Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization.

Area of Science:

• Biotechnology
• Polymer Science
• Enzymology

Background:

• Biocatalytic polyester recycling is advancing, prompting exploration of enzymatic deconstruction for other polymers like polyamides (PA).
• Nature possesses various amide-cleaving enzymes, making PA a potential target for enzymatic degradation.

Purpose of the Study:

• To screen a diverse set of nylon-hydrolyzing enzymes (nylonases) for their ability to depolymerize nylon-6 (PA6).
• To identify highly active nylonases and understand the limitations of enzymatic PA6 deconstruction.

Main Methods:

• Screening of 40 natural and engineered nylonases against PA6 films.
• Quantification of hydrolysis products using mass spectrometry.
• Time-course reactions at varying temperatures (40-70°C) to assess enzyme activity and depolymerization extent.

Main Results:

• Significant PA6 deconstruction activity was rare among the screened nylonases.
• A thermostabilized N-terminal nucleophile (Ntn) hydrolase variant, NylC_K-TS, showed the highest activity, hydrolyzing 0.67 wt% of a PA6 film.
• Enzyme addition after initial reaction failed to restart depolymerization, indicating substrate-based limitations.

Conclusions:

• Ntn hydrolases show potential for further development in PA6 enzymatic depolymerization.
• Key challenges for advancing PA6 enzymatic depolymerization include improving enzyme activity, product selectivity, and polymer accessibility.