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PreprintCited by 7Enzyme Catalysis and ImmobilizationEnzyme Production and Characterizationbiodegradable polymer synthesis and properties

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

Identification and characterization of substrate- and product-selective nylon hydrolases

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Drufva, Erin E., Cahill, John F., Saint-Vincent, Patricia M.B., Williams, Alexis N., Bocharova, Vera, Capra, Nikolas, Meilleur, Flora, Carper, Dana L., Bourgery, Célestin, Miyazaki, Kaito, Yonemura, Maina, Shiraishi, Yuki, Parks, Jerry M., Zhou, Muchu, Dishner, Isaiah T., Foster, Jeffrey C., Koehler, Stephen J., Valentino, Hannah R., Sedova, Ada, Kertesz, Vilmos, Vasileva, Delyana P., Hochanadel, Leah H., Figg, C. Adrian, Negoro, Seiji, Kato, Dai-ichiro, Chen, Serena H., Michener, Joshua K.·2024·Chem Catalysis
DOI 10.1101/2024.11.14.623603PMC PMC11601465

Preprint — published as 10.1016/j.checat.2025.101418 · Paper page

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Abstract

Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases. Significance: Nylons are common industrial polyamides with few recycling options. As an alternative to mechanical or chemical recycling, enzymes may provide a selective and energy-efficient route to deconstruct nylons from mixed waste. Several nylon hydrolases have been identified, most notably NylC, but these enzymes are all closely related and demonstrated similar activity and substrate range. In this work, we investigated a diverse set of enzymes and showed that nylon hydrolase activity is common, providing new insights into the evolution of microbial nylon hydrolysis. Unlike NylC, several enzymes demonstrated unprecedented substrate selectivity, preferentially hydrolyzing Nylon 6,6 compared to Nylon 6. These enzymes can be used to understand substrate selectivity in nylon hydrolysis and to engineer enzymes for nylon recycling from mixed waste.

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Metadata

Authors
Drufva, E. E., Cahill, J. F., Saint-Vincent, P. M. B., Williams, A. N., Bocharova, V., Capra, N., Meilleur, F., Carper, D. L., Bourgery, C., Miyazaki, K., Yonemura, M., Shiraishi, Y., Parks, J. M., Zhou, M., Dishner, I. T., Foster, J. C., Koehler, S. J., Valentino, H. R., Sedova, A., Kertesz, V., Vasileva, D. P., Hochanadel, L. H., Figg, C. A., Negoro, S., Kato, D.-I., Chen, S. H., & Michener, J. K.
Affiliations
  • Oak Ridge National Laboratory (US)
  • Kagoshima University (JP)
  • Virginia Tech (US)
  • University of Hyogo (JP)
Corresponding author
  • Chen, Serena H.
  • Michener, Joshua K.
ORCID
Venue
Chem Catalysis
Publisher
openRxiv
Published
2024-11-14
Volume
5
Issue
8
Pages
101418-101418
ISSN
2667-1093 (ISSN-L)
DOI
10.1101/2024.11.14.623603
PMC
PMC11601465
OpenAlex
W4411277758
Cited by
7
References
1
Citations by year
2026: 4 · 2025: 3
FWCI
2.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Chem Catalysis (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nd)
Funders
  • U.S. Department of Energy (AC05-00OR22725, DE--AC05--00OR22725, DE-AC05?, 00OR22725)
  • Battelle (DE-AC05- 00OR22725, DE-AC05)
  • UT-Battelle (DE-AC05, DEAC05-00OR22725, AC05-00OR22725)
  • National Institutes of Health (DE-AC05-00OR22725)
  • National Institute of General Medical Sciences (R24GM141256)
  • Oak Ridge National Laboratory (AC05-00OR22725, DE-AC05-00OR22725)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Physical Sciences › Materials Science › Biomaterials › biodegradable polymer synthesis and properties
Keywords
polyamide 66, polyamide 6, substrate specificity, enzyme evolution, enzyme crystal structure, oligomeric state, enzyme engineering

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