Whole-Cell Biocatalytic Synthesis of N-Acetyl-d-neuraminic Acid
COMMUNICATIONS
CACCCGCGCTCTTGCAT-3’ (BamHI cleavage site under-
lined) as primers based on the published nanA gene se-
quence (GenBank accession no. D00067). The 0.9-kb PCR
product was digested with NcoI and BamHI and purified
using Wizardꢂ PCR Preps DNA Purification System kit
4 mL toluene (to permeabilize the cells), and 16 mL 1M
Tris-HCl (pH=7.0). Then, deionized water was added to
bring the total volume to 400 mL. Reference batches with
only one type of cells contained the same amount of sub-
strates and other materials dispersed in water. One is Con-
trol A which contained only strain SDM, and the other is
Control B which contained only strain E. coli BL21 (DE3)
(pET15b-nanA). The reaction was carried out at 308C with
agitation (200 rpm) for 16–40 h. Triplicate experiments were
performed under the same conditions.
[3e,13]
(
Promega).
The purified fragment was inserted between
the NcoI and BamHI sites of pET15b (Novagen) to give
pET15b-nanA. The constructed plasmid pET15b-nanA was
transformed into E. coli BL21 (DE3) (Novagen). E. coli
BL21 (DE3) (pET15b-nanA) was cultured in 300 mL of
medium containing glucose 1%, Na HPO ·12H O 0.6%,
2
4
2
KH PO4 0.3%, NaCl 0.05%, NH Cl 0.5%, MgSO ·7H O
2
4
4
2
Isolation of Neu5Ac after Biotransformation
0
1
.055%, yeast extract 0.2%, and 100 mg/mL ampicillin in a
-liter flask at 250 rpm, 378C. After 1 h when the OD600 had
After biotransformation for 20 h, the reaction solution was
centrifuged at 5,000g for 10 min to remove pellets, and the
supernatant was adjusted to pH 4.5 with glacial acetic acid.
Then, the supernatant was loaded onto an anionic resin
column (800 mL) which had been pre-treated with 2M
formic acid. The unreacted GlcNAc and ManNAc were
washed clear with deionized water. Neu5Ac was eluted with
a linear gradient of 0–1M formic acid in 160 min with the
reached 1.5, 0.1 mM IPTG was added to induce Neu5Ac
adolase production for 5 h. Cells were harvested by centrifu-
gation at 5,000g for 10 min, and washed twice by 0.85%
NaCl solution. The activity of NanA was assayed according
[14]
to the previous work. One unit of NanA activity was de-
fined as the amount of NanA which catalyzed the produc-
tion of 1 mmol pyruvate per min at 378C.
À1
flow rate at 10 mLmin . The desired fraction was concen-
trated to approximately 125 g/liter Neu5Ac on a rotary
evaporator. Neu5Ac was crystallized by adding 5 volumes of
glacial acetic acid. After standing for 2 days at 48C, the
solid part was recovered by vacuum filtration and washed
with fresh glacial acetic acid before drying at 408C to a con-
stant weight.
Preparation of Pseudomonas stutzeri SDM Cells
The bacterial strain Pseudomonas stutzeri SDM, capable of
oxidizing lactate to pyruvate, was isolated from soil by our
laboratory and deposited at the China Center for Type Cul-
ture Collection (CCTCC No. M 206010). Cells of strain
SDM (18.1 g wet weight, about 907 U of LOX) were cul-
tured and harvested from 1,000 mL medium according to
[8a]
the previous study. One unit (U) of the LOX from lactate
was defined as the amount of LOX required for the forma-
tion of 1.0 mmol of pyruvate per minute under the test con-
dition.
Acknowledgements
The work was supported in part by the grant from the State
Major Basic Research Development Program (China) (Grant
Preparation of the ManNAc/GlcNAc Mixture
2
007CB707800) and the State Key Foundation of the Ministry
The procedure of base-catalyzed epimerization of GlcNAc
of Education of China (Grant no. 106102). We would like to
acknowledge the financial support and chemical analysis by
Shanghai Apple Flavor & Fragrance Co., Ltd. and Shanghai
Jiao Tong University (China).
[3b,e,15]
to ManNAc was adopted to prepare ManNAc.
Typical-
ly, 400 g of GlcNAc was dissolved in 1 L of NaOH (55 mM)
aqueous solution and allowed to stand at 258C for 48 h. The
[15a]
ManNAc:GlcNAc ratio was approximately 3.5:1.
The re-
sulting pale yellow solution was neutralized by the addition
of glacial acetic acid and then air-dried under vacuum. The
residue was reflux extracted by 500 mL methanol for 1 h. References
The extracted solution was allowed at 258C for 4 h, and fil-
tered to remove GlcNAc. The filtrate was concentrated
under reduced pressure to provide a mixture of GlcNAc/
[1] a) R. Schauer, Arch. Biochem. Biophys. 2004, 426, 132–
141.
[3e,15a]
ManNAc. The final mixture of ManNAc/GlcNAc
was
[2] a) M. Koketsu, H. Kawanami, L. J. Juneja, M. Jujiki, H.
Hatta, K. Nishimoto, M. Kim, N. Yamazaki, (Kaiyo
Kagaku Co., Ltd.), U.S. Patent 5,233,033, 1993; b) E. J.
McGuire, S. B. Binkley, Biochemistry 1964, 170, 247–
251; c) M. Shimatani, Y. Uchida, I. Matsuno, M.
Oyoshi, Y. Ishiyama, (Snow Brand Milk Products Co.
Ltd.), U.S. Patent 5,270,462, 1993.
analyzed, and the ratio of ManNAc:GlcNAc was about
5
.67:1. In each batch, 293.43Æ2.66 g GlcNAc was recovered.
The recovered GlcNAc could be reused in the next cycle.
About 95.37Æ10.52 g mixture containing 85.01Æ3.13% of
ManNAc was obtained from 400 g GlcNAc.
[
3] a) C. Auge, S. David, C. Gautheron, Tetrahedron Lett.
Synthesis of Neu5Ac
1
984, 25, 4663–4664; b) S. Blayer, J. M. Woodley, M. J.
The production of Neu5Ac was carried out in one pot. The
reaction mixture in a 3,000-mL flask contained cells of E.
coli BL21 (DE3) (pET15b-nanA) (2.1 g in wet weight,
about 625 U NanA), cells of strain SDM (14.5 g in wet
weight, about 727 U of LOX), 19.62 g ManNAc/GlcNAc
mixture, 26.25 g l-lactate (adjusted to pH 7.0 with NaOH),
Dawson, M. D. Lilly, Biotechnol. Bioeng. 1999, 66, 131–
136; c) M. J. Kim, W. A. J. Hennen, H. M. Sweers, C. H.
Wong, J. Am. Chem. Soc. 1988, 110, 6481–6484; d) U.
Kragl, D. Gygax, O. Ghisalba, C. Wandrey, Angew.
Chem. Int. Ed. 1991, 30, 827–828; e) M. Mahmoudian,
D. Noble, C. S. Drake, R. F. Middleton, D. S. Montgom-
Adv. Synth. Catal. 2007, 349, 1614 – 1618
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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