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

Author

Listed:
  • Elizabeth L. Bell

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Gloria Rosetto

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory)

  • Morgan A. Ingraham

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Kelsey J. Ramirez

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Clarissa Lincoln

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Ryan W. Clarke

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Japheth E. Gado

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Jacob L. Lilly

    (Battelle Memorial Institute)

  • Katarzyna H. Kucharzyk

    (Battelle Memorial Institute)

  • Erika Erickson

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

  • Gregg T. Beckham

    (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory
    BOTTLE Consortium)

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 °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 °C, 16.4 °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.

Suggested Citation

  • Elizabeth L. Bell & Gloria Rosetto & Morgan A. Ingraham & Kelsey J. Ramirez & Clarissa Lincoln & Ryan W. Clarke & Japheth E. Gado & Jacob L. Lilly & Katarzyna H. Kucharzyk & Erika Erickson & Gregg T. , 2024. "Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45523-5
    DOI: 10.1038/s41467-024-45523-5
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