TY - JOUR
T1 - Natural Diversity Screening, Assay Development, and Characterization of Nylon-6 Enzymatic Depolymerization
T2 - Article No. 1217
AU - Bell, Elizabeth
AU - Rosetto, Gloria
AU - Ingraham, Morgan
AU - Ramirez, Kelsey
AU - Lincoln, Clarissa
AU - Clarke, Ryan
AU - Gado, Japheth
AU - Lilly, Jacob
AU - Kucharzyk, Katarzyna
AU - Erickson, Erika
AU - Beckham, Gregg
PY - 2024
Y1 - 2024
N2 - 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 degrees 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 degrees C, 16.4 degrees 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.
AB - 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 degrees 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 degrees C, 16.4 degrees 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.
KW - amide-cleaving enzymes
KW - biocatalytic polyester recycling
KW - depolymerization
KW - enzymatic hydrolysis
KW - nylon deconstruction
KW - nylonases
U2 - 10.1038/s41467-024-45523-5
DO - 10.1038/s41467-024-45523-5
M3 - Article
SN - 2041-1723
VL - 15
JO - Nature Communications
JF - Nature Communications
ER -