Directed Evolution of IsPETase via Error-Prone PCR for Enhanced Thermal Stability and Polyethylene Terephthalate Microplastic Degradation at Elevated Temperatures

Polyethylene terephthalate (PET) accumulation represents a severe global environmental crisis, particularly in the form of microplastics. The PET-hydrolyzing enzyme from Ideonella sakaiensis (IsPETase) offers a promising biocatalytic solution, but its low thermal stability (melting temperature, T_m ≈ 48°C) limits its efficiency at elevated industrial process temperatures. In this study, we employed directed evolution via error-prone PCR to enhance the thermal stability of IsPETase for microplastic degradation. A library of IsPETase variants was generated and screened for residual esterase activity after incubation at 55°C. A double mutant, designated IsPETase-M2 (S121E/D186N), was identified, exhibiting a 9.2°C increase in melting temperature (T_m = 57.2°C) compared to the wild-type enzyme. IsPETase-M2 demonstrated a 4.1-fold increase in the degradation rate of PET microplastics at 50°C over a 72-hour period, releasing significantly higher levels of terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate (MHET). Structural modeling suggested that the introduced mutations stabilize flexible loop regions and optimize surface electrostatic interactions. This work highlights the potential of directed evolution to tailor plastic-degrading enzymes for robust bioremediation applications under thermally challenging conditions.