Applied Microbiology and Biotechnology

, Volume 99, Issue 19, pp 7997–8009 | Cite as

Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases

  • Christian Nyffenegger
  • Rune Thorbjørn Nordvang
  • Birgitte Zeuner
  • Mateusz Łężyk
  • Elisabetta Difilippo
  • Madelon J. Logtenberg
  • Henk A. Schols
  • Anne S. Meyer
  • Jørn Dalgaard Mikkelsen
Biotechnologically relevant enzymes and proteins

Abstract

This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both β-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors β-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases.

Keywords

Functional screening Protein expression Synthetic biology Chito-oligosaccharides Lacto-N-triose II 

Supplementary material

253_2015_6550_MOESM1_ESM.pdf (1 mb)
ESM 1(PDF 1049 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Christian Nyffenegger
    • 1
  • Rune Thorbjørn Nordvang
    • 1
  • Birgitte Zeuner
    • 1
  • Mateusz Łężyk
    • 1
  • Elisabetta Difilippo
    • 2
  • Madelon J. Logtenberg
    • 2
  • Henk A. Schols
    • 2
  • Anne S. Meyer
    • 1
  • Jørn Dalgaard Mikkelsen
    • 1
  1. 1.Center for BioProcess Engineering, Department of Chemical and Biochemical EngineeringTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Laboratory of Food ChemistryWageningen UniversityWageningenThe Netherlands

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