This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Forney, L J Articles by Wong, D C Search for Related Content PubMed PubMed Citation Articles by Forney, L J Articles by Wong, D C Agricola Articles by Forney, L J Articles by Wong, D C

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1989 October; 55(10): 2556-2560

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli.

Synergen, Inc., Boulder, Colorado 80301.

ABSTRACT

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

This article has been cited by other articles:

• Gabor, E. M., Janssen, D. B. (2004). Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis. Protein Eng Des Sel 17: 571-579 [Abstract] [Full Text]  
• Otten, L. G., Sio, C. F., Vrielink, J., Cool, R. H., Quax, W. J. (2002). Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the beta -Subunit. J. Biol. Chem. 277: 42121-42127 [Abstract] [Full Text]