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This study characterizes Acinetobacter sp. DL27, an APAP-degrading strain with broad temperature and pH adaptability, demonstrating its bioremediation potential in soil and wastewater. Through high-resolution mass spectrometry, we identified three novel metabolic intermediates, thereby refining the bacterial APAP degradation pathway. Multiomics analysis elucidated metabolic coordination and stress-tolerance mechanisms, leading to the identification of a novel amidase (AdA). Recombinant AdA exhibited activity over 10\u201360 \u00b0C and pH 4.0\u201310.0, with a Km of 8.96 \u00b1 1.03 \u03bcM and a kcat\/Km of 9.04 \u03bcM\u20131s\u20131, indicating higher catalytic efficiency than previously reported APAP amidases. Molecular dynamics simulations and site-directed mutagenesis confirmed that a Ser161\u2013Ser185\u2013Lys82 triad constitutes the catalytic center driving amide cleavage. These findings provide mechanistic insights into bacterial APAP biodegradation and highlight the practical application potential of strain DL27 and AdA.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c09241","type":"journal-article","created":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T09:59:59Z","timestamp":1787392799000},"page":"27779-27794","source":"Crossref","is-referenced-by-count":0,"title":["Functional Characterization and Catalytic Mechanism\nof an Amidase Involved in Acetaminophen Degradation from\n                    <i>Acinetobacter<\/i>\n                    sp."],"prefix":"10.1021","volume":"74","author":[{"given":"Boxiang","family":"Kou","sequence":"first","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yufei","family":"Yue","sequence":"additional","affiliation":[{"name":"Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences , , , ,","place":["Qingdao, Shandong, China, 266101"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0593-397X","authenticated-orcid":true,"given":"Dan","family":"Ma","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhongsen","family":"Sun","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuwei","family":"Liu","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zilian","family":"Zhang","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1173-3213","authenticated-orcid":true,"given":"Sijun","family":"Dong","sequence":"additional","affiliation":[{"name":"Hebei University , , , ,","place":["Baoding, Hebei, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"issue":"36","key":"2026090917081382700_cit1","doi-asserted-by":"publisher","first-page":"19146","DOI":"10.1021\/acs.est.5c05659","article-title":"Pharmaceutical and Personal Care\nProducts (PPCPs) in Global Surface Waters: Risk and Drivers","volume":"59","author":"Liu","year":"2025","journal-title":"Environ. 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