PETase - Plastic Degrading Enzymes

by Tyler Headley (VI), Maya Khan (V), Juliana Amorosi (IV), Sebastian Talarek (IV), Dr. D’Ausilio, Dr. Pousont

The United States of America only recycles about 35% of its total waste, sending nearly 150 million tons of trash to landfills each year. There, it takes up to 450 years to fully biodegrade. Often, it ends up invading oceans or forests, where it poses a serious threat to native species. Polyethylene terephthalate (PET) is one of the most widely used plastics in the United States, but only 31% of the 3.1 million tons of PET produced in America annually are recycled. It is necessary to find an efficient way to recycle plastic in order to mitigate the damage done to our planet. Our team works with PET depolymerase (PETase), an enzyme with the unique ability to break PET polymers into monomers, which can then be reassembled into new plastic. This nuanced recycling method is termed “biochemical recycling” and has become an increasing focus of research—however, it requires optimization before it can be implemented commercially. Because the severity and pervasiveness of plastic-related damage to the environment is growing, the scientific community is eagerly seeking to improve PETase through mutation–specifically, its efficiency and thermostability. Our goal is to identify and investigate various mutations, and assess the change in efficiency and thermostability that each mutation offers. After researching mutations that demonstrate promise for accomplishing these two goals through scientific literature, we obtained various PETase mutations through our partnership with Deerfield Academy. Deerfield created these mutations using site-directed mutagenesis, a common method to induce mutations, utilizing a primer to confer a mutation in double-stranded plasmid DNA. To replicate the mutated plasmid, we transform it into NEB 5-alpha cells and grow colonies on LB-AGAR plates with ampicillin. We harvest the colonies using inoculation loops before miniprepping: lysing the cells and discarding all contents besides the mutated plasmid. Then, we transform the mutated DNA into BL21 cells and induce protein expression with autoinduction to create copies of the mutated PETase enzyme. Finally, we perform cell lysis and purification to obtain the pure enzyme. To determine the efficiency of this mutated PETase enzyme, we plan to develop an assay. This assay will reveal whether, and how efficiently, our PETase mutants are capable of biodegrading PET. In the coming months, we hope to make more progress researching and documenting the effects of different mutations on PETase, which will further advance the scientific community’s knowledge of the enzyme, and contribute to the global effort to make recycling more efficient and widely used.

PETase Structure