Directed Evolution of the α-l-Fucosidase from Thermotoga maritima into an α-l-Transfucosidase

Abstract

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.

Synthesis of Fucosyl- N -Acetylglucosamine Disaccharides by Transfucosylation Using α-l-Fucosidases from Lactobacillus casei

“…The reaction efficiency of AlfB was similar to the efficiencies of other ␣-L-fucosidases found in the literature, which ranged from 3% to 30% (18,20,24). Interestingly, the 56% AlfC yield is the maximum percentage described for a wild-type ␣-L-fucosidase, showing that this enzyme has very high transfucosylation activity which resembles that of engineered ␣-L-fucosidases; i.e., yields of pNP-fucosyl-␣-1,2-galactose that ranged from 28% to 65% compared to 7% with wild-type enzyme have been obtained with mutants of an ␣-Lfucosidase from Thermotoga maritima (18). We have recently reported that probiotic bacterium L. casei strain BL23 can use Fuc-␣-1,3-GlcNAc as a carbon source and that AlfB is necessary for this.…”

“…Thus, ␣-L-fucosidases from some bifidobacteria are completely devoid of transfucosylation activity unless they are mutated at specific amino acid sites (19). The reaction efficiency of AlfB was similar to the efficiencies of other ␣-L-fucosidases found in the literature, which ranged from 3% to 30% (18,20,24). Interestingly, the 56% AlfC yield is the maximum percentage described for a wild-type ␣-L-fucosidase, showing that this enzyme has very high transfucosylation activity which resembles that of engineered ␣-L-fucosidases; i.e., yields of pNP-fucosyl-␣-1,2-galactose that ranged from 28% to 65% compared to 7% with wild-type enzyme have been obtained with mutants of an ␣-Lfucosidase from Thermotoga maritima (18).…”

“…In contrast, oligosaccharides have been largely produced using the transglycosylation activity of glycosidases and engineered glycosynthases (14,15). Only a few works describe the synthesis of FUS by transglycosylation using ␣-L-fucosidases; however, they cover a wide range of sources such as mammalian tissues (16), fungi (17), and bacteria (18,19). The transglycosylation activity of ␣-L-fucosidases is generally moderate compared with the hydrolysis activity, although it is variable and depends on the origin of the enzymes (20).…”

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains

“…However, as the regioselectivity and acceptor specificity of glycosidase-catalyzed transglycosylation are usually not that high, the reaction products are frequently obtained as mixtures of several oligosaccharides with varied linkages and sometimes varied lengths. These drawbacks become apparent in the synthesis of fucosyl oligosaccharides using ␣-L-fucosidases (22)(23)(24)(25) and ␣-L-fucosynthases (18) (details are described later) that belong to glycoside hydrolase family 29 (GH29) (26). Such a result prevents the use of these enzymes in the synthesis of oligosaccharides with defined structures.…”

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

“…2 and 3). Feng et al and Osanjo et al developed novel variant forms of glycosidase with high levels of transglycosylation activity through directed evolution based on the modulation of hydrolysis and transglycosylation (6,27). The regioselectivity of the P. furiosus TA mutant enzyme toward ␣-(1,4)-glycosidic linkages was unexpected because generally ␣-(1,6)-linked products are the major transfer products produced by CD-degrading enzymes with Asn and Glu residues at the positions corresponding to H414 and G415 of P. furiosus TA (1,14,28).…”