Arginine‐Induced Catalytic Microenvironment Engineering of ZrMOFs Enables Broad‐Spectrum Hydrolase Nanozymes for Biofilm Eradication

ABSTRACT Bacterial biofilms are protected by an extracellular polymeric substance (EPS) matrix primarily composed of extracellular DNA, proteins, and polysaccharides, rendering them highly resistant to conventional antimicrobial treatments. Although natural hydrolases efficiently degrade individual EPS components, their narrow substrate scope, poor stability, and high cost restrict effective biofilm eradication. Here, we report a catalytic microenvironment engineering strategy that enables broad‐spectrum hydrolase nanozymes through arginine‐directed modulation of Zr‐based metal–organic frameworks (ZrMOFs). Systematic amino acid‐directed defect engineering afforded a series of amino acid‐coordinated ZrMOFs, among which arginine‐coordinated ZrMOF (Arg‐ZrMOF) exhibited the highest hydrolase‐like catalytic efficiency ( k cat /K m = 1437.5 M −1 s −1 ). The enhanced catalytic performance originated from the engineered catalytic microenvironment, characterized by increased surface hydrophilicity, enhanced formation of nucleophilic Zr–OH species, and strengthened electrostatic interactions with negatively charged substrates. Arg‐ZrMOF efficiently hydrolyzed phosphodiester, peptide, and glycosidic bonds, enabling concerted degradation of extracellular DNA, proteins, and polysaccharides within the EPS matrix. This expanded hydrolytic substrate scope enabled superior inhibition and eradication of Escherichia coli and Staphylococcus aureus biofilms, outperforming the benchmark enzyme DNase I while retaining over 80% catalytic activity after 60 reuse cycles and excellent storage stability for more than 100 days. Furthermore, a spray‐on Arg‐ZrMOF formulation enabled effective biofilm removal from stainless‐steel, plastic, and glass surfaces, demonstrating its practical applicability for surface biofilm control. This work establishes catalytic microenvironment engineering as a versatile strategy for constructing hydrolase nanozymes capable of broad biomacromolecular degradation and practical biofilm eradication.