title: Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides.
abstract: This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2'-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Lezyk
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jers
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Louise
authors.initials: L
authors.lastname: Kjaerulff
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Gotfredsen
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Mikkelsen
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005643:Fucose / Q000378:metabolism; D006801:Humans; D054892:Metagenome / Q000235:genetics*; D008895:Milk, Human / Q000737:chemistry*; D009844:Oligosaccharides / Q000378:metabolism*; D005644:alpha-L-Fucosidase / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D005643:Fucose; D005644:alpha-L-Fucosidase