PETase Enzymes For Biorecycling
Miami University - Boock Laboratory
Miami University - Boock Laboratory
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.
Enzymes speed up reactions that would normally be slow. They are naturally produced, making them sustainable. They can speed up a wide variety of reactions at lower temperatures and without harsh chemicals making them green catalysts.
PETase variants were produced by incorporating the gene that encodes for them into a pET21 plasmid. We then transformed the plasmid into different strains of E. coli to see which could produce the most enzyme! We also screened production timing and different growth media with 4-nitrophenyl acetate. We found that a production time of 5 hours in the BL21 (DE3) strain produced the best results for LCC and PES-HY
All of the enzymes that we selected could be successfully produced and purified from E.coli. This is the first time leaf-compost cutinase and PES-HY have been made and characterized in our laboratory. We have shown them all to be active on the soluble, colorimetric substrate 4-nitrophenyl acetate. We are currently testing the activity of the purified enzymes with plastic samples. This will bring us closer to scaling up the enzyme production and use for global plastic remediation.
Esterases are a class of enzymes that use water to break ester bonds, like those found in PET plastic. The enzymes we worked with come from many different places. Some variants are developed in a lab and some come from tree compost or fungi. This table shows the percent identity each variant we worked with shares with the other variants. Each variant has different advantages and disadvantages and can be used in different applications for plastic decomposition.
Mia Angulo: First Year Chemical Engineering and Molecular Biology Student
Paloma Jolly: First Year Biomedical Engineering and Physics Student
Siena Madsen: Second Year Chemical Engineering and Energy Student
Dr. Boock: Assistant Professor. Chemical, Paper, and Biomedical Engineering