Hydrolase and Peroxidase Activity of Gold Nanocluster Composites with Lysozyme and Cross-Linked Lysozyme Crystals

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Lysozyme (Lyz) and cross-linked Lyz crystals (CLLC) are among the proteins and templates used in the bioinspired synthesis of subnanometer-sized gold nanoclusters (AuNCs) utilized in various biological applications as biosensors, bioimaging agents, and recyclable catalyst. While previous studies have explored the formation and stabilization of AuNCs in the presence of Lyz, little is known about the enzymatic activity of Lyz after AuNCs stabilization in both its free form (AuNC-Lyz) and within CLLC (AuNC-CLLC). In this study, we conducted a comprehensive analysis of AuNC-Lyz, including hydrodynamic characteristics, using multiwavelength fluorescence analytical ultracentrifugation and enzymatic activity assays. Results show that AuNC-Lyz retains bactericidal performance at pH 4.0 and exhibits enhanced hydrolytic activity compared with Lyz alone. Additionally, AuNC-Lyz showed peroxidase-like performance due to the catalytically active surface of AuNCs providing the bifunctional catalytic capabilities of AuNC-Lyz. In contrast, fluorescent-based hydrolytic activity assays of AuNC-CLLC revealed that morphological variations in CLLC affect the catalytic performance. The AuNC-CLLC enzyme activity depends on the size of the solvent channels and Lyz conformation within CLLC. As a consequence of the occluded solvent channels and limited breathing (in/out diffusion) of CLLC, AuNC-CLLC displayed a diminishing hydrolytic performance.