Industrial Biotransformations

N-Acetyl-d-neuraminic acid aldolase
Escherichia coli
Fig. 4.1.3.3 – 1
1) Reaction conditions
[1]: 0.9 M, 200 g · L –1 [221.21 g · mol –1 ]
pH: 7.5
T: 20°C
medium: aqueous
reaction type: C-C bond cleavage
catalyst: immobilized enzyme
enzyme: N-acetylneuraminate pyruvate-lyase (sialic aldolase)
strain: Escherichia coli
CAS (enzyme): [9027–60–5]
2) Remarks
● The chemical synthesis of Neu5Ac is costly since it requires complex protection and deprotection
steps.
● The enzyme is covalently immobilized on Eupergit C.
EC 4.1.3.3
GlaxoSmithKline
● Since N-acetyl-d-mannosamine is very expensive, it is synthesized from N-acetyl-d-glucosamine
by epimerization at C 2. The equilibrium of the epimerization is on the side of N-acetyld-glucosamine
(GlcNAc:ManNAc = 4:1). After neutralization and addition of isopropanol
GlcNAc precipitates. In the remaining solution a ratio of GlcNAc:ManNAc = 1:1 is reached.
After evaporation to dryness and extraction with methanol, a ratio of GlcNAc:ManNAc = 1:4 is
reached. In contrast to this chemical epimerization enzymatic epimerization using an epimerase
is carried out by another company, see pages 459 and 506.
● N-Acetyl-d-glucosamine is not a substrate for the aldolase, but it is an inhibitor and limits by
this way the applied maximal concentration of ManNAc.
● Non-converted GlcNAc can be recycled after down stream processing by epimerization to Man-
NAc.
● The natural direction of the aldolase-catalyzed reaction is the cleavage of Neu5Ac to pyruvate
and ManNAc. The K M for ManNAc is 700 mM. Therefore a very high ManNAc concentration
of up to 20 % w/V is used. By this means ManNAc itself drives the equilibrium. Pyruvate is
used in a 1.5 molar ratio.
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