Microplastic accumulation in terrestrial and aquatic environments is a growing environmental challenge. Biodegradation has shown promise as an intervention strategy for reducing the spread of microplastics. The wastewater treatment system is a key intervention point in microplastic biodegradation due to its pivotal role in the water cycle at the interface between human activity and the environmental. However, the best characterized microplastic degradation enzyme, PETase, lacks the stability to perform at scale in wastewater treatment. In this work, we show that genetic fusion of PETase to a silica binding peptide enables directed immobilization of the enzyme onto silica nanoparticles. PETase activity in simulated wastewater conditions is quantified by linear regression from time zero to the time of maximum fluorescence of a fluorescent oxidized product of PETase degradation of PET microfibers. Mesoporous silica is shown to be a superior support material to nonporous silica. The resulting biocatalytic nanomaterial has up to 2.5-fold enhanced stability and 6.2-fold increased activity compared to free enzyme in unbuffered, 40 °C simulated influent (ionic strength ∼15 mM). In unbuffered, 40 °C simulated effluent (ionic strength ∼700 μM), reaction velocity and overall catalytic activity were increased by the biocatalytic material 2.1-fold relative to free PETase. All reactions were performed in 0.2 mL volumes, and enzyme concentrations were normalized across both free and immobilized samples to 9 μg/mL. Site-directed mutagenesis is shown to be a complementary technique to directed immobilization, which may aid in optimization of the biomaterial for wastewater applications. PETase stabilization in application-relevant environments as shown here enables progress toward application of PETase for microplastic biodegradation in wastewater treatment.
Keywords: PETase; biocatalytic material; directed enzyme immobilization; fusion protein; nanomaterial; silica nanoparticles; wastewater treatment.