Mutation of Residue ßF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties.

Residue phenylalanine 71 of the ß-chain of penicillin acylase from E. coli is involved in substrate binding and chiral discrimination of its enantiomers. Different amino acid residues have been introduced at position ßF71, and the mutants were studied with respect to their enantioselectivity and substrate specificity. Some mutants demonstrated remarkably improved catalytic activity. Moreover, mutation of ßF71 residue allowed to enhance penicillin acylase enantioselectivity. The catalytic activity to the specific substrates was improved up to 36 times, most notably for K, R, and L mutants. Increased activity to a D-phenylglycine derivative - a valuable specificity improvement for biocatalytic synthesis of new penicillins and cephalosporins - was shown for ßF71R and ßF71L mutants. The synthetic capacity of penicillin acylase with 6-aminopenicillanic acid as an external nucleophile was especially sensitive to mutation of the ß71 residue in contrast to the synthesis with 7-aminodeacetoxycephalosporanic acid.