title: Production kinetics and stability properties of delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.
abstract: Delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis in Streptomyces clavuligerus. Time-course studies indicated that the S. clavuligerus ACV-synthetase was stable during the cephamycin C fermentation: the enzyme was produced early in the growth phase and its activity remained high up to 96 h of growth. The detection of crude ACV-synthetase activity in older cultures was best achieved with an assay medium supplemented with 5 mM phosphoenolpyruvate, at lower ATP concentrations. During storage at 4 degrees C, a progressive decrease in the stability of crude ACV-synthetase was observed with increasing culture age. Although a proteinolytic activity with a pH optimum at 8.2 was detected in crude cell-free extracts, no significant variation was observed in its activity with increasing culture age to account for the instability of ACV-synthetase in vitro. Addition of proteinase inhibitors did not improve the stability of the enzyme. However, a stabilization cocktail containing dithiothreitol, MgCl2, the three substrate amino acids, and glycerol increased the stability of the enzyme isolated from cultures grown for 30-40 h, which was shortly after the appearance of antibiotics in the culture fluid. This stabilized enzyme retained half of its initial activity after 6 days at 4 degrees C.
authors.affiliation: Department of Microbiology, University of Alberta, Edmonton, Canada.
authors.forename: T A
authors.initials: TA
authors.lastname: Kadima
authors.forename: S E
authors.initials: SE
authors.lastname: Jensen
authors.forename: M A
authors.initials: MA
authors.lastname: Pickard
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002513:Cephamycins / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005285:Fermentation; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology*; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D002513:Cephamycins; D011480:Protease Inhibitors; D010728:Phosphoenolpyruvate; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase; D004229:Dithiothreitol; C024925:cephamycin C