Optimization of expression and purification of MHETase-PETase in Escherichia coli

Abstract

    Polyethylene terephthalate (PET) is a widely used synthetic polymer with low biodegradability, causing tremendous environmental pollution. The discovery of PET-degrading enzymes PETase and MHETase from Ideonella sakaiensis 201-F6 has accelerated the development of biological approaches for PET waste treatment. However, expression and purification of the fusion protein MHETase–PETase in Escherichia coli remain suboptimal, requiring multiple complex steps. In this study, we optimized the expression conditions and streamlined the purification process of the MHETase–PETase enzyme to enhance production efficiency and scalability. Results indicated that E. coli BL21(DE3) was the most effective host, with optimal induction achieved by adding 0.1 mM IPTG at 30 ℃ for 6 hours. Using a single Ni–NTA affinity chromatography step, the purified enzyme reached 7.33 mg/mL in the elution peak. At 0.4 mg/mL of enzyme concentration, PET film degradation was observed after 7 days of incubation at 30 ^°C, and significantly increased by day 28. Notably, the fusion protein MHETase–PETase displayed superior activity to PETase alone, with PET degradation observed as early as 48 hours under the same conditions. These findings highlight the potential of combining an optimized host strain, tailored culture conditions, and a simplified purification protocol to improve production yield while reducing costs. This study paves the way for the application of enzyme-based biotechnology in PET processing, contributing to the development of sustainable plastic recycling solutions.