Gabriela de la Cruz Flores

Production of a fully functional, permuted single‐chain penicillin G acylase

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single‐polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single‐chain PGA not dependent on autoproteolytic processing. The mature sequence of the β‐domain was expressed as the N terminus of a new polypeptide, connected by a random tetra‐peptide to the α‐domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single‐chain variants were produced when using a Sec‐dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active‐site titration experiments showed that the single‐chain proteins displayed similar kcat values to the ones obtained with the wild‐type enzyme. Interestingly, the single‐chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.