Engineered Leaf-Branch Compost Cutinase with Enhanced PET Degradation Across a Broad Temperature Range.

Enzymatic recycling of poly(ethylene terephthalate) (PET) possesses several advantages over chemical and physical recycling methods. However, many currently identified PET degrading enzymes perform poorly under industrially relevant conditions. From a library of leaf and branch compost cutinase (LCC) variants engineered based on substitutions known to affect PET-degrading activity in homologous enzymes, a quadruple LCC variant (H218S-F243T-D238C-S283C) is discovered that hydrolyzes 90% of 9% crystalline PET microparticles in 17.0 ± 3.4 h at 70°C and also reaches >90% conversion at 55°C and 62°C. This novel variant produces 15.6 ± 0.3 g TPA L-1 h-1 at 70°C over 12 h and has a melting temperature of 82.1°C. Continuous evaluation of PET degradation used a pH stat to measure the progress of hydrolysis to nearly complete conversion or until enzyme activity plateaued. In contrast, initial rate measurements, which are commonly used to evaluate PETase variants, poorly predicted overall PET weight loss (R2 = 0.01 at 70°C, R2 = 0.01 at 62°C, R2 = 0.07 at 55°C). Future work must improve enzyme screening methodologies by optimizing reaction conditions to identify variants that achieve high PET conversion efficiencies.