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As promising alternatives to natural enzymes, nanoproteases have garnered substantial attention for overcoming their inherent limitations. However, the catalytic performance of most reported nanoproteases remains unsatisfactory, primarily due to their insufficient catalytic activity. In this work, a two-dimensional bimetallic metal\u2013organic framework (2D MOF, CeCuBDC) is developed as a highly efficient nanoprotease for peptide bond hydrolysis. The catalytic mechanism and the hydrolysis pathway are systematically investigated by comprehensive experiments and characterizations. The 2D MOF maximizes active site accessibility and reduces mass-transfer resistance. Meanwhile, bimetallic doping and the electron-withdrawing effect of the organic ligand further enhance the Lewis acidity of metal sites, thereby significantly promoting the catalytic performance toward peptide bond hydrolysis. Moreover, the organic ligand promotes protein conformational changes via hydrophobic interactions, exposing more protein cleavage sites and accelerating the hydrolysis rate. Consequently, the CeCuBDC nanoprotease exhibits a 3\u20135-fold enhancement in protein hydrolysis efficiency relative to conventional proteases reported in the literature. The as-prepared CeCuBDC exhibits excellent stability and recyclability during protein hydrolysis. Furthermore, it displays high efficiency toward the hydrolysis of various proteins and protein mixtures, while showing a preference for cleaving peptide bonds containing hydrophobic residues. This study enables the rational design of nanoproteases with superior hydrolytic activity and relative selective cleavage ability, offering new strategies for constructing high-performance nanoproteases toward applications in proteomic research.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c01164","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:48:18Z","timestamp":1788396498000},"page":"26397-26409","source":"Crossref","is-referenced-by-count":0,"title":["Bimetallic\n2D Metal\u2013Organic Framework Nanosheets\nas High-Performance Nanoproteases for Efficient Hydrolysis of Protein\nPeptide Bonds"],"prefix":"10.1021","volume":"98","author":[{"given":"Wenqian","family":"Jia","sequence":"first","affiliation":[{"name":"Beijing University of Chemical Technology , , ,","place":["Beijing, P. R. 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