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

Lysozyme (Lyz) and cross-linked Lyz crystals (CLLC) are among the proteins and templates used in the bio-inspired synthesis of subnanometer-sized gold nanoclusters (AuNCs) utilized in various biological applications as biosensors or bioimaging agents and recyclable catalyst, respectively. 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 stabilization of AuNCs with the protein (AuNC@Lyz) and in 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. AuNC@Lyz exhibited bactericidal performance as efficiently as Lyz at pH 4.0, with enzymatic activity assays indicating more efficient hydrolytic activity of AuNC@Lyz compared to Lyz under the same conditions. Additionally, AuNC@Lyz showed peroxidase-like performance due to the catalytically active surface of AuNCs in the presence of H2O2 and a chromogenic substrate. The multifunctional catalytic capabilities of AuNC@Lyz, including lytic activity and mimic peroxidase function, were highlighted. Additionally, we conducted fluorescent-based hydrolytic activity assays of CLLC, revealing that morphological variations of CLLC influence the catalytic performance of the enzyme crystals depending on the size of solvent channels and Lyz conformation within the CLLC. Furthermore, the bio-inspired synthesis of AuNCs in CLLC resulted in size distributions of AuNCs correlated with pore sizes. As a consequence of the occluded solvent channels and limited breathing of CLLC, AuNC@CLLC displayed a diminishing hydrolytic performance.