To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.