PET Plastic Biodegradation: Impacts of Mutating the Bacterial Enzyme Is-PETase

We live in a sea of plastics. Plastics are an integral part of our lives from the #5-plastic toothbrush covered with toothpaste from a #2-plastic tube with which we brush our teeth in the morning until we lay down at night on our #7-foam-filled pillows under our recycled #1-fleece blankets. However, increased use of plastics has not come with an increased ability to reuse or remove these plastics from the environment. It is estimated that only 25% of #1 PET plastic water and soda bottles are actually recycled. Even with recycling, many plastics have a limited number of reuses before they are discarded. In 2016, researchers discovered Ideonella sakaiensis in soil near a plastic bottle recycling plant. This bacterium degrades polyethylene terephthalate (PET). Under lab conditions, the enzyme Is-PETase is able to almost completely degrade PET film over 42 days at 30° C. Scientists around the world are now working with Is-PETase, making genetic modifications to increase the ability of this enzyme to degrade PET. In this study, I used the computer program FoldIt to model the impact of changing two amino acids near the catalytic site of the enzyme. Commercially purchased plasmids were mutated using site-directed mutagenesis in parallel and transformed into Escherichia coli cells for growth. Is-PETase was purified and used in a bulk absorbance assay to assess the impact of amino acid substitutions at positions 208, 279 and 208/279 on the enzyme’s ability to degrade PET pellets. Kinetic activity was compared against the wild-type Is-PETase