Silica immobilized PETase for microplastic bioremediation: Influence of linker peptides on activity

Environmental microplastic leads to bioaccumulation in humans, animals, and plants with potential toxicity. Polyethylene terephthalate (PET) degrading enzymes (PETases) present an opportunity to depolymerize PET in key intervention points, such as wastewater treatment. While PETase has been extensively studied since its discovery and modified for enhanced performance (especially thermostability), knowledge on immobilization for reusability remains limited. This study investigated the effect of linker peptides between functional, active, stable, and tolerant (FAST) PETase and a silica binding protein for immobilization onto silica and the reusability of the enzyme. Linker peptides and a silica binding protein were assembled onto FAST-PETase and expressed in Escherichia coli. The activity of the constructs was tested on PET before and after binding to silica. In the free system, repeating (GGGGS)₃ flexible linker achieved the same activity as FAST-PETase parent enzyme after 48 h of degradation. Once immobilized to silica, repeating (GGGGS)₃ flexible linker preserved 50 % of enzymatic activity, compared to free FAST-PETase, and 80 % compared to its free form. Silica-immobilized enzyme constructs all retain at least 15 % of relative activity compared to the first cycle of use after 5 reuse cycles. Integration of linker peptides between the enzyme and the silica binding peptide had a significant effect on the overall catalytic activity of FAST-PETase and advances our understanding of immobilized PETase for potential recovery and reuse in applications such as wastewater treatment. SYNOPSIS: Minimal research exists on the immobilization of polyethylene terephthalate degrading enzymes for reuse in environmental systems. This study reports the ability of silica immobilized enzyme, with aid of linker peptides, to minimize microplastic contamination from wastewater treatment plants.