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The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides. [ABSTRACT FROM AUTHOR]

Three novel bacterial a-L-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-a-L-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively a1,6-linked fucose and Eo3812 released only a1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca2+, Mn2+ and Zn2+ ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-a-L-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15%transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-L-Fucosidases catalyze the hydrolysis of α-L-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-L-fucosidase from Thermotoga maritima (TmaFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-Ltransfucosidase starting with the α-L-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs. [ABSTRACT FROM AUTHOR]