Improved screening for beta-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate.
The very sensitive and specific method for the detection of beta-lactam antibiotics using DD-carboxypeptidase (DDCase) from Actinomadura strain R39 has been improved to meet the requirements of a high-throughput beta-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate N alpha-acetyl-N epsilon-4-(7-nitrobenzofurazanyl)-L-lysyl-D-al anyl-D-alanine (ANLA2) which is converted by DDCase into ANLA1 with only one D-alanine residue left. Both compounds are intensely yellow as well as highly fluorescent and can be separated by thin-layer chromatography. This allows easy determination of residual DDCase activity after reaction with beta-lactams by simple visual inspection of chromatograms. Also, many assays can be run at a time without sophisticated instrumentation. Details of the method as well as some results of a beta-lactam screening performed with this type of assay are described.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000900:Anti-Bacterial Agents / Q000302:isolation & purification*; D002856:Chromatophores; D004150:Dipeptidases; D010450:Endopeptidases; D008722:Methods; D009842:Oligopeptides; D010069:Oxadiazoles; D011480:Protease Inhibitors; D013056:Spectrophotometry, Ultraviolet; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D009842:Oligopeptides; D010069:Oxadiazoles; D011480:Protease Inhibitors; D047090:beta-Lactams; C048203:N(alpha)-acetyl-N(epsilon)-4-(7-nitrobenzofurazanyl)lysyl-alanyl-alanine; D010450:Endopeptidases; D004150:Dipeptidases; C019989:DD-carboxypeptidase-endopeptidase; D009327:4-Chloro-7-nitrobenzofurazan