X. Li et al.
FULL PAPER
(the mutation site is printed in bold italics). After several selection
cycles, the plasmids of the positive clone were extracted and trans-
formed into E. coli AS1.1739 cells, which were then inoculated onto
LBAmp (Lysogeny broth containing ampicillin) plates and were
grown at 37 °C overnight. Several single colonies were selected for
further 12 h cultivation with 5 mL of LBAmp broth at 37 °C. The
cultivation volume was sequentially amplified to 100 mL (12 h) and
4000 mL (28 h) in LBAmp broth at the same temperature with 1%
inoculum concentration. The protein production was induced by
adding lactose (1% w/v) to the 4000 mL of fermentation broth
(300 rpm, in sterile air). The induced cells were harvested by centri-
fugation (6000 rpm, 15 min) and the pellet was resuspended in a
phosphate buffer (50 mm, pH 6.6). Cell disruption was carried out
by ultrasonication (in ice bath) and the cell debris was removed by
centrifugation. The cell lysate was heated by immersion in an 80 °C
water bath for 15 min and the supernatant was recovered after cen-
trifugation (12000 rpm, 30 min, and 4 °C). The precipitate, which
included TnG-E338A, was obtained from the supernatant by frac-
tional precipitation with the addition of ammonium sulfate to a
final concentration of 60% degree of saturation. The precipitate
was recovered by centrifugation, dissolved in phosphate buffer
(50 mm, pH 6.6), and then subjected to dialysis (in the same buffer)
by using a cellulose membrane tube (MWCO, 8–14 kDa). After
lyophilization, the crude enzyme was redissolved in a small volume
of the buffer, and the solution was passed through a hydrophilic
syringe filter (0.22 μm). The filtrate was loaded to a diethylamino-
ethyl cellulose (DEAE-cellulose) column on a fast protein liquid
chromatography (FPLC) purification system, which was then
eluted with a gradient of buffer A (50 mm phosphate buffer) and
buffer B (buffer A containing 1 m NaCl, 0Ǟ30% gradient) in total
five column volumes. The fractions containing TnG-E338A were
identified by SDS-PAGE (Figure S1, in the Supporting Infor-
mation) and were collected. The resulting solution was lyophilized,
dissolved in the phosphate buffer, and then subjected to dialysis.
Finally, TnG-E338A was isolated by lyophilization in about
10 mgL–1 yield. The purity was assessed to be greater than 95% by
SDS-PAGE.
21, 22, 23, 26, and 40, as shown in Figure 2). HPLC conditions:
Agilent Eclipse XDB-C18 column (250ϫ4.6 mm), eluent CH3CN/
H2O (8:92 for 70 min at 1.5 mLmin–1), UV detection at 301 nm.
Supporting Information (see footnote on the first page of this arti-
cle): Preparation of the TnG-E338A mutant, optimization of the
TnG-E338A-catalyzed reactions, determination of active donors
and acceptors, chemical and enzymatic synthesis (protocols and
data), NMR and MS spectra, HPLC chromatograms.
Acknowledgments
This work was financially supported by the Ministry of Science
and Technology, China (MOST) (973 Program, 2012CB518803),
the National Natural Science Foundation, China (NSFC) (grant
number 81273381), and the Chinese Academy of Sciences (CAS)
(grant number KSCX2-EW-J-6).
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General Procedure for Preparative Scale Reactions Catalyzed by
TnG-E338A Glycosynthase: A solution of glycosyl donor (50 mm),
acceptor (50 mm), and TnG-E338A mutant (180 μg) in a glycine–
NaOH buffer (100 mm, pH 8.8, 1 mL) was incubated at 45 °C. If
necessary, DMF was added to the mixture (25% v/v) to improve
the aqueous solubility of hydrophobic acceptors. Once TLC indi-
cated that the reaction was complete (about 2 days), the mixtures
were then filtered (Nylon 66, 13 mmϫ0.22 μm) and subjected to
preparative HPLC purification (or to gel filtration for compound
27). The fractions containing oligosaccharide or glycoside products
were collected and lyophilized. The isolated yields for the products
were 10–91%, as summarized in Table 1. All products were charac-
terized by NMR spectroscopy and MS [new compounds were fur-
ther analyzed by HRMS(ESI)]. The relevant data are provided in
the Supporting Information.
One-Pot, Parallel Reaction Catalyzed by TnG-E338A Glyco-
synthase:
A mixture containing glucosyl donor 1 (250 mm,
5 equiv.), TnG-E338A mutant (900 μg), acceptors 4, 7, 10, 11, and
13 (50 mm each) in 100 mm glycine–NaOH buffer (pH 8.8, 1 mL)
was incubated at 45 °C. After reacting for 1, 3, 5, 7, 9, 11, 22, 24,
and 26 h, the aliquots of reaction mixture (after removal of the
enzyme by ultrafiltration) were subjected to HPLC analysis. The
time course of reaction traced by HPLC is shown in Figure 2. The
HPLC monitoring indicated that all the glycosyl transfers were
complete within 24 h, and the oligosaccharide product yields
ranged from 9 to 91% (the products were determined as 5, 15, 19,
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