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

• Published in: Applied microbiology and biotechnology 2015-10, Vol.99 (19), p.7997-8009
• Main Authors: Nyffenegger, Christian, Nordvang, Rune Thorbjørn, Zeuner, Birgitte, Łężyk, Mateusz, Difilippo, Elisabetta, Logtenberg, Madelon J., Schols, Henk A., Meyer, Anne S., Mikkelsen, Jørn Dalgaard
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria - classification; Bacteria - enzymology; Bacteria - genetics; Bacteria - isolation & purification; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - genetics; beta-N-Acetylhexosaminidases - metabolism; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Enzyme Stability; Escherichia coli; Glycosylation; Humans; Life Sciences; Metagenomics; Microbial Genetics and Genomics; Microbiology; Milk, Human - chemistry; Milk, Human - metabolism; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Phylogeny; Sequence Alignment; Soil Microbiology; Substrate Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6550-0

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 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 β- N -acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para -nitrophenyl-β- N -acetylglucosamine ( p NP-GlcNAc) as well as para -nitrophenyl-β- N -acetylgalactosamine ( p NP-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.