Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

Summary Penicillin G acylase of Escherichia coli ATCC 11105 catalyzes hydrolysis as wellas synthesis of penicillin G. In this work a recombinant penicillin G acylase genewas mutagenized in vivo. A mutant with altered penicillin G acylase was selectedby its ability to grow with phthalyl-L-leucine as sole source of leucine. Themutant enzyme obtained was deficient in hydrolyzing penicillin G. A mutation ofGly359 to aspartic acid was mapped first by construction of chimeric pac genescomposed of wild type and mutant DNA, followed by nucleotide sequencing.