Examining the Autocatalytic Cleavage and Reassembly of Nylonases (TvgC, TslC and TbcC) for Nylon Degradation

Plastics play a crucial role in modern society, yet their durability and resistance to degradation result in challenges for waste management and environmental sustainability. Current estimates indicate that global plastic production exceeds 400 million tonnes annually, with only 9% of waste recycled. The biocatalytic degradation of plastic is an emerging sustainable solution to mitigate plastic pollution. Advances in this field show promising results for the degradation of Polyethylene Terephthalate (PET); however, effective strategies to degrade other plastics, such as nylon, a polymer used in the textile, automotive, and packaging industries, remain limited. To date, a class of nylonases called 6-aminohexanoate oligomer endohydrolases (NylCs) have been identified, but these enzymes have been insufficient to fully degrade nylon at an industrial scale. TvgC, a novel NylC found through a sequence similarity network (SSN) with known NylCs, shows remarkable catalytic activity towards the degradation of nylon-6 and nylon 6-6. TvgC functions through an autocatalytic cleavage mechanism to release Thr234 as its N-terminal nucleophile before assembling into the active protein, characteristic of the N-terminal nucleophile (Ntn) hydrolase family. This generates an N- and a C-terminal fragment, which assemble via non-covalent interactions to form a heterodimer. A recent study shows that the autocatalytic cleavage of TvgC requires 24 hours of heating at 50°C, which is inefficient for industrial applications. Two other NylCs have been identified from the same SSN, TslC and TbcC, which are expected to degrade nylon with an analogous mechanism to TvgC. The current research project aims to determine if independently expressed N-terminal and C-terminal halves of TvgC, TslC and TbcC can reassemble into an active, nylon-degrading protein, eliminating the time required for autocatalytic cleavage. Addressing this question will expand our understanding of the autocatalytic cleavage and reassembly mechanisms and support the development of nylonases that can recycle plastic industrially with increased efficiency.