Semi-rational design of transglycosidase
7
uncoated fused silica capillary, 47 cm) (Teze and Dion 2013; Teze
et al. 2014). 200 µL of media containing 10 mM imidazole (used as
an internal standard), 10 mM α-Gal-pNP and the enzyme (enzyme
concentrations are indicated in Tables II–IV) was incubated at 30°C
in phosphate buffer 0.1 M, pH 7, within the capillary electrophoresis
apparatus and analyzed every 24 min for 2.8 h then each hour for the
remaining 7 h. If specified, α-galactosyl-fluoride (α-Gal-F) 10 mM or
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β-galactosyl-azide (β-Gal-N
α-Gal-pNP. If specified, 1-thio-β-D-galactopyranoside (Gal-S-Ph)
0 mM was used as an acceptor, in which case the given transglycosyla-
tion yields refer to the synthesis of phenyl-(α-D-galactopyranosyl)-
1,6)-1-thio-β-D-galactopyranoside (GalGal-S-Ph). Separations were
3
) 10 mM was used as a donor instead of
1
(
performed at 17 kV with 50 mM Borax, pH 9.5, as running buffer.
Donors (except α-Gal-F), acceptor and products were detected by
UV absorbance at 214 nm and quantified by comparison with imid-
azole. Four experiments with the native enzyme and the N334A mu-
tant gave maximum yields within a percent precision. As an example,
thermostability, native enzyme and also AgaB variants are fully stable
in the timescale of experiments.
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glycosynthases for oligosaccharides synthesis. In: Cellulases. 1st ed. Vol.
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Tellier C, Rabiller C. 2001. Modulation of the regioselectivity of a Bacillus
alpha-galactosidase by directed evolution. Glycoconjugate J. 18:215–223.
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tification by saturation mutagenesis of a single residue involved in the
α-galactosidase AgaB regioselectivity. Glycoconjugate J. 18:457–464.
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Converting a β-glycosidase into a β-transglycosidase by directed evolution.
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Svensson B. 2011. Crystal structure of α-galactosidase from Lactobacillus
acidophilus NCFM insight into tetramer formation and substrate binding.
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Funding
This work was supported by the Pays de la Loire and Bretagne regions (Glyconet
Network) and the French Agence Nationale de la Recherche (ANR-10-
BLAN-0718).
Acknowledgments
The authors are grateful to Genadi Chikov, Corentin Léger, Amina Fateh and
Claude Solleux for their help and technical support.
Conflict of interest statement
None declared.
Abbreviations
AgaB: Geobacillus stearothermophilus α-galactosidase B; GalGal-pNP:
4
-nitrophenyl-(α-D-galactopyranosyl)-(1,6)-α-D-galactopyranoside; GalGal-S-Ph:
phenyl-(α-D-galactopyranosyl)-(1,6)-1-thio-β-D-galactopyranoside; Gal-S-
Ph: 1-thio-β-D-galactopyranoside; GH36: glycoside hydrolase family 36;
TmαGal: Thermotoga maritima α-galactosidase; PDB, protein data bank;
WT: wild type; α-Gal-F: α-Galactosyl-fluoride; α-Gal-pNP: 4-nitrophenyl-
Ghazarian H, Idoni B, Oppenheimer SB. 2011. A glycobiology review: Carbo-
hydrates, lectins and implications in cancer therapeutics. Acta Histochem.
1
13:236–247.
Hachem MA, Fredslund F, Andersen JM, Larsen RJ, Majumder A, Ejby M, Van
Zanten G, Lahtinen SJ, Barrangou R, Klaenhammer TR, et al. 2012. Raf-
finose family oligosaccharide utilisation by probiotic bacteria: Insight into
substrate recognition, molecular architecture and diversity of GH36
α-galactosidases. Biocatal Biotransform. 30:316–325.
3
α-D-galactopyranosyl; β-Gal-N : β-galactosyl-azide.
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