title: CMP-N-acetyl neuraminic-acid synthetase from Escherichia coli: fermentative production and application for the preparative synthesis of CMP-neuraminic acid.
abstract: 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.
authors.affiliation: Ciba-Geigy Ltd, Pharmaceuticals Division, Basel, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Kittelmann
authors.forename: T
authors.initials: T
authors.lastname: Klein
authors.forename: U
authors.initials: U
authors.lastname: Kragl
authors.forename: C
authors.initials: C
authors.lastname: Wandrey
authors.forename: O
authors.initials: O
authors.lastname: Ghisalba
mesh_terms: 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
chemical_list: D003470:Culture Media; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase