No abstract

β-N-Acetylhexosaminidases (GH20; EC 3.2.1.52) are exo-glycosidases with a dual activity for cleaving both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis of N-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous β-N-acetylhexosaminidase from Talaromyces flavus (TfHex), which, as a wild type, exhibits a GalNAcase/ GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed in Pichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild type TfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailored N-acetylhexosamines.