Industrial Biotransformations

N-Acetyl-d-neuraminic acid aldolase
Escherichia coli
HO
HO
HO
1) Reaction conditions
[1]: 0.8 M, 177 g · L –1 [221.21 g · mol –1 ]
pH: 7.2
T: 30 °C
medium: aqueous
reaction type: C-C bond cleavage
catalyst: solubilized enzyme
enzyme: N-acetylneuraminate pyruvate-lyase (sialic aldolase)
strain: Escherichia coli
CAS (enzyme): [9027–60–5]
2) Remarks
epimerase
1 2
1 = N-acetyl-D-glucosamine (GlcNAc)
2 = N-acetyl-D-mannosamine (ManNAc)
3 = pyruvic acid
3 = N-acetyl-D-neuraminic acid (Neu5Ac)
Fig. 4.1.3.3 – 1
O
OH
NHAc
HO HO
HO
NHAc
O
OH
EC 4.1.3.3
Marukin Shoyu Company, Ltd.
Forschungszentrum Jülich, GmbH
● N-Acetyl-d-neuraminic acid aldolase from E. coli K-12 and N-acetyl-d-glucosamine epimerense
from porcine kidney have been cloned and overexpressed in E. coli.
● The enzyme catalyzed aldol condensation to N-acetylneuraminic acid is combined in a one-vessel
synthesis with the enzyme-catalyzed epimerization of N-acetyl-glucosamine (GlcNAc), see
page 385.
● The production of Neu5Ac on a multi ton scale is carried out by Glaxo utilizing chemical epimerization
(see page 338). In contrast to this synthesis here the native, non-immobilized
enzyme is applied.
● Both enzymes (epimerase and aldolase) can be used in a pH range of 7.0 to 8.0. For the biotransformation
pH 7.2 was chosen.
● Since excess amounts of pyruvate (educt for the aldolase) inhibit the epimerase, a fed batch in
regard to pyruvate is performed. After the start of the reaction with a ratio of pyruvate to
GlcNAc of 1:0.6, two times pyruvate is added twice up to a total amount of 251 mol (ratio of
pyruvate to GlcNAc at start: 1:0.6; after first addition: 1:1.5; after second addition 1:2).
● Before the product is purified by crystallization (initiated by the addition of 5 volumes of glacial
acetic acid), the enzymes are denaturated by heating to 80 °C for 5 minutes, afterwards the
reaction solution is filtered.
+
O
3
COOH
E
HO
OH
COOH
HO
AcHN
O OH
HO
4
459