Rapid Screening of Plastic-Degrading Enzymes Using an Optimized Cell-Free Protein Synthesis Platform
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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 timeconsuming 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 37o C. 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.
Abstract
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 timeconsuming 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 37o C. 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.