Performing Department
(N/A)
Non Technical Summary
Over the past 50 years, the global production of polyethylene terephthalate (PET) plastic has surged to 80 million metric tons annually due to its durability and cost-effectiveness. However, less than 20% of PET is recycled, with the rest ending up in landfills or incinerated, contributing significantly to plastic pollution. PET breaks down into microplastics, posing environmental risks, including contamination and toxicity that affect plant and soil health. A promising solution involves the enzyme PETase (PETase), which can break down PET, producing water-soluble byproducts with minimal environmental impact. While research has explored PETase's mechanisms and enhancements, practical applications remain limited.This research focuses on immobilizing PETase onto solid supports like activated charcoal (AC), commonly used in water purification, to improve its stability and reusability in extreme conditions. This approach aims to enhance microplastic degradation in water treatment systems, addressing a significant research gap in the application of PET hydrolase enzymes for bioremediation. Key objectives include optimizing the immobilization conditions and demonstrating the effectiveness of immobilized PETase in degrading microplastics in simulated water systems. This research aims to provide practical solutions for integrating PETase into industrial water treatment, ultimately improving water quality and reducing environmental plastic pollution.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
The goal of this project is to conduct research on biological methods to remove polyethylene terephthalate (PET) and develop an activated charcoal (AC) immobilized PET degrading enzyme (PETase) for use in municipal water treatment plants.Objective 1: Preliminary Research. Determine optimal binding and immobilization conditions for PETase. Identify the reusability and recovery of immobilized PETase.Objective 2: Application Research. Create a simulated water treatment filtration system to demonstrate the use of immobilized PETase. Evaluate the enzymatic activity within this simulated filtration system and on polyester-based consumer plastics that may be found in the environment.
Project Methods
Objective 1: PETase target sequence and AC binding protein will be put into a commercial vector. Polymerase chain reaction and High Fidelity Gibson Assembly Method will amplify and join DNA fragments for transformation into E. coli DH5α. The DNA sequence will be sequenced after purification using QIAprep Spin Miniprep Kit. Purified DNA will be transformed into an expression strain of E. coli, BL21(DE3). Protein expression will be carried out in Luria Bertani supplemented with Ampicillin (LB-Amp) broth and then inoculated at into auto-induction media and expressed overnight. Protein purification will take place at 4°C and will be purified by His-Pur Cobalt Resin following manufacturer's instructions. Eluted proteins will be applied to desalting columns to remove imidazole. Protein content of concentrated proteins will be measured by Bicinchoninic Acid Assay (BCA) and put in varying conditions to test AC immobilization capacity. A range of binding time, temperature, and pH will be tested. Enzyme activity is determined by High-Performance Liquid Chromatography (HPLC) of degradation products. pH and temperature stability of free and immobilized enzymes will be compared using Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Once binding conditions have been optimized, reusability of PETase will be tested evaluating the residual activity in the washes. Recovery of immobilized enzymes will be evaluated to quantify recovery yields using BCA and SDS-PAGE. Objective 1 will result in (1) Verification of genetic modifications via Sanger Sequencing. (2) Confirmation of purified enzyme and affinity towards AC. (3) Quantification of free and immobilized PETase activity through HPLC analysis of PET degradation products. (4) Demonstration of immobilized PETase reusability and recovery.Objective 2: Building from Objective 1, purified enzyme will be used for application on AC filters. Using the optimal binding conditions determined in Objective 1, PETase will be immobilize onto the AC filters. Binding efficiency onto AC filters, in simulated environments will be tested. Flowthrough will be collected and tested for protein content using BCA to determine immobilized enzyme content. Contact time required by PETase for PET degradation will also be tested on a bench scale continuous flow system with manually incorporated PET powder and flowthrough evaluated via HPLC analysis. Lastly, I will collect a variety of postconsumer polyester-based plastics to test the hydrolytic capacity of PETase. Post-consumer plastic degradation will carried out for at least 10 days. Objective 2 will result in (1) Confirmation of enzyme immobilization onto AC filters. (2) Collection and analysis of flowthrough for enzyme binding capacity. (3) Application of continuous flow system to test PET hydrolytic capacity through a filter. (4) Inspection of degraded postconsumer plastics.