Abstract
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.
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Acknowledgments
P. B. and V. K. acknowledge support by mobility projects no. LTC18038 and LTC18041 (MEYS, the Ministry of Education, Youth and Sports of the Czech Republic). N. K. acknowledges access to the computing and storage facilities provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085 under the program “Projects of Large Research, Development, and Innovations Infrastructures.”
Funding
This study was funded by the Ministry of Education, Youth, and Sports of the Czech Republic mobility projects nos. LTC18038 and LTC18041, by CESNET (LM2015042), and by CERIT Scientific Cloud (LM2015085) under the program “Projects of Large Research, Development, and Innovations Infrastructures.”
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Bojarová, P., Kulik, N., Slámová, K. et al. Selective β-N-acetylhexosaminidase from Aspergillus versicolor—a tool for producing bioactive carbohydrates. Appl Microbiol Biotechnol 103, 1737–1753 (2019). https://doi.org/10.1007/s00253-018-9534-z
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DOI: https://doi.org/10.1007/s00253-018-9534-z