Production and Evaluation of Fluorophore-Doped Polymer Substrates to Screen for Plastic-Degrading Enzymes

Fast and sensitive analytical methods are the key to
efficient
screening of plastic-degrading enzymes. Here, we present a streamlined
and affordable approach to assess the enzymatic deconstruction of
insoluble synthetic polymers by blending them with a fluorescent dye,
rhodamine 6G, and we evaluate this screening method using poly(ethylene
terephthalate) (PET) as a model material. Our results indicate that
enzymatic depolymerization of the rhodamine-doped PET can be observed
in a high-throughput fashion by following release of the fluorophore.
The fluorescence data obtained during the hydrolysis of rhodamine-doped
PET by 14 PET hydrolases, produced with a robotic platform, correlated
with the quantitative chromatographic analysis of PET degradation
products. Remarkably, the use of the rhodamine-loaded PET substrate
resulted in negligibly low background signals even when detecting
PETase activity in crude cell lysates, suggesting suitability for
screening of a wide variety of samples. Encouraged by these results,
we next produced a selection of polyethylene- and nylon-based materials
loaded with rhodamine 6G. While rapid leaching of fluorophore observed
with nylon substrates limits the utility of the method for detecting
nylonase activity, the rhodamine-loaded polyethylene showed promising
performance in passive diffusion tests, indicating that this latter
substrate may be used to screen for polyolefin-degrading enzymes.