caa a22 4500 605498881 CHVBK 20210128100548.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6550-0 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6550-0 Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β- N -acetylhexosaminidases [Elektronische Daten] [Christian Nyffenegger, Rune Nordvang, Birgitte Zeuner, Mateusz Łężyk, Elisabetta Difilippo, Madelon Logtenberg, Henk Schols, Anne Meyer, Jørn Mikkelsen] 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. Springer-Verlag Berlin Heidelberg, 2015 Functional screening nationallicence Protein expression nationallicence Synthetic biology nationallicence Chito-oligosaccharides nationallicence Lacto- N -triose II nationallicence Nyffenegger Christian Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Nordvang Rune Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Zeuner Birgitte Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Łężyk Mateusz Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Difilippo Elisabetta Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut Logtenberg Madelon Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut Schols Henk Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut Meyer Anne Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Mikkelsen Jørn Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7997-8009 0175-7598 99:19<7997 2015 99 253 https://doi.org/10.1007/s00253-015-6550-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6550-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nyffenegger Christian Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 700 1- Nordvang Rune Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 700 1- Zeuner Birgitte Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 700 1- Łężyk Mateusz Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 700 1- Difilippo Elisabetta Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Logtenberg Madelon Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Schols Henk Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708, Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Meyer Anne Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 700 1- Mikkelsen Jørn Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, Søltofts Plads, 2800, Kgs. Lyngby, Denmark aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7997-8009 0175-7598 99:19<7997 2015 99 253