Cloning of penicillin acylase from Escherichia coli: Catalytic properties of recombinant enzymes

The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer strain that is an analogue of strain ATCC 11105. Optimization of the cultivation conditions made it possible to obtain up to 130 mg of active enzyme per liter of culture broth. A number of single, double, and triple mutants were obtained by the method of site-specific mutagenesis using PCR. As a result of isolation and purification procedures, homogeneous preparations of the wild-type enzyme and its mutants were obtained. Studies showed that (1) the obtained enzymes have the correctly folded structure; (2) complexing agents and metal cations do not inhibit their catalytic activity; (3) mutant PAs, like the wild type, are efficiently inactivated by phenylmethylsulfonyl fluoride (PMSF), which makes it possible to titrate their active sites; and (4) the obtained mutants are characterized by a greater specificity constant in the reaction of hydrolysis of a colorimetric substrate; however, they are inferior to the wild type in the synthesis of ampicillin by acyl transfer.