Engineering Hyperthermostable Nylonase, TvgC, to Improve Catalytic Efficiency for Degradation

Nylons are too inert to be recycled in a cost-effective manner. The Howe Group’s recent discovery of TvgC, a hyperthermostable amidase with the capability to degrade nylon, hints at an exciting possibility: sustainable biocatalytic nylon recycling. While experiments showed that TvgC degraded nylon at rates even greater than those achieved by the most highly engineered enzymes, significant improvements are still required to improve the catalytic efficiency of TvgC such that the protein can enable biocatalytic recycling on an industrial scale. This proposal will develop the directed evolution pipeline to facilitate the production of TvgC variants with increased catalytic efficiencies. We have already identified a method to force E. coli cells to overexpress and secrete TvgC into the surrounding media. This project will use nylon-doped agar plates harbouring many E. coli colonies, with each expressing one member of a TvgC mutant library generated by error- prone PCR and site-saturation mutagenesis. State-of-the-art ambient ionization mass spectrometry techniques will then be used to analyze the nylon-doped surface in the vicinity of each colony. Cells expressing TvgC variants with higher activities will exhibit more nylon degradation, and these cells can be isolated, so that variants of interest can be identified and further analyzed. By developing this on-plate selection method, this proposal has the potential to enable biocatalytic nylon recycling and to transform how directed evolution campaigns are carried out.