Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes

  Hendrik and Christoph Copyright: © biotec Hendrik Pütz and Christoph Janknecht

Hendrik Puetz, Christoph Janknecht, Francisca Contreras, Mariia Vorobii, Tetiana Kurkina, and Ulrich Schwaneberg, ACS Sustainable Chemistry & Engineering,

A sensitive high-throughput screening system enables directed depolymerase evolution to leverage sustainable polyamide and polyurethane recycling.

The accumulation of synthetic polymer waste poses a significant challenge to both society and the environment. Enzymes offer a promising avenue to foster a circular plastic economy by facilitating polymer degradation into monomers, enabling value-preserving and sustainable recycling. Recent years have shown impressive progress in using enzymes to break down PET. However, the lack of efficient enzymes and screening systems for discovering and engineering viable biocatalysts for other major polycondensates like PA and PUR impedes progress in this field. In this work, we report the first highly sensitive colorimetric amine screening system for directed polyamidase and polyurethanase evolution (AMIDE). We used this system to screen the breakdown of PA 6 and PA 6,6 on polymer film and granules and detect common PUR degradation products (2,4-TDA, 2,6-TDA, HMDA, MDA) down to the nanomolar range. We highlighted the applicability of AMIDE for directed depolymerase evolution by kinetically improving the polyamidase NylC. This was achieved through the screening of a small random mutagenesis library. We believe the AMIDE method not only helps to improve existing depolymerases for PA and PUR but also may be applied to screen e.g., metagenomic libraries on authentic polymeric substrates. This could lead to the discovery of novel enzymes to contribute to a circular synthetic polymer economy.

Hendrik Pütz and Christoph Janknecht are financially supported by the German Federal Ministry for Education and Research ([FKZ: 031B0867E], [FKZ: 031B0506C], [FKZ: 031B1342C], [FKZ: 031B1159CX]).

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Puetz, H.; Janknecht, C.; Contreras, F.; Vorobii, M.; Kurkina, T.; Schwaneberg, U. Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes. ACS Sustainable Chemistry & Engineering 2023.

  AMIDE high-throughput screening method Copyright: © ACS Sustainable Chemistry & Engineering

Figure 1. Synthetic polymer degradation efficiency of enzyme libraries is screened on authentic substrates and released products can be labeled and quantified. This allows the selection of improved enzyme variants in directed polyamidase and polyurethanase evolution campaigns.