Thermostable protein-stabilized gold nanoclusters as a peroxidase mimic

Protein-stabilized gold nanoclusters (AuNCs) are fascinating nanostructures with exciting properties owing to their ultra-small sizes and functional shell. However, their applications under extreme conditions are still complicated, waiting for programmable solutions. Therefore, the design of a multi-functional protein stabilizer for specific purposes gains attention to improve the stability and functionality of AuNCs. Herein, we exploited the thermostability of genetically engineered KlenTaq DNA polymerase containing five cysteine residues (KTQ5C) to synthesize heat-stable AuNCs (AuNC@KTQ5C) and characterize optical, structural, and hydrodynamic properties. Besides their excellent photophysical properties, AuNC@KTQ5C also exhibit superior peroxidase-like (POD-like) catalytic activity following typical Michaelis-Menten kinetics together with a high affinity towards the POD substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS). Moreover, FTIR and relative catalytic activity analysis of AuNC@KTQ5C reveal that KTQ5C is resistant to changes in protein secondary structure while the AuNCs conserve 70-80% of their catalytic performance after heat treatments up to more than 80 °C. Our findings show that stabilizing AuNCs with thermostable KTQ5C not only preserves the advantages of protein-stabilized AuNCs but can also promote the resistance of AuNCs against aggregation due to protein denaturation under extreme reaction temperatures, protecting their fluorescent emission or catalytic activity.