation activity, with these functional contributions further validated by site-directed mutagenesis. Molecular dynamics simulation results indicated that AuHex92-pNP-GlcNAc exhibited a stable, compact conformation, which enhanced its substrate affinity and transglycosylation activity. AuHex92-(GlcNAc)2 achieved both-donor usage by utilizing a flexible catalytic groove region to guide the entry of the larger (GlcNAc)2 donor. The unique biochemical properties and structural features of AuHex92 establish a foundation for producing (GlcNAc)3 in the food, chemical, and pharmaceutical industries.