New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase

Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization.We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell.The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.Penicillin G acylases (PGA, also known as PA, benzylpenicillin acylase, penicillin amidase, penamidase or acyl transferase), are enzymes widely distributed among micro-organisms in the living world. The physiological role of these enzymes is still unclear, but in Escherichia coli the PGA expression is regulated both by temperature and by phenylacetic acid, suggesting that the enzyme could be involved in vivo in the assimilation of aromatic compounds to be used as carbon source [1]. In E. coli,