β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

Jan Muschiol, Marlene Vuillemin, Anne S. Meyer*, Birgitte Zeuner

*Corresponding author for this work

Research output: Contribution to journalReviewResearchpeer-review

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Abstract

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis of terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial β-N-acetylhexosaminidases are mainly involved in cell wall peptidoglycan synthesis, analogously, fungal β-N-acetylhexosaminidases act on cell wall chitin. The enzymes work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, the β-N-acetylhexosaminidases possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis of functional carbohydrates, including biomimetic synthesis of human milk oligosaccharides and other glycan-functionalized compounds. In this review, we summarize the reaction engineering approaches (donor substrate activation, additives, and reaction conditions) that have proven useful for enhancing trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews of reported synthesis reactions with GH20 enzymes, including tables that list the specific enzyme used, donor and acceptor substrates, reaction conditions, and details of the products and yields obtained. We also describe the active site traits and mutations that appear to favor trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. Finally, we discuss novel protein engineering strategies and suggest potential “hotspots” for mutations to promote trans-glycosylation activity in GH20 for efficient synthesis of specific functional carbohydrates and other glyco-engineered products.
Original languageEnglish
Article number365
JournalCatalysts
Volume10
Issue number4
Number of pages34
ISSN2073-4344
DOIs
Publication statusPublished - 2020

Keywords

  • Glycoside hydrolase family 20
  • GH20
  • Lcto-N-biosidase
  • Human milk oligosaccharides
  • Reverse hydrolysis
  • Enzyme engineering
  • Reaction engineering
  • Regioselectivity
  • N-acetylglucosamine
  • Oxazoline

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