Novel approach for untreated household PET waste depolymerization: recombinant extracellular thermostable hydrolases

Plastics represent a global environmental challenge due to their widespread use and persistent accumulation. This study is focused on the enzymatic depolymerization of non-pretreated household PET waste using two plastic-degrading esterases, LCCICCG and IsPETaseW159H/F229Y, leveraging the advantages of the extracellular expression in a Pichia pastoris system. The simple recovery and improved stability enabled by this expression system are crucial foundations in the development of biocatalytic remediation processes. Expression in batch cultures resulted in esterase activity levels of 86.3 ± 3.7 IU/mg for LCCICCG and 16.4 ± 0.3 IU/mg for IsPETaseW159H/F229Y after 48 hours of induction, and these enzymes kept their catalytic activity after at least 30 days at 4 and 25 °C. LCCICCG successfully degraded non-pretreated PET (87.6 ± 10.7 gPET h-1 genzyme-1), while IsPETaseW159H/F229Y exhibited a tenfold increase in depolymerization efficiency over previous reports (1.71 ± 0.3 gPET h-1 genzyme-1). LCCICCG exhibited enhanced activity at high temperatures, associated with the glycosylations introduced during expression in P. pastoris, making it a promising candidate for industrial applications requiring high-temperature operations. It is outstanding the specific space-time yield achieved by LCCICCG (183.1 mMTAeq h-1 mgenzyme-1), which results higher than those previously reported. These results encourage reducing both time and costs associated with conditioning pretreatments for non-pretreated household plastic wastes or biocatalyst downstream processing. They also underscore the potential of exploring a promising pathway towards large-scale, environmentally sustainable PET waste management.