Measuring PETase enzyme kinetics by single-molecule microscopy

Abstract

Polyethylene terephthalate (PET) is one of the most widely produced man-made polymers and is a significant contributor to microplastics pollution. The environmental and human health impacts of microplastics pollution have motivated a concerted effort to develop microbe- and enzyme-based strategies to degrade PET and similar plastics. A PETase derived from the bacteria Ideonella sakaiensis was previously shown to enzymatically degrade PET, triggering multidisciplinary efforts to improve the robustness and activity of this and other PETases. However, because these enzymes only erode the surface of the insoluble PET substrate, it is difficult to measure standard kinetic parameters, such as kon, koff and kcat, complicating interpretation of the activity of mutants using traditional enzyme kinetics frameworks. To address this challenge, we developed a single-molecule microscopy assay that quantifies the landing rate and binding duration of quantum dot-labeled PETase enzymes interacting with a surface-immobilized PET film. Wild-type PETase binding durations were well fit by a biexponential with a fast population having a 2.7 s time constant, interpreted as active binding events, and a slow population interpreted as non-specific binding interactions that last tens of seconds. A previously described hyperactive mutant, S238F/W159H had both a faster on-rate and a slower off-rate than wild-type PETase, potentially explaining its enhanced activity. Because this single-molecule approach provides a more detailed mechanistic picture of PETase enzymatic activity than standard bulk assays, it should aid future efforts to engineer more robust and active PETases to combat global microplastics pollution. Statement of significancePlastic pollution is a global environmental and human health problem. PETases are recently discovered enzymes that degrade the ubiquitous plastic polyethylene terephthalate (PET). A push is underway to understand and optimize these enzymes to enable large-scale microplastics remediation. Here, we use single-molecule fluorescence microscopy to visualize the interactions of PETase enzyme molecules with a thin film of PET. We identify specific binding interactions of a few seconds that differ between wild-type and PETase mutants that have been previously shown to have altered activities. These single-molecule investigations provide a new window into the mechanism and activity of PETase enzymes, and provide a platform for characterizing and optimizing novel PETases with improved function and stability.