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

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

doi:10.1016/j.checat.2025.101418

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

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

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 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, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1%, necessitating further optimization before potential applications. 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 nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.

Original language	English
Article number	101418
Journal	Chem Catalysis
Volume	5
Issue number	8
DOIs	https://doi.org/10.1016/j.checat.2025.101418
State	Published - Aug 21 2025

Funding

Research was sponsored by the Laboratory Directed Research and Development Program at Oak Ridge National Laboratory (ORNL), which is managed by UT-Battelle LLC under contract no. DE-AC05-00OR22725 for the U.S. Department of Energy (DOE). The U.S. government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. government purposes. The DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ). We thank Philip Gray for assisting with the design of Figure 1 . This work used resources of the Compute and Data Environment for Science (CADES) at ORNL. Crystallization screening at the National Crystallization Center at HWI was supported through NIH grant R24GM141256 .

Keywords

NylC
SDG6: Clean water and sanitation
SDG9: Industry, innovation, and infrastructure
nylon
nylon hydrolase
nylon hydrolysis
nylonase
polyamide
recycling

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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., ... Michener, J. K. (2025). Identification and characterization of substrate- and product-selective nylon hydrolases. Chem Catalysis, 5(8), Article 101418. https://doi.org/10.1016/j.checat.2025.101418

@article{60512c038bb04970ae87fb808009a696,

title = "Identification and characterization of substrate- and product-selective nylon hydrolases",

abstract = "Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 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, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1\%, necessitating further optimization before potential applications. 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 nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.",

keywords = "NylC, SDG6: Clean water and sanitation, SDG9: Industry, innovation, and infrastructure, nylon, nylon hydrolase, nylon hydrolysis, nylonase, polyamide, recycling",

author = "Drufva, \{Erin E.\} and Cahill, \{John F.\} and Saint-Vincent, \{Patricia M.B.\} and Williams, \{Alexis N.\} and Vera Bocharova and Nikolas Capra and Flora Meilleur and Carper, \{Dana L.\} and C{\'e}lestin Bourgery and Kaito Miyazaki and Maina Yonemura and Yuki Shiraishi and Parks, \{Jerry M.\} and Muchu Zhou and Dishner, \{Isaiah T.\} and Foster, \{Jeffrey C.\} and Koehler, \{Stephen J.\} and Valentino, \{Hannah R.\} and Ada Sedova and Vilmos Kertesz and Vasileva, \{Delyana P.\} and Hochanadel, \{Leah H.\} and Figg, \{C. Adrian\} and Seiji Negoro and Kato, \{Dai ichiro\} and Chen, \{Serena H.\} and Michener, \{Joshua K.\}",

note = "Publisher Copyright: {\textcopyright} 2025 Elsevier Inc.",

year = "2025",

month = aug,

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doi = "10.1016/j.checat.2025.101418",

language = "English",

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Drufva, EE, Cahill, JF, Saint-Vincent, PMB, Williams, AN , Bocharova, V , Capra, N, Meilleur, F, Carper, DL , Bourgery, C, Miyazaki, K, Yonemura, M, Shiraishi, Y, Parks, JM , Zhou, M , Dishner, IT , Foster, JC, Koehler, SJ, Valentino, HR, Sedova, A , Kertesz, V , Vasileva, DP, Hochanadel, LH, Figg, CA, Negoro, S, Kato, DI, Chen, SH & Michener, JK 2025, 'Identification and characterization of substrate- and product-selective nylon hydrolases', Chem Catalysis, vol. 5, no. 8, 101418. https://doi.org/10.1016/j.checat.2025.101418

TY - JOUR

T1 - Identification and characterization of substrate- and product-selective nylon hydrolases

AU - Drufva, Erin E.

AU - Cahill, John F.

AU - Saint-Vincent, Patricia M.B.

AU - Williams, Alexis N.

AU - Bocharova, Vera

AU - Capra, Nikolas

AU - Meilleur, Flora

AU - Carper, Dana L.

AU - Bourgery, Célestin

AU - Miyazaki, Kaito

AU - Yonemura, Maina

AU - Shiraishi, Yuki

AU - Parks, Jerry M.

AU - Zhou, Muchu

AU - Dishner, Isaiah T.

AU - Foster, Jeffrey C.

AU - Koehler, Stephen J.

AU - Valentino, Hannah R.

AU - Sedova, Ada

AU - Kertesz, Vilmos

AU - Vasileva, Delyana P.

AU - Hochanadel, Leah H.

AU - Figg, C. Adrian

AU - Negoro, Seiji

AU - Kato, Dai ichiro

AU - Chen, Serena H.

AU - Michener, Joshua K.

PY - 2025/8/21

Y1 - 2025/8/21

N2 - Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 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, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1%, necessitating further optimization before potential applications. 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 nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.

AB - Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 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, but there was no relationship between phylogeny and activity, nor any evidence of prior evolutionary selection for nylon hydrolysis. Several newly identified hydrolases showed substrate selectivity, generating up to 20-fold higher product titers with nylon-6,6 versus nylon-6. However, the yield was still less than 1%, necessitating further optimization before potential applications. 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 nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.

KW - NylC

KW - SDG6: Clean water and sanitation

KW - SDG9: Industry, innovation, and infrastructure

KW - nylon

KW - nylon hydrolase

KW - nylon hydrolysis

KW - nylonase

KW - polyamide

KW - recycling

UR - https://www.scopus.com/pages/publications/105008088418

U2 - 10.1016/j.checat.2025.101418

DO - 10.1016/j.checat.2025.101418

M3 - Article

AN - SCOPUS:105008088418

SN - 2667-1107

VL - 5

JO - Chem Catalysis

JF - Chem Catalysis

IS - 8

M1 - 101418

ER -

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

Abstract

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Keywords

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