Mutation of Residue βF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties

Abstract

residue phenylalanine 71 of the β-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 βF71, and the mutants were studied with respect to their enantioselectivity and substrate specificity. Some mutants demonstrated remarkably improved catalytic activity. Moreover, mutation of βF71 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 βF71r and βF71L mutants. the synthetic capacity of penicillin acylase with 6-aminopenicillanic acid as an external nucleophile was especially sensitive to mutation of the β71 residue in contrast to the synthesis with 7-aminodeacetoxycephalosporanic acid.

Specificity of Penicillin Acylases in Deprotection of N-Benzyloxycarbonyl Derivatives of Amino Acids

“…5 ); by introducing mutations in the enzyme structure, one can enhance the catalytic activity to these nonspecific substrates. The experience in studying penicillin acylase from Escherichia coli shows that both the catalytic activity and the affinity for the substrates can be improved by protein engineering [ 22 ].…”

“…Отличие ацильных групп на один атом кислорода приводит к существенному изменению в эффек- активность к неспецифическим субстратам. Опыт изучения пенициллинацилазы из E. coli показывает, что методами белковой инженерии удается повысить как каталитическую активность, так и сродство фермента к субстратам [22].…”