Selective β-N-acetylhexosaminidase from Aspergillus versicolor—a tool for producing bioactive carbohydrates

  • Pavla BojarováEmail author
  • Natallia Kulik
  • Kristýna Slámová
  • Martin Hubálek
  • Michael Kotik
  • Josef Cvačka
  • Helena Pelantová
  • Vladimír Křen
Biotechnologically relevant enzymes and proteins


β-N-Acetylhexosaminidases (EC 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.


Aspergillus versicolor β-N-Acetylhexosaminidase Glycosidase Homology modeling Heterologous expression Pichia pastoris 



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.”


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.”

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2018_9534_MOESM1_ESM.pdf (1.5 mb)
ESM 1 (PDF 1485 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Pavla Bojarová
    • 1
    Email author
  • Natallia Kulik
    • 2
  • Kristýna Slámová
    • 1
  • Martin Hubálek
    • 3
  • Michael Kotik
    • 1
  • Josef Cvačka
    • 3
  • Helena Pelantová
    • 1
  • Vladimír Křen
    • 1
  1. 1.Laboratory of Biotransformation, Institute of MicrobiologyCzech Academy of SciencesPrague 4Czech Republic
  2. 2.Laboratory of Structure and Function of Proteins, Institute of MicrobiologyCzech Academy of SciencesNové HradyCzech Republic
  3. 3.Institute of Organic Chemistry and BiochemistryCzech Academy of SciencesPrague 6Czech Republic

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