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H-6aB), 3.74 (d, 1H, H-2B), 3.72 (d, 1H, H-6bB), 3.71 (d, 1H, H-
6bA), 3.58 (dd, 1H, H-3B), 3.56 (t, 1H, H-4A); 13C NMR (500
MHz, D2O): d 99.2 (C-1B), 97.7 (C-1A), 74.5 (C-3A), 73.2 (C-5B),
72.2 (C-2A), 72.2 (C-5A), 70.8 (C-4A), 70.8 (C-3B), 70.5 (C-4B),
69.6 (C-2B), 66.9 (C-6A), 62.4 (C-6B).
Hydrolytic reactions
Hydrolytic activity of the enzymes TmGalA and TmGalA-HisTag
were determined by spectrophotometrical quantication of pNP
liberated by the hydrolysis of different pNP-substrates: pNP-a-
Fuc, pNP-b-Fuc, pNP-a-Gal, pNP-b-Gal, pNP-a-Glc, pNP-b-Glc,
pNP-a-Man, pNP- b-D-Man, pNP-b-GalNAc and pNP-b-GlcNAc.
Reactions were carried out using a solution of pNP-substrate
5 mM in sodium acetate buffer 50 mM, pH 5 in 300 ml during
5 minutes at 65 ꢁC and 10 mg of enzyme. Reactions were stopped
by addition of 300 ml of sodium carbonate 0.5 M and its
absorbance measured at 410 nm. Each assay was determined at
least three times with standard deviation under 5% of the
average of samples. One enzyme unit (U), was dened as the
amount (mg) of protein that hydrolyzes 1.0 mmol of substrate
per minute.
1
Gal-a(1–6)-Man-a-pNP. H NMR (500 MHz, D2O): d 5.87 (bs,
1H, H-1A), 4.95 (d, 1H, JH-1,H-2 ¼ 3.8 Hz, H-1B), 4.30 (dd, 1H, H-
2A), 4.14 (dd, 1H, H-3A), 4.00 (d, 1H, H-4B), 3.98 (t, 1H, H-5A),
3.97 (dd, 1H, H-6aA), 3.90 (t, 1H, H-5B), 3.89 (t, 1H, H-4A),
3.82 (dd, 1H, H-6aB), 3.81 (dd, 1H, H-2B), 3.78 (dd, 1H, H-
6bB), 3.74 (dd, 1H, H-6bA), 3.66 (dd, 1H, H-3B); 13C NMR (500
MHz, D2O): d 99.3 (C-1B), 99.2 (C-1A), 73.8 (C-5B), 72.3 (C-5A),
72.0 (C-3A), 71.1 (C-2A), 71.0 (C-3B), 70.6 (C-4B), 69.8 (C-2B),
68.0 (C-4A), 67.0 (C-6A), 62.4 (C-6B).
Acknowledgements
Transgalactosylation
This work was supported by two research projects: one of the
´
˜
Spanish MICINN (Ministerio de Ciencia e Innovacion de Espana
CTQ2012-32042) and one of the Agenzia Spaziale Italiana
(Project MoMa. 1/014/06/0).
pNP-a-Gal and Gal-b-N3 (14 mM) were used as donors for the
reactions with TmGalA-HisTag and TmGal D327G-HisTag,
respectively. Different substrates were used as acceptors (14
mM): Glc, Man, Gal, Fuc, Fru, GalNAc, GlcNAc, pNP-a-Fuc, pNP-
b-Fuc, pNP-a-Gal, pNP-b-Gal, pNP-b-Glc, pNP-a-Man, pNP-b-
Man, pNP-b-GalNAc and pNP-b-GlcNAc. Substrates were dis-
solved in 100 ml of buffer sodium acetate 50 mM, pH 5. In
reactions with SDB, they were added to a nal concentration of
2 M. In reaction with ILs, they were added to a 30% concen-
Notes and references
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ꢁ
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ꢁ
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¨
The scaled-up reaction mixture was composed by b-Gal-N3 14
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