ISSN:
1476-5535
Keywords:
Streptomyces clavuligerus
;
ACV-synthetase activity profile
;
ACV-synthetase stability
;
Cephalosporins
;
Cephamycins
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Summary ϖ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis inStreptomyces clavuligerus. Time-course studies indicated that theS. 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°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°C.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01570129
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