Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Natural Diversity Screening, Assay Development, and Characterization of Nylon-6 Enzymatic Depolymerization

Authors:

Gregg T. Beckham1,2* (gregg.beckham@nrel.gov), Elizabeth L. Bell1,2, Gloria Rosetto1, Morgan A. Ingraham1,2, Kelsey J. Ramirez1,2, Clarissa Lincoln1,2, Ryan W. Clarke1,2, Japheth E. Gado1,2, Jacob L. Lilly3, Kate H. Kucharzyk3, Erika Erickson1,2

Institutions:

1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory; 2BOTTLE Consortium; 3Battelle Memorial Institute

Goals

Motivated by the achievements in biocatalytic PolyEthylene Terephthalate (PET) recycling, there is a growing interest in investigating the enzymatic recycling of various other man-made polymers. Polyamides (nylons) have emerged as a logical focus due to the extensive range of naturally occurring amide-active enzymes. In this pursuit, researchers aimed to assess a selection of biocatalysts for their propensity for nylon-6 hydrolysis. The team assessed 40 potential nylon-deconstructing enzymes (nylonases) for their ability to depolymerize solid nylon-6 films. Initially considering enzymes with various catalytic mechanisms, such as nylon oligomer hydrolases, amidases, serine hydrolases, and proteases, researchers also strategically thermostabilized some of the most promising candidates to enhance nylon-6 hydrolysis at high temperatures. The testing of such a range of enzymes should allow researchers to select the best candidate biocatalysts for further interrogation.

Abstract

Approach and Activities: A high-throughput LC-MS/MS method was devised to analyze the products of nylon-6 hydrolysis reactions, simultaneously identifying, and quantifying eight potential polymer deconstruction products from a single sample. The 40 potential nylonases were then assessed in time-course reactions spanning 40 to 70°C using nylon-6 film as the substrate, and the described LC-MS/MS based analytical method to quantify the extent of nylon deconstruction. These activities were coupled with rigorous assessments of the nylon substrate pre-and post-enzymatic deconstruction using a range of materials characterization techniques such as DSC, TGA, GPC and SEM. The best candidate enzymes were then examined more rigorously by altering reaction pH, substrate loadings, and enzyme loadings.

Results and Lessons Learned: Analysis of the products following enzymatic hydrolysis of solid nylon-6 unveiled notable differences in product selectivity among the studied enzyme types. Despite extensive testing, all the nylonases showed low depolymerization extents, hinting at the rarity of robust nylon deconstruction activity amongst natural enzymes. Within the examined enzyme set, a rationally thermostabilized N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, exhibited the highest activity, suitable for depolymerization reactions up to 80°C. However, this enzyme only deconstructed 0.7 wt% of a nylon-6 film with reactions levelling off after 7 days. Inability to restart reactions after adding fresh enzyme led the team to hypothesize a substrate-based limitation in further nylon-6 deconstruction, possibly due to the lack of remaining enzyme- accessible amide bonds. In conclusion, this research expands the knowledge of nylonase activity distribution among diverse enzyme types, highlights the promise of Ntn-hydrolases for deeper exploration, and identifies crucial pathways for advancing the enzymatic depolymerization of nylon-6. These pathways include further enzyme engineering, refining product selectivity, and augmenting polymer accessibility.