CMP-N-acetyl neuraminic-acid synthetase from Escherichia coli: fermentative production and application for the preparative synthesis of CMP-neuraminic acid.
In an optimized sorbitol/yeast extract/mineral salt medium up to 12 U/l CMP-N-acetyl-neuraminic-acid (Neu5Ac) synthetase was produced by Escherichia coli K-235 in shake-flask culture. A colony mutant of this strain, E. coli K-235/CS1, was isolated with improved enzyme formation: in shake flasks with a yield of up to 20.8 U/l and 54 mU/mg protein in the cell extract. With this strain 26500 U CMP-Neu5Ac synthetase was produced with a high specific activity (0.128 U/mg) by fed-batch fermentation on 230-l scale. On a 10-1 scale the enzyme yield was 191 U/l culture medium. The enzyme was partially purified by precipitation with polyethyleneglycol resulting in a three- to fourfold enrichment and a recovery rate of more than 80%; most of the CTP hydrolysing enzymes were removed. The native synthetase was deactivated completely by incubation at 45 degrees C for 10 min, but could be stabilized remarkably by glycerol and different salts. The enzyme was used for the preparative synthesis of CMP-Neu5Ac with a conversion yield of 87% based on CTP.
Kittelmann
M
M
Ciba-Geigy Ltd, Pharmaceuticals Division, Basel, Switzerland.
Klein
T
T
Kragl
U
U
Wandrey
C
C
Ghisalba
O
O
D003470:Culture Media; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D000216:N-Acylneuraminate Cytidylyltransferase / Q000096:biosynthesis* / Q000302:isolation & purification
D003470:Culture Media; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase