014: Silica Immobilized PETase for Microplastic Bioremediation

Introduction

In the past three decades PET plastic use has doubled, especially in single-use food packaging, breaking down into microplastics during and after use. The widespread existence of microplastics poses physical and toxicological risks to organisms and the environment. To address this, PETase is employed to target environmental PET microplastics. Previously, computational strategies have created PETase mutants with improved thermostability and PET-degrading activity and immobilization strategies have focused on magnetic nanoparticles or nanoflowers. This study modifies PETase with a SBP and linker proteins, to provide conformational space between PETase and SBP, to determine PETase stability and reusability when immobilized onto silica.

Methods

In this study DNA of PETase mutants were modified through PCR and Gibson Assembly Hi-Fi. Wild-type PETase, PETase with SBP, and PETase with SBP separated by protein linkers were expressed in an E. coli system then purified using affinity chromatography and evaluated using SDS-PAGE for purity. Purified proteins were bound to silica then silica binding efficiency and PET degradation activity of bound and unbound enzymes were evaluated.

Results

Modified DNA was confirmed with 100% pairwise alignment to template using whole plasmid sequencing. The purity of modified DNA seen on SDS-PAGE showed a 5 kDa increase in molecular weight after SBP addition. PETase activity was quantified by PET degradation using HPLC. PETase with SBP unbound to silica showed similar or lower enzymatic activity after 48 hours. Once bound to silica, degradation of PET powder and PET disk by PETase with SBP increased 5-fold and 1.5-fold, respectively, at 24 hours. PETase with a short flexible linker bound to silica showed the greatest PET degradation after 24-hour incubation with PET disk, similar to unbound activity, and retaining 10-20% activity after 5 cycles of reuse. After 5 cycles of reuse, the monomer products formed from PETase with a short flexible linker were approximately equal to cycle 1 of PETase with SBP.

Significance

PET microplastics are continually accumulating in the environment and adversely impacting human safety, water, and agriculture. Silica-immobilized PETase aids in industrial (e.g. wastewater treatment) viability through improving PETase reusability and stability. The greater use of PETase can ultimately reduce PET microplastic environmental pollution.

Authors: Sonia Su, Julie M. Goddard