title: Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases.
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.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Nyffenegger
authors.forename: Rune Thorbjørn
authors.initials: RT
authors.lastname: Nordvang
authors.forename: Birgitte
authors.initials: B
authors.lastname: Zeuner
authors.forename: Mateusz
authors.initials: M
authors.lastname: Łężyk
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Difilippo
authors.forename: Madelon J
authors.initials: MJ
authors.lastname: Logtenberg
authors.forename: Henk A
authors.initials: HA
authors.lastname: Schols
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.forename: Jørn Dalgaard
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D056186:Metagenomics; D008895:Milk, Human / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D010802:Phylogeny; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D001619:beta-N-Acetylhexosaminidases