Poster K5 - Thermal Stabilization of a Plastic-Degrading Enzyme by Tethering to a Mixed Polymer Brush

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Caroline Rucker

Undergraduate Researcher
University of South Carolina
Powhatan, Virginia, United States

Co-Author(s)

David Kelaita, BS

Graduate Research Assistant
University of Colorado Boulder, United States

Primary Investigator(s)

DS

Daniel Schwartz

Glenn L. Murphy Professor of Engineering
University of Colorado Boulder, United States

Introduction: The improper disposal of synthetic polymer products leads to the introduction of microplastics into the environment. Microplastic exposure through ingestion or inhalation can have adverse effects on various organ systems in the body. To combat this threat to human health, it is crucial to develop more effective plastics recycling systems. PETase is an enzyme that hydrolyzes poly(ethylene) terephthalate (PET), one of the most abundantly manufactured polyesters. Since its discovery in 2016, many groups have been interested in engineering PETase for biocatalysis applications. However, challenges remain in maximizing the enzyme’s thermostability and storage stability. Enzyme immobilization is a promising strategy to address this issue. Previous work in our group explored the use of copolymer brushes composed of sulfobetaine methacrylate (SBMA) and poly(ethylene glycol) methacrylate (PEGMA) as immobilization supports. Based on experimental data from a range of enzymes, an algorithm called Hi-patch was developed to predict the optimally stabilizing SBMA/PEGMA composition based on protein surface hydrophobicity for a novel enzyme. In the present study, we utilize Hi-patch to predict an optimal brush composition for PETase and evaluate the stability of the immobilized enzyme under thermally denaturing conditions for potential applications in recycling processes.

Materials and

Methods: Hi-patch was used to calculate the free energy of solvation per unit area of PETase. Based on this value, previous experimental data was used to predict an optimal support composition of 50% PEGMA (Fig. 1A). SBMA/PEGMA brushes were grafted from the surface of (p-Chloromethyl)phenyltrichlorosilane-modified silica nanoparticles via green light-activated atom-transfer radical polymerization. Glycidyl methacrylate, an epoxide-containing monomer, was included to allow for covalent attachment through reactions with nucleophilic groups on the enzyme surface. The polymer-functionalized nanospheres were incubated with a solution of PETase using a revolving orbital shaker to enable immobilization (Fig. 1B). After separating the particles via centrifugation, enzyme loading was determined using a mass balance based on the relative activity of PETase in the supernatant. Activity assays were conducted using a fluorogenic substrate. To determine the effect of immobilization on thermal stability, the activity of immobilized PETase relative to its maximum was measured at a range of temperatures and compared to that of free PETase. To compare the time stability of free and immobilized PETase, the activity of each case relative to the initial measurement was measured at various time points after incubation at a set temperature. To evaluate the ability of immobilized PETase to degrade PET, free and immobilized enzyme were incubated with PET discs from a commercial drinking bottle, and degradation products were quantified using analytical high-performance liquid chromatography (HPLC).

Results, Conclusions, and Discussions: The immobilization of PETase on a 50% SBMA/PEGMA support resulted in an enzyme loading of 0.349 mg/m2. Immobilization was found to increase the temperature optimum of PETase from 30 °C to 35 °C. Furthermore, while free PETase retained less than 10% of its activity beyond 50 °C, immobilized PETase retained around 50% of its activity at the same temperature (Fig. 1C). Due to stabilizing interactions between PETase and its polymer support, we anticipate that immobilized PETase will retain more of its activity over time compared to free PETase. Additionally, we expect that immobilization will increase the ability of PETase to degrade PET, particularly at higher temperatures. These results demonstrate the potential of our immobilization strategy to enhance PETase stability at higher temperatures and longer timescales. This could allow PETase to be used for more efficient plastics recycling and ultimately reduce the health impacts of microplastics. Future work will focus on optimizing conditions such as support composition and reaction parameters to maximize immobilized PETase activity.

Acknowledgements (Optional): This research was funded in part by NSF REU grant #2348856.
We thank the Shared Instruments Pool (RRID: SCR_018986) of the Department of Biochemistry at the University of Colorado Boulder for the use of its fluorimeter and Dr. Annette Erbse for providing training and support.