Novel α-L-fucosidases from a soil metagenome for production of fucosylated human milk oligosaccharides
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Novel α-L-fucosidases from a soil metagenome for production of fucosylated human milk oligosaccharides. / Lezyk, Mateusz Jakub; Jers, Carsten; Kjærulff, Louise; Gotfredsen, Charlotte H.; Mikkelsen, Maria Dalgaard; Mikkelsen, Jørn D.
In: P L o S One, Vol. 11, No. 1, e0147438, 2016.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Novel α-L-fucosidases from a soil metagenome for production of fucosylated human milk oligosaccharides
AU - Lezyk, Mateusz Jakub
AU - Jers, Carsten
AU - Kjærulff, Louise
AU - Gotfredsen, Charlotte H.
AU - Mikkelsen, Maria Dalgaard
AU - Mikkelsen, Jørn D.
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
KW - Escherichia coli
KW - Fucose
KW - Humans
KW - Metagenome
KW - Milk, Human
KW - Oligosaccharides
KW - alpha-L-Fucosidase
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1371/journal.pone.0147438
DO - 10.1371/journal.pone.0147438
M3 - Journal article
C2 - 26800369
VL - 11
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 1
M1 - e0147438
ER -
ID: 169136946