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

Abstract

Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60°C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45°C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the K M and 2.8 mmol/(mg*h) for the V max . Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the β-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2′-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome

“…Moreover, considering all the fucosylated glycans produced by both enzymes, Fuc5372 synthesizes lower amounts of FUS than Fuc2358. Different levels of regioselectivity have been already observed among the GH29A family and non-HMO oligosaccharides (α-1,1-, α-1,3-, α-1,4-, or α-1,6-fucosylated) have been synthesized when trying to obtain α-1,2-fucosylated products (Perna et al 2023 ; Thogersen et al 2020 ; Usvalampi et al 2020 ; Zeuner and Meyer 2020 ; Zeuner et al 2018a ). Although studies to evaluate their potential beneficial effects should be performed, these non-HMOs could have a biotechnological application as antiadhesive antimicrobials, as previously observed for other human milk glycans (Newburg et al 2005 ; Ray et al 2019 ), but with the putative advantage of not being metabolized by gut bacteria.…”

An In Silico Approach to Enzymatic Synthesis of Fucooligosaccharides Using α-l-Fucosidase from Thermotoga maritima

“…On the other hand, the effect of the fucosyl-donors on the transfucosylation process can be related to the reactivity showed in the HOMO-LUMO gap, because previous in vitro studies have shown the effective transference of pNP-fucose to lactose to synthesize FucOS [5,6], while other studies have found low yields or long process when fucose itself is used as the donor [7,8]. Thus, ethyl-fucose could show similar results to those obtained with fucose mainly due to both molecules showing similar reactivity.…”

“…Unfortunately, this approach presents the inconvenience of requiring nucleotide sugars as fucosyl-donors, which are more expensive than those used for fucosidases [1,3]. Consequently, FucOS synthesis by fucosylhydrolases like the α-L-fucosidase from Thermotoga maritima has gained importance, as this pathway allows the use either of less expensive fucosyl-donors or even agro-industrial waste [3][4][5][6][7][8]. However, this enzymatic route provides lower yields than the transferase, or involves the release of toxic compounds such as p-nitrophenol [5][6][7][8].…”

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

“…GH29 comprises α- l -fucosidases (EC 3.2.1.51), α-1,3/4- l -fucosidases (EC 3.2.1.111) and a single α-1,2- l -fucosidase (EC 3.2.1.63), which all employ a retaining double-displacement reaction mechanism. Several GH29 members have been employed for transfucosylation [ 6 , 7 , 8 , 13 , 14 , 15 ] and for fucosylation by reverse hydrolysis [ 16 ]. GH29 has been divided into subfamilies GH29A and GH29B based on sequence homology and the resulting substrate specificity: members of GH29B are regiospecific α-1,3/4- l -fucosidases targeting only α-1,3- or α-1,4-linkages with a branched galactose (Gal) residue, whereas members of GH29A have a more relaxed fucosyl substrate specificity [ 17 ].…”