Hypertransglycosylating Variants of the GH20 β-N-Acetylhexosaminidase for the Synthesis of Chitooligomers
Fungal β-N-acetylhexosaminidases of the CAZy family 20 of glycoside hydrolases are wellestablished tools for the enzymatic synthesis of a wide variety of natural and modified oligosaccharides and glycoconjugates. In order to increase their synthetic efficiency, the β-N-acetylhexosaminidase from Aspergillus oryzae (AoHex) was employed as a model enzyme for enzyme engineering aiming at shifting the reaction course from hydrolysis toward transglycosylation. Specifically, nine mutant variants of AoHex were designed by molecular modeling based on its crystal structure and molecular dynamics simulations. The selected mutation hotspots included the tyrosine residue at the active site, which stabilizes the transition state of the reaction, and two residues at the aglycone-binding site, which were replaced by tryptophan residues to increase the hydrophobicity of this subsite. Besides the individual mutants, combined double-mutant variants were also prepared and characterized. As a result, eight out of the studied new AoHex variants had transglycosidase activity, with V306W/Y445N AoHex being a superior transglycosidase with a transglycosylation-to-hydrolysis ratio greater than 110, which is entirely unique among the hypertransglycosylating glycosidase mutants including the GH20 β-N-acetylhexosaminidases.
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