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The signal of \u03b2-1,2-N-acetylglucosamine is a key component within complex biantennary N-glycans on the cell surface, leaving related research in its early exploratory stages. Currently, there is a lack of efficient tools and methods capable of directly modifying terminal N-acetylglucosamine on living cells. In this study, we identified a novel \u03b2-N-acetylglucosaminidase from Elizabethkingia meningoseptica FMS-007, which specifically cleaves nonreducing terminal \u03b2-1,2-N-acetylglucosamine, enabling direct removal from native glycoproteins and\/or free oligosaccharides. More importantly, we demonstrate for the first time the successful removal of \u03b2-1,2-N-acetylglucosamine from the living cell surface using this enzyme. Based on its isolation and newly characterized cell-surface glycan-editing activity, we designated it as EmNag, also termed cell-surface glycan-editing N-acetylglucosaminidase (csgeNag). It showed favorable enzymatic stability between pH 5\u20138 and from 4 to 37 \u00b0C. Sequence analysis and alanine-scanning mutagenesis identified a conserved active pocket containing the catalytic residue pair Asp317-Glu318, with Asp317 being essential for activity. In summary, our findings provide an effective and reliable method for the targeted removal of \u03b2-1,2-N-acetylglucosamine from the living cell surface, establishing a foundation for further functional studies and practical applications.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00631","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T08:01:45Z","timestamp":1790928105000},"page":"3206-3216","source":"Crossref","is-referenced-by-count":0,"title":["Identification and Characterization of EmNag, a Novel\n\u03b2-1,2-N-Acetylglucosaminidase from\n                    <i>Elizabethkingia meningoseptica<\/i>"],"prefix":"10.1021","volume":"65","author":[{"given":"Shaoxian","family":"Lyu","sequence":"first","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongliang","family":"Tong","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiange","family":"Lin","sequence":"additional","affiliation":[{"name":"Zhejiang Chinese Medical University , , , ,","place":["Hangzhou, Zhejiang, China, 310053"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yilin","family":"Ye","sequence":"additional","affiliation":[{"name":"Zhejiang Chinese Medical University , , , ,","place":["Hangzhou, Zhejiang, China, 310053"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinliu","family":"Geng","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cuiying","family":"Chen","sequence":"additional","affiliation":[{"name":"Sysdiagno (Nanjing) Biotech Co., Ltd , , , ,","place":["Nanjing, Jiangsu, China, 210008"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinrong","family":"Lu","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guiqin","family":"Sun","sequence":"additional","affiliation":[{"name":"Zhejiang Chinese Medical University , , , ,","place":["Hangzhou, Zhejiang, China, 310053"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7006-4443","authenticated-orcid":true,"given":"Li","family":"Chen","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, China, 200032"]},{"name":"Fudan Zhangjiang Institute , , ,","place":["Shanghai, China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,2]]},"reference":[{"key":"2026102007161323800_cit1","doi-asserted-by":"publisher","first-page":"129","DOI":"10.3390\/jof6030129","article-title":"N-Acetylglucosamine\n(GlcNAc). sensing, utilization, and functions in Candida albicans","volume":"6","author":"Du","year":"2020","journal-title":"J. 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Neuroinflammation"},{"key":"2026102007161323800_cit5","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.62645","article-title":"Hyaluronic\nacid fuels pancreatic cancer cell growth","volume":"10","author":"Kim","year":"2021","journal-title":"Elife"},{"key":"2026102007161323800_cit6","doi-asserted-by":"publisher","first-page":"1366","DOI":"10.1016\/j.xphs.2018.11.029","article-title":"The Mechanistic Impact\nof N-Glycosylation on stability, pharmacokinetics,\nand immunogenicity of therapeutic proteins","volume":"108","author":"Zhou","year":"2019","journal-title":"J. Pharm. 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