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

Lysozyme\n(Lyz) and cross-linked Lyz crystals (CLLC) are among the\nproteins and templates used in the bioinspired synthesis of subnanometer-sized\ngold nanoclusters (AuNCs) utilized in various biological applications\nas biosensors, bioimaging agents, and recyclable catalyst. While previous\nstudies have explored the formation and stabilization of AuNCs in\nthe presence of Lyz, little is known about the enzymatic activity\nof Lyz after AuNCs stabilization in both its free form (AuNC-Lyz)\nand within CLLC (AuNC-CLLC). In this study, we conducted a comprehensive\nanalysis of AuNC-Lyz, including hydrodynamic characteristics, using\nmultiwavelength fluorescence analytical ultracentrifugation and enzymatic\nactivity assays. Results show that AuNC-Lyz retains bactericidal performance\nat pH 4.0 and exhibits enhanced hydrolytic activity compared with\nLyz alone. Additionally, AuNC-Lyz showed peroxidase-like performance\ndue to the catalytically active surface of AuNCs providing the bifunctional\ncatalytic capabilities of AuNC-Lyz. In contrast, fluorescent-based\nhydrolytic activity assays of AuNC-CLLC revealed that morphological\nvariations in CLLC affect the catalytic performance. The AuNC-CLLC\nenzyme activity depends on the size of the solvent channels and Lyz\nconformation within CLLC. As a consequence of the occluded solvent\nchannels and limited breathing (in/out diffusion) of CLLC, AuNC-CLLC\ndisplayed a diminishing hydrolytic performance.