Engineering a PET-degrading Enzyme to Increase Stability
Current waste management techniques, including recycling methods, are ill-equipped to handle the enormous problem that is global plastic pollution. Poly(ethyleneterephthalate) (PET) is a common plastic pollutant whose current recycling methods are environmentally unfriendly and degrade plastic quality. In contrast, enzymatic recycling of PET is environmentally friendly and retains plastic quality. However, all known PET depolymerases are too slow to for industrial use. Leaf and branch compost cutinase (LCC) is an enzyme that shows promise as a template for an industrially-viable PET depolymerase. Due to the high thermostability of LCC, PET depolymerization can be sped up by heating the reaction without immediately denaturing the enzyme. Still, the enzyme is too slow and lacks the stability necessary for industrial use. While engineering attempts of LCC have increased stability, further improvements are necessary. Here, I describe the kinetic and thermal stability of 12 novel LCC variants, one of which is more stable. This variant, F243W/N246M, increases the melting temperature by 4 ℃ compared to wild type LCC without significantly affecting catalysis. I also identify a variant containing a homologous disulfide bond that, while much less stable, improves activity against PET by approximately 20% compared to wild type LCC. Characterization of these two variants adds to the list of possible beneficial mutations that could be used in future attempts to make LCC an industrially-competitive PET depolymerase.