Fluorogenic assay for penicillin G acylase activity

A simple, highly sensitive, and rapid assay for high-throughput screening o f penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through c leavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as variou s penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate w as used for rapid assay of amidase activity in nondenaturing polyacrylamide gels. (C) 2001 Academic Press.