Rapid Screening of Plastic-Degrading Enzymes Using an Optimized Cell-Free Protein Synthesis Platform

The accumulation of plastic waste poses a significant environmental challenge, necessitating the development of efficient plastic-degrading enzymes for bioremediation and recycling. However, traditional enzyme engineering approaches rely on microbial expression systems and are time-consuming and prone to unintended interactions between host cells and recombinant circuits. To address these limitations, a cell-free protein synthesis (CFPS) platform was developed for rapidly screening plastic-degrading enzymes. Using CFPS, cutinase and PET-degrading enzymes (PETases) were successfully synthesized, and their catalytic activities were assessed using polymer-containing agar plates. Clear degradation halos were observed for cutinase and PETase on polycaprolactone (PCL)-containing and bis (2-hydroxyethyl) terephthalate (BHET)-containing plates, respectively. The optimization of CFPS conditions revealed that enzyme synthesis efficacy was higher at room temperature than at 37^oC. The screening of PETase variants (C3 N1377, Mipa-P, and C5 N1251), synthesized using the CFPS platform, demonstrated that the catalytic activity of Mipa-P was the highest and surpassed that of IsPETase. This finding was further validated through purified enzyme analysis. Our results establish CFPS as a rapid, scalable, and cell-free alternative platform for screening and optimizing plastic-degrading enzymes, facilitating advancements in enzymatic plastic recycling.