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 k cat 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.