Improving Yield and Thermostability of PETase as a Maltose Binding Protein Fusion in the Periplasm of Escherichia coli

Polyethylene terephthalate (PET) waste accumulation requires sustainable recycling alternatives. While Ideonella sakaiensis PETase offers a green solution, its industrial application is hindered by low solubility and poor thermostability. In this study, we systematically evaluated the synergistic effects of maltose-binding protein (MBP) fusion and periplasmic translocation strategies to optimize PETase production in Escherichia coli. Our results demonstrate that MBP acts as a potent solubilizing partner for PETase, with the cytosolic MBP–PETase variant achieving a high purification yield of 8.4 mg per gram of wet cell weight–a significant improvement over the PelB–PETase control (1.1 mg per gram of wet cell weight). Furthermore, the periplasmic MalE–MBP–PETase construct provided an optimal intermediate compromise between the yield, thermal stability, and catalytic activity by leveraging the oxidative environment of the periplasm for critical disulfide bond formation. Although PelB–PETase exhibited higher specific activity, its low yield limits industrial scalability. This study establishes a robust plug-and-play platform for high-throughput PET depolymerization, providing a foundational step toward a circular plastic economy.