IMPROVED SCREENING FOR β-LACTAM ANTIBIOTICS
A SENSITIVE, HIGH-THROUGHPUT ASSAY USING DD-CARBOXYPEPTIDASE AND A NOVEL CHROMOPHORE-LABELED SUBSTRATE
1986 年 39 巻 1 号 p. 53-57
詳細
1986 年 39 巻 1 号 p. 53-57
The very sensitive and specific method for the detection of β-lactam antibiotics using DD-carboxypeptidase (DDCase) fromActinomadura strain R39 has been improved to meet the requirements of a high-throughput β-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate Nα-acetyl-Nε-4-(7-nitrobenzofurazanyl)-L-lysyl-D-alanyl-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 β-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 β-lactam screening performed with this type of assay are described.
