Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

Abstract

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α-l-fucosidases is a new route for manufacturing biomimetic HMOs. Seven α-l-fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α-l-1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto-N-fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto-N-tetraose as acceptor with a 39% yield. α-l-Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2'-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively.

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

“…Furthermore, this new product could also be used as a standard when studying unknown enzymatic activities. In this context it is important to note, that a fucosyllactose other than the well-known 2'-FL and 3-FL was found as a translycosylation product in another study [14].…”

“…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].…”

“…Moreover, some α-fucosidases are known to have also transglycosylation activity, adding fucose units to a saccharide structure. All of the transglycosylating α-fucosidases belong to the GH29 family [12][13][14]. Transglycosylation activities are of interest as they can be used to synthesize oligosaccharides without the need for nucleotide-activated sugars as donor molecules.…”

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

“…It has been reported that the presence of an aromatic amino acid in the active site of the fucosidase from T. maritima induces a preference for monosaccharide acceptors, in particular, pNP‐monosaccharides. In fact, several monosaccharides, or their methyl, pNP, or allyl derivatives were used as acceptors in transfucosylation reactions . Ajisaka and Shirakabe and Murata et al.…”

“…Furthermore, transfucosylation reactions demand an activated donor substrate, which means that the fucose moiety that is to be transferred, must form a high energy bond with a good leaving group, so that the energy provided by cleaving this bond contributes to lowering the activation energy in the transglycosylation reaction, for example, mono or disaccharides, a methyl group, a nitrophenyl group, and even some halogens, such as F. By far, the most reported substrate in transfucosylation reactions is pNP‐Fuc . One reason is that many fucosidases recognize this compound as a substrate.…”