Impact of PETase’s Active Site Disulfide Bond on PET Biodegradation

Abstract

Plastic pollution is one of the largest problems globally, with polyethylene terephthalate (PET) plastic as one of the main sources. Effective depolymerization of PET to its monomers for upcycling is a challenge. PETase is reported to be an effective enzyme for biodegradation of PET via C-O bond cleavage of ester linkage. The role of the disulfide bond, present in PETase’s active site sequence, is unknown in the cleavage of PET’s ester linkage. To understand the role of this bond, two separate versions of PETase – one containing the disulfide bond, and the other without the disulfide bond - were modeled using PyMol™, synthesized, and tested for degradation of PET surrogate compound, bis (2-hydroxyethyl) terephthalate (BHET). Several experiments were performed in the presence and absence of phenylmethylsulfonyl fluoride (PMSF), a serine protease. The results reveal that the role of the disulfide bond in the degradation of BHET’s ester linkage is insignificant and the variation in the results (ethylene glycol yields, BHET degradation per microgram of enzyme) are within the experimental uncertainty. This finding is a stepping-stone to further modifying PETase and improving its activity towards commercial adaption of this technology for PET upcycling and creating a circular carbon economy, improving the world’s carbon footprint, and mitigating ocean and environmental plastic pollution.