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.

Production and characterization of Aspergillus niger GH29 family α-fucosidase and production of a novel non-reducing 1-fucosyllactose

“…To our knowledge, there are no previous reports using free fucose as substrate for fucosyllactose production with any enzyme. However, there are α-fucosidases, Mfuc5 from a soil metagenome and FgFCO1 from Fusarium graminearum that have been shown to use xyloglucan as fucose donor [8,14,26].…”

“…org [7]). Currently, the GH151 family contains only three characterized members, and they seem to have poor activity on fucosylated substrates questioning whether they are genuine αfucosidases [7][8][9]. Enzymes of the GH95 family, are highly specific for the hydrolysis of non-reducing terminal L-fucose linked to D-galactose residue by an α-1,2-linkage and are characterized by an inverting mechanism of hydrolysis [10].…”

Employment of fucosidases for the synthesis of fucosylated oligosaccharides with biological potential

“…An important property of the fucosidases of the GH29 family is that in addition to the hydrolysis of fucosyl bonds, they are capable of transferring fucose from a donor substrate to substrates such as methyl, p ‐nitrophenol (pNP), and oligosaccharides or to allyls derived from the latter .…”

“…Unlike the fucosidases of the GH29 family, fucosidases of the GH95 family have not been shown to be capable of synthesizing oligosaccharides .…”

Transglycosylating β‐d‐galactosidase and α‐l‐fucosidase from Paenibacillus sp. 3179 from a hot spring in East Greenland

“…Pichia kluyveri using lactose as a substrate Lezyk et al (2016). created a metagenome library from soil and discovered several putative α-fucosidases, which were recombinantly expressed and proven to have hydrolytic as well as transfucosylating activities, potentially suitable for formation of fucosylated oligosaccharides for supplementing instant formula to mimic the natural composition of human breast milk.…”