Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic [beta]-N-acetylhexosaminidases

• Published in: Applied microbiology and biotechnology Vol. 99; no. 19; pp. 7997 - 8009
• Main Authors: Nyffenegger, Christian, Nordvang, Rune Thorbjørn, Zeuner, Birgitte, Lezyk, Mateusz, Difilippo, Elisabetta, Logtenberg, Madelon J, Schols, Henk A, Meyer, Anne S, Mikkelsen, Jørn Dalgaard
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01.10.2015; Springer Nature B.V
• Subjects: Analysis; Biotechnology; Chemical synthesis; Chitin; Crustaceans; E coli; Enzymes; Glycosylation; Human body; Influence; Laboratories; Lactose; Libraries; Microorganisms; Milk; Molecular weight; Nickel; Oligomers; Oligosaccharides; Plasmids; Polymerization; Prebiotics; Protein expression; Studies; Synthetic biology
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6550-0

This paper describes the discovery and characterization of two novel [beta]-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as [beta]-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The [beta]-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His.sub.6-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 [beta]-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-[beta]-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-[beta]-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 [beta]-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.