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- Title
Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β- N-acetylhexosaminidases.
- Authors
Nyffenegger, Christian; Nordvang, Rune; Zeuner, Birgitte; Łężyk, Mateusz; Difilippo, Elisabetta; Logtenberg, Madelon; Schols, Henk; Meyer, Anne; Mikkelsen, Jørn
- 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 His-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.
- Subjects
BREAST milk; OLIGOSACCHARIDES; GLYCOSYLATION; METAGENOMICS; PROTEIN expression; SYNTHETIC biology; GALACTOSE
- Publication
Applied Microbiology & Biotechnology, 2015, Vol 99, Issue 19, p7997
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-015-6550-0