B Cobucci-Ponzano et al.
Enzymatic characterization
50% for 15 min for reactions III and IV. For all the disacchar-
ides isolated except for the product 3, the structural determi-
nation was obtained by 1H mono-dimensional and
homonuclear (1H, 1H) and heteronuclear (1H, 13C) two-
dimensional NMR experiments and by methylation analysis.
The α-galactosidase activity assays, the steady-state kinetic
studies and the azide rescue reactions were performed as
reported previously (1 µg of wild type and 10 µg of
Asp327Gly mutant), but at 65°C and without added bovine
serum albumin (Comfort et al. 2007). Suitable blanks, con-
taining all the reagents with the exception of enzyme, were
always used to take into account the negligible spontaneous
hydrolysis of the substrates. One enzymatic unit is defined as
the amount of enzyme catalyzing the conversion of 1 µmol
substrate into product in 1 min, under the indicated con-
ditions. All kinetic data were calculated as the average of at
least two experiments and were plotted and refined with the
program GraFit (Leatherbarrow 1992).
The effect of temperature on the activity was analyzed by
measuring the α-galactosidase activity of wild type and
Asp327Gly mutant (1 and 10 µg, respectively) in 50 mM
sodium acetate buffer, pH 5.0, in the temperature range 37–
80°C. Blanks with no enzyme were used at each temperature
to subtract the spontaneous hydrolysis of the substrate.
Assays of TmGalA wild type and mutants on permeabilized
cells of E. coli BL21(DE3) were performed by the method of
Zhang and Bremer (1995) at 65°C in 50 mM sodium acetate
buffer, pH 5.0, 2.5 mM 4NP-α-Gal and 0.1–2.0 M sodium
azide.
1
Product 1. H NMR (600 MHz, D2O, 308K): δ 5.87 (d, 1H,
JH-1,H-2 = 3.6 Hz, H-1A), 4.88 (d, 1H, JH-1,H-2 = 3.7 Hz,
H-1B), 3.95 (t, 1H, H-3A), 3.93 (d, 1H, H-4B), 3.90 (t, 1H,
H-5A), 3.87 (d,1H, H-6aA), 3.87 (d, 1H, H-5B), 3.82 (dd,
1H, H-2A), 3.75 (d, 1H, 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 (150 MHz, D2O): δ 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).
1
Product 2. H NMR (600 MHz, D2O, 298K): δ 5.93 (d, 1H,
JH-1,H-2 = 3.3 Hz, H-1A), 5.00 (d, 1H, JH-1,H-2 = 3.8 Hz,
H-1B), 4.09 (t, 1H, H-5B), 3.90 (d, 1H, H-3A), 3.90 (d, 1H,
H-4B), 3.82 (dd,1H, H-3B), 3.79 (dd, 1H, H-2A), 3.69 (t, 1H,
H-5aA), 3.68 (d, 1H, H-2B), 3.67(m, 1H, H-4A), 3.64 (d, 2H,
H-6a,bB), 3.48 (t, 1H, H-5bA); 13C NMR (150 MHz, D2O): δ
97.5 (C-1B), 95.3 (C-1A), 76.0 (C-2A), 72.7 (C-3A), 72.3
(C-5B), 70.3 (C-4B), 70.2 (C-3B), 70.0 (C-4A), 69.3 (C-2B),
63.3 (C-5A), 62.2 (C-6B).
Transgalactosylation trials
1
The glycosynthetic reactions were performed by incubating
Asp327Gly (10 µg) for 16 h at 65°C in 0.1 mL of 50 mM
sodium acetate buffer pH 5.0 at the indicated concentrations
of β-Gal-N3 (donor) and the suitable acceptor. Blank mixtures
without enzyme were always prepared. The products distri-
bution was evaluated by TLC as reported previously
(Cobucci-Ponzano et al. 2009). The transgalactosylation effi-
ciency of Asp327Gly mutant was measured by the use of a
HPAEC-PAD equipped with a PA200 column (Dionex, USA).
Samples were eluted with 20 mM NaOH at a flow rate of 0.5
mL min−1 and the data were analyzed as reported previously
(Cobucci-Ponzano et al. 2009). In particular, to measure the
total amount of galactose enzymatically transferred (total
transgalactosylation efficiency shown in Table II), 1/10 of the
reaction mixtures were incubated for 90 min at 65°C in the
presence of 5.2 µg TmGalA wild type. The efficiency of the
transgalactosylation reaction was calculated as: total amount of
galactose transferred − moles of galactose transferred to water/
total amount of galactose transferred × 100.
Product 4. H NMR (600 MHz, D2O, 298K): δ 5.12 (d, 1H,
J
H-1,H-2 = 7.7 Hz, H-1A), 5.10 (d, 1H, JH-1,H-2 = 3.7 Hz,
H-1B), 4.20 (dd, 1H, H-5aA), 3.87 (dd, 1H, H-3B), 3.86 (t,
1H, H-5B), 3.75 (d, 1H, H-4B), 3.74 (dd,1H, H-2B), 3.69 (t,
1H, H-3A), 3.68 (dd, 1H, H-4A), 3.65 (dd, 2H, H-6a,bB),
3.56 (t, 1H, H-2A), 3.56 (m, 1H, H-5bA); 13C NMR (150
MHz, D2O): δ 101.5 (C-1B), 101.0 (C-1A), 78.7 (C-4A),
75.5 (C-3A), 73.6 (C-2A), 72.7 (C-5B), 70.5 (C-3B), 70.4
(C-4B), 69.7 (C-2B), 65.6 (C-5A), 62.4 (C-6B).
Product 5. 1H NMR (600 MHz, D2O, 315K): δ 5.87 (bs,
1H, H-1A), 4.95 (d, 1H, JH-1,H-2 = 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 (150 MHz, D2O): δ 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).
Characterization of the galactosylated oligosaccharides
1
The glycosynthetic reactions were performed under the same
conditions described in Transgalactosylation trials section in a
total volume of 2 mL. The transgalactosylation products were
purified by reverse phase chromatography (Polar-RP 80A,
Phenomenex, 4 µm, 250 × 10 mm) on an Agilent HPLC
instrument 1100 series and revealed by UV at 220 nm.
Samples were eluted using the following conditions: 40% of
methanol in water for reaction I; 40% of methanol in water
for 5 min, 40–50% for 30 min, 50% for 15 min for reaction
II; 30% methanol in water for 5 min, 30–50% for 30 min,
Product 6. H NMR (600 MHz, D2O, 298K): δ 5.66 (bs, 1H,
H-1A), 5.23 (d, 1H, JH-1,H-2 = 4.0 Hz, H-1B), 4.28 (dd, 1H,
H-2A), 4.10 (dd, 1H, H-3A), 4.00 (dd, 1H, H-5B), 3.90 (d,
1H, H-4B), 3.85 (dd,1H, H-3B), 3.84 (dd, 1H, H-4A), 3.73
(dd, 1H, H-2B), 3.63 (dd, 1H, H-6aA), 3.65 (dd, 1H, H-6aB),
3.67 (dd, 1H, H-6bB), 3.68 (dd, 1H, H-6bA), 3.56 (dd, 1H,
H-5A); 13C NMR (150 MHz, D2O): δ 101.9 (C-1B), 98.9
(C-1A), 79.3 (C-3A), 75.0 (C-5A), 72.7 (C-5B), 70.7 (C-2A),
70.7 (C-3B), 70.5 (C-4B), 69.5 (C-2B), 66.6 (C-4A), 62.2
(C-6B), 61.7 (C-6A).
454