Abstract
Plastics have become a familiar aspect of daily life, as they are strong, flexible, and cheap materials to manufacture. One of the most common plastics, polyethylene terephthalate (PET) is found in plastic water bottles, textiles, and other films. As a result of their strength and durability, plastics are not biodegradable and therefore pose a huge threat to our planet in both landfills and oceans. Recently, a new species of bacteria Ideonella sakaiensis, was found in a Japanese recycling facility which can use PET as its main carbon source. The enzyme responsible for the initial degradation of PET was identified as a hydrolase with an alpha/beta fold capable of turning PET into its monomers. While this enzyme shares a similar fold and chemistry with many other hydrolases, how this novel function evolved is still unknown. To answer this question, I made a phylogeny of 101 PETase homologs, which included thermophilic enzymes, cutinases, and other enzymes which have partial PETase activity. I then used ancestral sequence reconstruction (ASR) to identify a loop insertion and a critical phenylalanine to serine mutation which together, work to substantially enlarge the substrate binding pocket, allowing PETase to bind to the bulky PET. I expressed and characterized the activity and stability of the novel PETase enzyme to compare with the reconstructed ancestors. I also successfully expressed several ancestral PETase and hydrolase constructs and I made homology models to predict their structure. Future work will involve mutagenesis to study the functional evolution of the ancestors. I also studied the evolution of active site dynamics in another enzyme family, the lactate and malate dehydrogenases to further understand evolutionary mechanisms of novel substrate specificity.