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.

Fig 1. Phylogenetic tree of novel α-L-fucosidase sequences, Thma and characterised representatives of GH 29 family.

Phylogeny was inferred using the neighbour-joining method. The percentage of replicate trees (>50%) in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. Bacterial α-L-fucosidases are shown in red, eukaryotic in blue and the archeal α-L-fucosidase in purple. Accession numbers for the enzymes included in the analysis are provided in the materials and methods section.

Fig 2. Structure of reaction products from enzymatic trans-fucosylation.

In addition to an undetermined FL, 1: the HMO 2’-FL and the self-condensation products 2: Fucp-(1–2)-Fucp-pNP, 3: Fucp-(1–3)-Fucp-pNP, 4: Fucp-(1–4)-Fucp-pNP were synthesised.

Fig 3. Time study of trans-fucosylation catalysed by Thma and Mfuc5.

Reactions were done using 25 mM pNP-Fuc as donor and 100 mM lactose as acceptor at 30°C and optimal pH. The concentrations were determined by HPAEC-PAD and for pNP by spectrophotometry. L-fucose (■), pNP (⧫), pNP-Fuc (X), 2’-FL (▲), and 3-FL (●).

Fig 4. Time study of trans-fucosylation with xyloglucan as fucosyl donor catalyzed by Mfuc5.

Reaction was done using 5.5 mM xyloglucan-bound fucose as donor and 140 mM lactose as acceptor at 30°C and pH 7. Concentration of L-fucose (■), and FL (●) was determined by HPAEC-PAD.