# A Modular Biosensor Platform for the Detection of Plastic Monomers and the Engineering of Promiscuous Amidases Toward Challenging Substrates

## TL;DR

A new biosensor platform detects plastic monomers and helps engineer enzymes to break down challenging plastic materials like polyurethanes.

## Contribution

The study introduces a first-of-its-kind biosensor platform for real-time amidase and urethanase activity monitoring and enzyme improvement.

## Key findings

- The biosensor platform enables real-time detection of amidase and urethanase activity without chromogenic/fluorogenic molecules.
- Engineered enzyme variants showed up to 5.5-fold increased activity toward difficult substrates like N-substituted decanamides and polyether dicarbamates.
- The platform expands bacterial luciferase detection to include plastic monomers like polyols, aiding PU recycling.

## Abstract

Stable chemical bonds dictate the properties of industrial chemicals and materials. Particularly the persistence of synthetic polymers like polyurethanes (PUs) contributes to the global issues of waste accumulation and environmental pollution. To accelerate the discovery and engineering of plastic‐degrading biocatalysts, a genetically encoded biosensor platform is established that enzymatically converts polyfunctionalized monomers into (aliphatic) aldehydes and allows their robust detection by a bacterial luciferase. In vivo, in vitro, and hybrid applications of the investigated biosensor system facilitate the bioluminescence‐based assessment of the promiscuous esterase, amidase, and urethanase activity of amidase signature family enzymes, circumventing chromatographic analysis. Furthermore, the biosensor platform guides the selection of improved variants in a site‐saturated enzyme library, exhibiting up to 5.5‐fold enhanced activity toward difficult to hydrolyze screening molecules, including N‐substituted decanamides, a representative polyether dicarbamate, and a commercial polyester‐PU. The latter contain polyols like diethylene glycol, for which biosensor applications are scarce. Hence, this biosensor platform is not only the first to enable the monitoring of amidase and urethanase activity independent of chromogenic/fluorogenic molecules in real‐time. It expands the detection scope of bacterial luciferases toward plastic monomers like polyols, which will aid advancing current recycling strategies for PU waste and beyond.

A luciferase is used as biosensor for aldehydes, produced enzymatically from different monomeric building blocks of plastics like polyurethanes (PUs). The screening platform is used to monitor esterase, amidase, and urethanase activity and to guide the selection of enzyme variants with improved hydrolytic activities toward difficult to hydrolyze substrates, including N‐substituted amides, polyether‐dicarbamates, and a commercial polyester‐PU.