Improved screening for .BETA.-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate.

Abstract

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.