Inactivation of PETase at Interfaces Inhibits PET Plastic Depolymerization.

Enzymatic depolymerization of polyethylene terephthalate (PET) is a promising alternative to traditional recycling showing promise in addressing issues with high energy demands and challenges associated with mixed-waste streams. While engineered, thermostable PETase variants exist, achieving high yields under industrially relevant process conditions remains a significant hurdle. Poor thermostability and PET crystallinity are often blamed for low conversions, but these factors do not fully account for the low productivity observed in all cases. This suggests that other poorly understood phenomena may be limiting the catalytic efficiency of PETase warranting further investigation. This work systematically explores the impact of mixing, and in particular, the presence of the air-water and the solid-liquid interfaces, on PETase stability. We found that PET degradation plateaus while soluble PETase activity is lost at increasing mixing rates when an air-water interface is present. It is hypothesized that the enzyme adsorbs to the air-water interface leading to denaturation, aggregation and precipitation. Additional studies demonstrate that the hydrophobic PET film negatively impacts enzyme stability and the rate of enzyme inactivation is also proportional to the PET surface area. Traditional approaches to mitigate enzyme inactivation at interfaces, such as using blocking agents, were effective in preserving enzyme activity, but resulted in significantly reduced PET degradation rates. In contrast, PEGylation of PETase yielded improved stability and enhanced PET degradation during mixing. These findings help explain the significant conversion discrepancies reported in previous studies and demonstrate a robust strategy for improving PETase performance under industrially relevant conditions.