Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics

Penicillin acylase (PA) from Escherichia coli can catalyze the acylation of 6-aminopenicillanic acid (6-APA), a conversion that is applicable in the biocatalytic preparation of semi-synthetic β-lactam antibiotics such as ampicillin. The efficiency of this kinetically controlled conversion, in which an amide or ester acts as the acyl donor, is dependent on the kinetic properties of the enzyme. To further improve the synthetic properties of PAs, family gene shuffling was performed with the PA-encoding genes of the PAs from E. coli, Kluyvera cryocrescens and Providencia rettgeri. Of these three PAs, the E. coli enzyme possessed the best properties for the synthesis of ampicillin. Shuffled recombinant libraries were pre-screened for activity by growth selection, followed by testing the catalytic performance in ampicillin synthesis using HPLC. Three clones with improved synthetic properties were selected and sequence analysis showed that the shuffled genes were hybrids of the PA-encoding genes from E. coli and K. cryocrescens, with additional point mutations. The hybrid enzymes displayed a 40–90% increase in the relative rate of acyl transfer to the β-lactam nucleus during ampicillin synthesis. This increase was not accompanied by a reduction of synthetic activity that has previously been reported for mutants of E. coli PA constructed by site-directed mutagenesis. Similar improvements in acyl transfer were obtained for the synthesis of amoxicillin, cephalexin and cefadroxil, making the new hybrid enzymes interesting candidates for the biocatalytic synthesis of several β-lactam antibiotics.