The preparation of CMP-sialic acids by using CMP-acylneuraminate synthase from frog liver immobilized on sepharose 4B.
A preparation of frog liver CMP-acylneuraminate synthase (2-10-fold enriched over the homogenate) obtained from DEAE-Sephadex A-50 chromatography of a 105,000g liver supernatant was bound to Sepharose 4B by the CNBr method. The enzyme retained 80-100% activity on binding and showed similar properties to the purified soluble enzyme from the same source with respect to Km, pH optimum and inhibition. The bound enzyme was stable to temperatures above 40 degrees C, in contrast with the soluble enzyme, and could be stored for 4 months at 2 degrees C with loss of 20% activity. The bound enzyme was used preparatively for the synthesis of radioactive and non-radioactive CMP-N-acetylneuraminic acid and CMP-N-glycolloylneuraminic acid. With suitable substrate concentrations and ratios, yields of 80% and over can be achieved.
D000818:Animals; D001001:Anura; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000138:chemical synthesis; D004355:Drug Stability; D004800:Enzymes, Immobilized; D008099:Liver / Q000201:enzymology; D000216:N-Acylneuraminate Cytidylyltransferase; D009713:Nucleotidyltransferases; D012685:Sepharose; D012794:Sialic Acids / Q000138:chemical synthesis*
D004800:Enzymes, Immobilized; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012685:Sepharose; D009713:Nucleotidyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase