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Y. Zou et al. / Carbohydrate Research 373 (2013) 76–81
then lysed in an ice bath by sonication for 10 min at 400 W. After
centrifugation at 4 °C, 12,000g for 30 min, the supernatant was
gathered and loaded onto the HisTrap affinity column, which was
previously equilibrated with start buffer. The target His-tagged
proteins were eluted with elution buffer (500 mM NaCl, 20 mM
phosphate buffer, pH 8.0, 500 mM imidazole). The portions con-
taining the purified proteins were collected and desalted by desalt-
ing column with buffer comprising 50 mM Tris–HCl, and then
concentrated in Tris–HCl buffer by centrifugation at 3500g for
30 min. The purity and molecular weight was analyzed by 12%
SDS–PAGE as described by Laemmli.35 The concentration of the
purified proteins was detected by Bradford method.36 The purified
enzymes were stored in 20% glycerol at ꢀ20 °C for further analysis.
1 mg/mL SpGalU. The reaction was incubated at 42 °C for 24 h.
When no further formation of UDP-Glc was observed by CE and
TLC, the reaction was terminated.
4.6. Purification of UDP-Glc
The reaction was terminated by heating in boiling water bath for
3 min, and subsequently centrifuged at 13,400 rpm at room temper-
ature for 30 min. The supernatant was collected, concentrated and
then loaded onto the top of the Bio-Gel P-2 column (2.5 ꢁ 100 cm)
gel bed, which was preprocessed by degassed deionized water.
The product UDP-Glc was eluted by deionized water and collected
in 10 mL tubes using a fraction collector.36 CE was used to detect
which fractions contain UDP-Glc. The fractions which only comprise
the sugar nucleotide UDP-Glc were gathered in a 50 mL centrifuge
tube. The freeze-dryer was used to lyophilize the solution to a white
powder. Then the UDP-Glc was characterized by mass spectrometry
(negative mode) and 1H NMR.
4.3. Enzymatic assay of SpGalK and SpGalU
The activity of SpGalK toward Gal was measured by the forma-
tion of the product (Gal-1-P). The reactions were carried out in a
final volume of 50 lL, containing 100 mM Tris–HCl (pH 8.5),
4.7. Synthesis of UDP-Glc/Gal derivatives by one-pot three-
enzyme system
10 mM MgCl2, 8 mM ATP, 8 mM Gal, and recombinant SpGalK
(0.5 mg/mL). After incubated at 45 °C for 180 min, the reactions
were terminated by heating in a boiling water bath for 3 min. Sub-
sequently, the reactions were centrifugated at 13,400 rpm for
30 min to remove the precipitation. Then the supernatant was col-
lected and subjected to thin-layer chromatography.37
The activity of GalU was analyzed by product (UDP-Glc) forma-
tion. The reaction was executed in a 50 lL mixture, comprising
100 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 10 mM Glc-1-P, 10 mM
UTP, 1 unit yeast inorganic pyrophosphatase, and recombinant
GalU (0.5 mg/mL). After 180 min incubation at 37 °C, the product
UDP-Glc was detected by thin-layer chromatography.
We chose 14 monosaccharides which could be used by GalK in
previous report36 as substrates for one-pot three-enzyme system
to synthesize various sugar nucleotides. The reactions were carried
out in the system containing 50 mM Tris–HCl (pH 8.0), 10 mM
MgCl2, various monosaccharides, 10 mM ATP, 10 mM UTP,
0.5 mg/mL SpGalK, 0.5 mg/mL SpGalU, and 0.1 U PPase. The reac-
tions were incubated at 42 °C for 24 h. The reactions were termi-
nated by heating at 100 °C for 5 min, followed by centrifugated
at 13,400 rpm at room temperature for 30 min to remove protein
precipitation. The products were determined by TLC, CE and MS.
For CE analysis, 5 lL of each sample was diluted into 50 lL and
subjected to CE analysis as described above.
4.4. Optimization of the reaction conditions
The one-pot reaction was carried out in a final volume of 50 lL.
4.8. ESI-MS Analysis
The reaction mixture contained 100 mM Tris–HCl, 10 mM MgCl2,
10 mM ATP, 10 mM UTP, 10 mM Glc, 1 unit yeast inorganic pyro-
phosphatase, 0.5 mg/mL SpGalK, and 0.5 mg/mL SpGalU. The opti-
mum conditions of the one-pot reaction were measured with
various buffers, at different temperatures and with different concen-
trations of bivalent cation Mg2+. For the optimal pH experiments,
100 mM of each buffer (PBS/Tris–HCl/Glycine–NaOH) from pH 5.6
to pH 9.3 was added into the reaction system. The optimal temper-
ature assays were executed under standard conditions except that
the reactions were incubated under different temperatures (from
0 °C to 52 °C) for 180 min. For the experiments aimed at defining
the optimal Mg2+ concentration, assays were carried out under stan-
dard assay conditions except that different concentrations of Mg2+
(from 0 to 50 mM) were added into the reaction system. After incu-
bation, the reactions were quenched by heating at 100 °C for 3 min.
The amount of product UDP-Glc was detected by a Bechman Coulter
P/ACE MDQ Capillary Electrophoresis (CE) system equipped with a
ESI-MS analysis was carried out using a Shimadzu LCMS-IT-TOF
mass spectrometer. The parameters used were as follows: electro-
spray voltage of 3.5 kV for the negative mode, CDL temperature
200 °C, flow rate of nebulizer gas was 1.5 L/min, and CID MS/MS
collision energy of 50%. A mobile phase which contained 50% aque-
ous acetonitrile and 0.1% formic acid was delivered by a Shimadzu
LC-20 AB pump with 50 lL/min flow rate. The sample (5 lg) was
injected and analyzed in the negative ion mode.
4.9. 600 MHz 1H NMR spectroscopy identification
6 mg UDP-Glc was dissolved in 500 lL D2O at rt for the NMR as-
say. 1H NMR spectra was recorded with the Bruker Avance
600 MHz spectrometer. The chemical shifts were measured by ref-
erence to internal DHO (d = 4.700).
Acknowledgements
UV detector and a 50 cm capillary tubing (75 lm I.D.). Assays were
run at 25 kV with 25 mM sodium borate buffer (pH 9.4) for 20 min.
Percent conversions were calculated from peak areas of UDP-sugar
and UTP monitored by UV absorbance at 262 nm. All assays were
carried out in duplicate.
This work was supported by National Major Scientific and Tech-
nological Special Project for ‘Significant New Drugs Development’
(2012ZX09502001-005), 863 Program (No. 2012AA021504), NSFC
(No. 31070824, 31270983, 31100039), and SRF for ROCS, SEM.
L.C. acknowledges support from University of South Carolina
Salkehatchie.
4.5. Production of UDP-Glc in preparation scale
To demonstrate the application of this one-pot three-enzyme
catalytic system in the synthesis of monosaccharide nucleotides,
UDP-Glc was produced in preparation scale in vitro. The reaction
was performed in 10 mL reaction mixture comprising 100 mM
Tris–HCl, 10 mM MgCl2, 20 mM ATP, 20 mM UTP, 16 mM Glc, 50
units of yeast inorganic pyrophosphatase, 1 mg/mL SpGalK, and
Supplementary data
Supplementary data associated with this article can be found, in
Zou, Yang
