Novel alpha-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

作者：Lezyk Mateusz; Jers Carsten^*; Kjaerulff Louise; Gotfredsen Charlotte H; Mikkelsen Maria D; Mikkelsen Jorn D

来源：PLos One, 2016, 11(1): e0147438.

DOI：10.1371/journal.pone.0147438

This paper describes the discovery of novel alpha-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel alpha-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 alpha-L-fucosidases were substrate -inhibited, moderately thermostable and most hydrolytically active in the pH range 6-7, when tested with para-nitrophenyl-aL-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 alpha-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.

• 出版日期2016-1-22