beads, thereby being the main reason for the observed decrease
of overall yield. Addition of purified Agm1 in the reaction could
partially restore the whole activity and increase the yield of
UDP-GlcNAc to 78%. After repeated reactions, the deactivated
enzymes were removed from the nickel beads. The agarose
resins were recharged for further uses.
In summary, the UDP-GlcNAc production beads have been
generated by co-immobilization of recombinant enzymes along
the biosynthetic pathway of UDP-GlcNAc on Ni–NTA agarose
resins. We demonstrated that these beads could be used as a
common reagent in the synthesis of UDP-GlcNAc with
relatively high stability. Work is in progress to introduce
recombinant N-acetylglucosaminyltransferases into the multi-
ple-enzyme system. These glycosyltransferases utilize UDP-
GlcNAc as a substrate. The resulting UDP will be re-
phosphorylated to UTP by pyruvate kinase, which is then
consumed to generate another molecule of UDP-GlcNAc by the
immobilized enzymes. Thus, glycoconjugates can be synthe-
sized efficiently without the purification of sugar nucleotides. It
is anticipated that the so-called sugar nucleotide regeneration
beads will become versatile tools for the production of
glycoconjugates and their derivatives with GlcNAc residues.
Fig. 1 Capillary electrophoresis profiles of the enzymatic reaction at 16 h.
Electrophoresis was run in 75 mm 3 50 cm (40 cm to detector) bare fused
silica capillary, under 22 kV with UV detection at 262 nm. UDP-GlcNAc
has a retention time of 14.7 ± 0.2 min. The identities of each peak are as
follows: 1. UDP-GlcNAc; 2. ATP; 3. UTP; 4. ADP; 5. UDP.
Notes and references
† Uridine 5A-diphosphate N-acetylglucosamine (1.18 g, 92%). 1H NMR
(D2O, 40 0 MHz): d 7.90 (d, H-6B, J 8.1 Hz), 5.93 (d, H-1A, J 3.3 Hz), 5.92
(d, H-5B, J 8.1 Hz), 5.46 (dd, H-1, J 7.3, 3.3 Hz), 4.32 (m, 2H, H-2A, H-3A),
4.11–4.23 (m, 3H, H-4A, H-5Aa,b), 3.72–3.97 (m, 5H, H-5, H-4, H-3, H-
6a,b), 3.49 (t, 1H, H-2, J 8.7 Hz), 2.02 (s, 3H, Ac); 13C NMR (D2O, 100
MHz): d 174.99, 166.50, 152.04, 141.86, 102.86, 94.74, 88.71, 83.42,
74.02, 72.23, 71.15, 69.85, 69.70, 65.22, 61.57, 60.52, 59.62, 53.94, 53.86,
22.30; 31P NMR (D2O, 162 MHz): d 210.24, 211.91; ESI-MS (m/z):
607.76 (M + H+), 629.83 (M + Na+), 651.80 ((M 2 H) + 2Na+), 673.80
Fig. 2 The time course for UDP-GlcNAc production by multi-immobilized
enzymes. Reaction mixture consisted of 20 mM GlcNAc, 20 mM UTP, 1
mM ATP, 40 mM Glc-1,6-dPi, 20 mM PEP, 10 mM MgCl2, 50 mM KCl and
50 mM Tris-HCl (pH 7.0) in a total volume of 100 mL.
((M 2 2H) + 3Na+), 302.57 (M 2 2H)22
,
605.96 (M 2 H)2, 627.96
G-15 gel filtration column with water as the mobile phase. The
product containing fractions were pooled and lyophilized to
give 1.18 g sugar nucleotide. NMR spectroscopy and mass
spectrometry (ESI-MS) were utilized to identify the produced
UDP-GlcNAc.†
(M 2 2H + Na+)2.
1 J. E. Heidlas, K. W. William and G. M. Whitesides, Acc. Chem. Res.,
1992, 25, 307–314; J. E. Pallanca and N. J. Turner, J. Chem. Soc. Perkin
Trans. 1, 1993, 23, 3017–3022; V. Wittmann and C.-H. Wong, J. Org.
Chem., 1997, 62, 2144–2147.
The stability of multiple enzyme beads was demonstrated by
repeated synthesis of UDP-GlcNAc. As expected, these beads
were recyclable, but lost some enzymatic activities during the
reactions. A 50% yield of product can still be achieved after five
20 h reaction cycles (Fig. 3). Further enzymatic assays revealed
that GlcNAc phosphate mutase was the least stable enzyme on
2 J. E. Heidlas, W. J. Lees, P. Pale and G. M. Whitesides, J. Org. Chem.,
1992, 57, 146–151; B. Leiting, K. D. Pryor, S. S. Eveland and M. S.
Anderson, Anal. Biochem., 1998, 256, 185–191.
3 K. Okuyama, T. Hamamoto, K. Ishige, K. Takenouchi and T. Noguchi,
Biosci. Biotechnol. Biochem., 2000, 64, 386–92; K. Tabata, S. Koizumi,
T. Endo and A. Ozaki, Biotechnol. lett., 2000, 22, 479–483.
4 Z.-Y. Liu, J.-B. Zhang, X. Chen and P. G. Wang, ChemBioChem, 2002,
3, 348–355; L. Revers, R. M. Bill, I. B. Wilson, G. M. Watt and S. L.
Flitsch, Biochim. Biophys. Acta., 1999, 1428, 88–98; X. Chen, J. Fang, J.
Zhang, Z. Liu, J. Shao, P. Kowal, P. Andreana and P. G. Wang, J. Am.
Chem. Soc., 2001, 123, 2081–2082; S. Nishiguchi, K. Yamada, Y. Fuji,
S. Shibatani, A. Toda and S. Nishimura, Chem. Commun., 2001, 19,
1944–1945.
5 T. Yamada–Okabe, Y. Sakamori, T. Mio and H. Yamada-Okabe, Eur. J.
Biochem., 2001, 268, 2498–2505.
6 M. Hofmann, E. Boles and F. K. Zimmermann, Eur. J. Biochem., 1994,
221, 741–747.
7 K. Brown, F. Pompeo, S. Dixon, D. Mengin-Lecreulx, C. Cambillau and
Y. Bourne, EMBO J., 1999, 18, 4096–4107.
8 E. Ponce, N. Flores, A. Martinez, F. Valle and F. Bolivar, J. Bacteriol.,
1995, 177, 5719–5722.
Fig. 3 Recyclability of immobilized recombinant enzymes for the synthesis
of UDP-GlcNAc. All reactions were run at 30 °C for 20 h.
9 R. Lahti, T. Pitkaranta, E. Valve, I. Ilta, E. Kukko-Kalske and J.
Heinonen, J. Bacteriol., 1988, 170, 5901–5907.
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