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<i>N</i>-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by &#946;-<i>N</i>-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. &#946;-<i>N</i>-Acetylhexosaminidases exhibit activity towards both <i>N</i>-acetylglucosamine (GlcNAc) and <i>N</i>-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of &#946;-<i>N</i>-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the &#946;-<i>N</i>-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAc&#946;4GlcNAc disaccharide, a selective ligand of galectin-3.