Mentors: Jason Boock, Ph.D., Jason Berberich, Ph.D.

Accumulation of plastic is a global problem, with plastic buildup occurring in the environment and even in organisms. Finding strategies to remediate plastic is necessary to shorten its half-life and prevent environmental issues. A wide variety of esterase enzymes have been discovered and developed to break down polyethylene terephthalate (PET) plastic, offering a sustainable, biological route for plastic remediation and potentially recycling. Our research sought to produce and characterize several of these enzymes, with the goal of comparing performance and being able to make more protein as needed for future experiments. Our focus was on making and producing leaf-compost cutinase (LCC) and PES-HY, as our research laboratory has not worked with them previously. Genes that encoded for each protein were ordered and cloned into inducible plasmids for production in E. coli. We evaluated growth medium, strain, and production time to find those that maximized expression, finding that 5 hours of production using the strain BL21(DE3) was best for LCC and PES-HY. Proteins were made and purified prior to further activity assessment. Along with LCC and PES-HY, we tested the variants Hot PETase, FAST PETase, and commercially available Humicola isolens cutinase (HiC). We determined the Michaelis-Menten kinetic parameters on the soluble, colorimetric substrate 4-nitrophenyl acetate. Further, we measured soluble products produced over time based on the degradation of plastic films. Taken together, this study has shown how we can vary culture and reaction conditions to maximize enzyme production and activity. By experimenting with the different types of esterase enzymes, growth medium, and production time, we have taken steps to improve the processes of plastic remediation. With a better understanding of the production and activity of these enzymes, global plastic accumulation may soon become an issue of the past.

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