Incorporation of Nonstandard Amino Acids and Development of a High-throughput Screen for Enzymatic Plastic Oxidation

• Arnav Bedekar, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Plastic pollution is harmful to environmental and human health. LDPE is a major contributor to this issue, making up a quarter of plastics produced annually. Many techniques exist to degrade LDPE, but often these techniques require expensive conditions due to maintaining high temperatures and/or pressures and can yield a wide array of products that have to be separated after treatment. Biocatalytic methods represent an avenue for plastic degradation that may be more cost-effective, as only ambient conditions are needed. Enzyme specificity also allows for the creation of specific, value-added products. In order for this breakdown to occur, non-terminal oxidation to an ester followed by ester cleavage would result in value added alcohols and carboxylic acids which could be further metabolized to create value added products. Currently, two enzyme families have shown promise in this area: dye decolorizing peroxidases (DyP’s), which can oxidize long-chain alkanes into ketones, and Baeyer-Villiger Monooxygenases (BVMO’s), which can oxidize long-chain ketones into esters. These long-chain esters can then be degraded by existing esterases and subsequently be converted into valorized products. However, most candidate DyP’s show weak activity on LDPE substrates. This is hypothesized to be due to the weak ability of native amino acids to harbor a radical necessary for oxidation. By incorporating nonstandard amino acids (nsAA’s) at the active site, the radical can be stabilized far beyond what is naturally possible. Herein, we report successful incorporation and synthesis of DyPs containing nsAA’s. In regards to BVMOs, analyzing activity is slow due to inefficient and single-stream methods. We have developed a multiplexed, high-throughput colorimetric assay that detects long-chain esters formed during oxidation to identify promising candidate BVMOs far more rapidly than traditional methods. Both enzymes represent a promising path toward plastic oxidation followed by eventual degradation, converting plastic waste into specific value added products.