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

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.

doi:10.1016/j.checat.2025.101418

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

Journal Article · Fri Jun 13 00:00:00 EDT 2025 · Chem Catalysis

DOI:https://doi.org/10.1016/j.checat.2025.101418· OSTI ID:3002770

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Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Sciences (CNMS)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); North Carolina State University, Raleigh, NC (United States)
Kagoshima University (Japan)
Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
University of Hyogo, Himeji (Japan)

Enzymes can rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but few have been shown to hydrolyze synthetic polyamides. Here, 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.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 3002770

Journal Information:: Chem Catalysis, Journal Name: Chem Catalysis Journal Issue: 8 Vol. 5; ISSN 2667-1093

Publisher:: Cell PressCopyright Statement

Country of Publication:: United States

Language:: English

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