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Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes
Summary
Researchers used a rapid lab technique called cell-free expression to screen enzymes that might break down polyester polyurethane plastics, sourcing the enzymes from bacteria found growing on aircraft and vehicle surfaces. They identified 10 enzymes with measurable plastic-degrading activity, though none performed as well as an established plastic-eating enzyme. This work advances the search for biological tools that could help break down microplastic pollution in the environment.
Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.