Characterization and Engineering of a Fungal Poly(ethylene terephthalate) Hydrolyzing Enzyme from Aspergillus fumigatiaffinis

As our environmental pollution awareness increases, effective poly(ethylene terephthalate) (PET) waste management has become an important global goal, and biorecycling is considered as one of the strategies for PET recycling. Although several bacterial cutinases have been investigated with respect to PET hydrolysis, fungal cutinases and their potential still need to be explored. Here, we screened 15 fungal cutinases and discovered a cutinase from Aspergillus fumigatiaffinis (AfC), a potent PET hydrolase satisfying PET hydrolytic activity and thermal stability. Structural analysis of AfC revealed that the enzyme has an additional disulfide bond compared to known fungal cutinases. We also developed the AfC^{T37V/A84K/N117E/N124E/A171P/N182E} variant (AfC^6p), which showed enhanced thermal stability and a 4-fold increase in PET hydrolytic activity at 60 °C compared with AfC^Wild‑type. AfC^6p completely decomposed the postconsumer PET film within 12 days at 60 °C.