Preparative enzymatic synthesis of activated neuraminic acid by using a microbial enzyme.
Using Escherichia coli K-235 as a production strain in a fed-batch fermentation process with an optimized sorbitol/yeast extract medium, we were able to produce 640 U of CMP-Neu5Ac synthetase in 10 l scale (64 U/l) and 9200 U (total enzyme) in 200 l scale (390 U/kg wet weight). By simple one-step purification procedures, enzyme preparations were obtained that could be used efficiently for the synthesis of CMP-Neu5Ac from CTP and Neu5Ac with over 90% yield, from Neu5Ac, CMP, and ATP or phosphoenolpyruvate by in situ generation of CTP, and from CTP, pyruvate, and ManNAc or GlcNAc by in situ generation of Neu5Ac.
Kittelmann
M
M
CIBA-GEIGY Limited, Pharmaceutical Division, Basel, Switzerland.
Klein
T
T
Kragl
U
U
Wandrey
C
C
Ghisalba
O
O
D001426:Bacterial Proteins / Q000302:isolation & purification; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D019158:N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism*
D001426:Bacterial Proteins; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D003570:Cytidine Triphosphate; D000216:N-Acylneuraminate Cytidylyltransferase; D019158:N-Acetylneuraminic Acid