Natural Diversity Screening, Assay Development, and Characterization of Nylon-6 Enzymatic Depolymerization: Article No. 1217

Elizabeth Bell, Gloria Rosetto, Morgan Ingraham, Kelsey Ramirez, Clarissa Lincoln, Ryan Clarke, Japheth Gado, Jacob Lilly, Katarzyna Kucharzyk, Erika Erickson, Gregg Beckham

Research output: Contribution to journalArticlepeer-review

8 Scopus Citations

Abstract

Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 degrees C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylCK variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, Tm = 87.4 degrees C, 16.4 degrees C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.
Original languageAmerican English
Number of pages17
JournalNature Communications
Volume15
DOIs
StatePublished - 2024

NREL Publication Number

  • NREL/JA-2A00-88295

Keywords

  • amide-cleaving enzymes
  • biocatalytic polyester recycling
  • depolymerization
  • enzymatic hydrolysis
  • nylon deconstruction
  • nylonases

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