Directed Immobilization of PETase on Mesoporous Silica Enables Sustained Depolymerase Activity in Synthetic Wastewater Conditions

Abstract

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.

Synthesis of Mesoporous Silica Using the Sol–Gel Approach: Adjusting Architecture and Composition for Novel Applications

“…Therefore, there is a requirement to immobilize plastic-degrading enzymes on the surfaces of nanomaterials [ 79 ], especially on the surface of mesoporous silica by the sol–gel method [ 80 ]. Goddard et al reported [ 81 ] the direct immobilization of the enzyme on the surface of mesoporous silica by grafting the polyethylene terephthalate (PET) enzyme binding with a peptide (SBP), as shown in Figure 10 a. Furthermore, the introduction of PET enzyme-fixed mesoporous silica into the lining material of wastewater treatment tanks was found to enhance the degradation of microplastic waste ( Figure 10 b).…”

“… ( a ) The schematic illustration of the immobilization process of SBP-tagged PETase on mesoporous silica. ( b ) Proposed intervention of immobilized PETase for microplastic degradation in wastewater treatment tanks [ 81 ]. Copyright 2022, American Chemical Society.…”

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling

“…In this regard, immobilization of Is PETase on Co 3 (PO 4 ) 2 nanoparticles has been shown to increase the enzyme lifetime by 75% [ 154 ]. Furthermore, the silica-immobilised PETase was successfully applied for wastewater treatment [ 154 , 155 ], whereas magnetic nanoparticles-tagged PETase was used for removal of PET microplastic [ 156 ]. Protein surface display represents another strategy for enzyme immobilisation, which is based on a functional display of target enzymes through fusion to various secreted proteins [ 59 , 123 , 124 , 127 , 129 ] ( Figure 3 ).…”

Simulation Study of Polyethylene Terephthalate Hydrolase Adsorption on Self-Assembled Monolayers

“…Jia et al 29 immobilized IsPETase on a metal support by genetically fusing the enzyme to multiple histidine tags for metal binding, thereby increasing its stability. Additionally, Zurier et al 30 utilized genetic fusion of IsPETase with silica-binding peptides to immobilize the enzyme on silica nanoparticles, resulting in improved enzyme activity and stability. Chen et al 31 coimmobilized DuraPETase and MHETase in calcium phosphate nanocrystals (CaP) using the SpyTag/SpyCatcher system.…”