Investigation of Enzymes for the Depolymerization of Nylon

Abstract

Plastic waste is ubiquitous in modern society, and a problem that every person interfaces with daily. As well as mismanagement of plastic waste posing a threat to ecosystems, the loss of plastic material to the environment or landfill represents loss of a valuable resource for new materials and other products. While current recycling practices are limited and produce lower-quality materials, biocatalysis offers a promising route to recover the value of plastic waste through generation of new materials through a more environmentally-compatible process. However, while enzymes have been identified and optimized to degrade ester-based polymers efficiently, other types of polymers pose a greater challenge.

Nylon is a useful and ubiquitous plastic, and forms a large component of ocean plastic waste in the form of discarded fishing equipment. Nylon is not easily recycled through traditional means, and is therefore an attractive target for biocatalytic degradation. In this thesis, we explore enzymes with the potential to degrade nylon, via two distinct approaches. First, we investigate Bacillus subtilis transglutaminase (bTG), which belongs to a class of enzymes known to be able to form and break aliphatic amide bonds. We discover that the activity of bTG is highly substrate-selective, and does not demonstrate the desired amide hydrolysis activity for this project. Second, we explore a known class of nylon oligomer hydrolases (NylCs) and develop assays to improve the efficiency of engineering campaigns for this and related enzymes. Our first class of substrate analogues are soluble, chromogenic substrates that can rapidly detect acyltransfer activity in NylC enzymes. Our second class of substrates are insoluble linear amide analogues which more closely resemble nylon, which are readily hydrolyzed by NylCs. We demonstrate the applicability of our assays to high-throughput screens, while exploring the substrate scope and specificity of this class of enzyme.