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Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase.

Abstract : A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the –1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26–57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.
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Submitted on : Tuesday, May 26, 2015 - 2:57:24 PM
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  • HAL Id : hal-01114459, version 1
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David Teze, Franck Daligault, Vincent Ferrières, Yves-Henri Sanejouand, Charles Tellier. Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase.. Glycobiology, Oxford University Press (OUP), 2015, 25 (6), pp.420-427. ⟨hal-01114459⟩



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