UItrastable Silver-Nanozymes surface-functionalized with artificial catalytic-triad biomimic esterase hydrolisis activity

Natural enzymes are essential catalysts for a wide range of reactions and they can have many applications in medicine, industry and research fields, but have some limitations such as sensitivity to environmental conditions, difficulty of isolation and lack of stability. The development of artificial catalysts represents a promising research field for catalysis. The use of nanomaterials offers many advantages, including the reduction of production costs, ease of large-scale production, increased stability, high surface reactivity and the ability to fine-tune the catalytic properties by changing the composition of the nanomaterial and of the linker used. In particular, catalytic nanosystems based on nanoparticles, also known as nanozymes, offer a promising alternative to traditional enzymes. Nanozymes based on metal nanoparticles may combine the stability and reproducibility of inorganic materials with the catalytic properties of the ligands composing the nanoparticles. This research project focused on obtaining nanozymes with hydrolytic activity of ester substrates, exploiting highly stable silver nanoclusters functionalized with various artificial ligands inspired by the catalytic triad of esterases. Thiol-protected ultrastable silver nanoclusters were synthesized through chemical reduction. They were then functionalized with artificial catalytic-triad biomimetic peptides synthesized in solid phase. Several nanozyme candidates have been created, using different peptide ligands. Notably, one approach involved peptides containing the entire catalytic triad within a single unit, while another catalytic construct was designed by assembling two distinct helical peptides, each containing a part of the catalytic triad. Furthermore, nanozymes were enriched with alkyl chains to enhance their catalytic performance. Indeed they create hydrophobic pockets, similar to those found in natural enzymes, to accommodate the catalytic residues, improving the catalytic efficiency of hydrolytic reactions on ester substrates and preventing the inactivation of the catalyst. These nanozymes underwent to detailed analysis of their physical and chemical properties, including the determination of the size and shape of the functionalized nanoclusters. Furthermore, the catalytic activity of the nanozymes was evaluated by specific tests using model ester substrates. Key factors influencing the catalytic performance, such as nanozymes concentration, pH and reaction temperature, were investigated. Furthermore, the effect of hydrophobic environment on the catalytic performance of nanozymes was investigated, in order to optimize their selectivity and stability in an environment similar to that of native esterases. The kinetic results obtained by UV-visible spectrometry, which monitored the spectrum of the reaction product, demonstrated that the designed nanozymes exhibited high catalytic activity in ester conversion. This success suggests that such nanozymes may represent a promising alternative to natural esterases for ester hydrolysis, opening new perspectives for bio-catalytics and nanomedicine.