β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.

• Klíčová slova
• Aspergillus versicolor, Glycosidase, Heterologous expression, Homology modeling, Pichia pastoris, β-N-Acetylhexosaminidase,
• MeSH
• Aspergillus enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• chromatografie iontoměničová MeSH
• disacharidy metabolismus   MeSH
• exprese genu MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• Pichia genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Talaromyces enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH
• disacharidy MeSH
• rekombinantní proteiny MeSH