Development of a High-Throughput Platform for the Kinetic Analysis of Enzymatic Depolymerization

Polymers, such as poly(ethylene terephthalate) (PET), are valued for their excellent material properties, but their end-of-life is poorly controlled. Conventional recycling methods have been largely unable to mitigate this issue, as they are energy intensive and yield lower-quality materials leading to non-circularity. Enzymatic degradation is a promising, sustainable, low-energy recycling approach, but progress is constrained by the lack of standardized methods for evaluating enzyme performance. This bottleneck prevents comparisons across studies and limits understanding of how substrate properties shape degradation outcomes. Here, we present a semi-automated protocol for optimizing polymer-degrading enzyme expression via a previously established high-throughput workflow, followed by evaluation of activity using highly controlled PET nanoparticle substrates with tunable crystallinity and surface area. Enzyme activity was measured by sampling the degradation reaction over time and monitoring fluorescence, enabling the kinetic profiling of degradation products. This approach provides detailed insight into structure–function relationships and reveals how substrate properties impact enzymatic degradation. Overall, this work establishes a robust platform for generating datasets that capture the kinetics of enzymatic polymer degradation while accounting for key substrate properties, offering new insight into enzyme–substrate interactions and degradation mechanisms.