New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes

The discovery and engineering of novel biocatalysts capable of
depolymerizing polyethylene terephthalate (PET) have gained significant
attention since the need for green technologies to combat plastic
pollution has become increasingly urgent. This study focuses on the
development of novel substrates that can indicate enzymes with PET
hydrolytic activity, streamlining the process of enzyme evaluation
and selection. Four novel substrates, mimicking the structure of PET,
were chemically synthesized and labeled with fluorogenic or chromogenic
moieties, enabling the direct analysis of candidate enzymes without
complex preparatory or analysis steps. The fluorogenic substrates,
mUPET1, mUPET2, and mUPET3, not only identify enzymes capable of PET
breakdown but also differentiate those with exceptional performance
on the polymer, such as the benchmark PETase, LCCICCG.
Among the substrates, the chromogenic p-NPhPET3 stands
out as a reliable tool for screening both pure and crude enzymes,
offering advantages over fluorogenic substrates such as ease of assay
using UV–vis spectroscopy and compatibility with crude enzyme
samples. However, ferulic acid esterases and mono-(2-hydroxyethyl)
terephthalate esterases (MHETases), which exhibit remarkably high
affinity for PET oligomers, also show high catalytic activity on these
substrates. The substrates introduced in this study hold significant
value in the function-based screening and characterization of enzymes
that degrade PET, as well as the the potential to be used in screening
mutant libraries derived from directed evolution experiments. Following
this approach, a rapid and dependable assay method can be carried
out using basic laboratory infrastructure, eliminating the necessity
for intricate preparatory procedures before analysis.