Refinement of Synthetization Parameters for High Laccase-Like Activity of Imidazole-Copper (II) Nitrate Trihydrate Nanozyme Towards an Efficient Biomimetic Nanozyme

Laccase's industrial application is hindered by its sensitivity and low stability to extreme conditions. To overcome these limitations, the development of biomimetic nanozymes is gaining momentum. Nevertheless, developing multifunctional nanozymes with high laccase-like activity poses several challenges. This study focused on optimizing the synthesis of imidazole-copper (II) nitrate trihydrate (I-Cu) nanozymes and characterizing its physicochemical properties. Key synthesis parameters (precursor amount, incubation time, and oven temperature) were optimized. I-Cu nanozymes were synthesized in a Teflon-lined autoclave via water-induced precipitation of Cu2+ and imidazole, mimicking the N-Cu coordination found in laccase's active sites. Initial screenings revealed the superior catalytic activity of I-Cu nanozymes synthesized using methanol compared to ethanol, and a smaller nano-scale size than laccase. FTIR analysis confirmed the presence of similar chemical components as laccase (C44H69N11O20), verifying I-Cu nanozyme's capability to degrade phenolic compounds, and imidazole did not decompose throughout the synthesis process. The optimized I-Cu nanozyme demonstrated higher catalytic activity (6.569 UA), oxidation efficiency (Vmax of 0.00893 mM/min and Km of 2.4020 mM), and greater stability under varying pH, temperature, and storage conditions, compared to laccase. Conclusively, the optimized I-Cu nanozyme, with a 6.00-fold increase in catalytic activity compared to previous studies, as well as 1.69-fold higher Km, and 2.08-fold higher Vmax compared to laccase, shows promise as a wastewater treatment alternative. Its enhanced performance, achieved with fewer precursors through synthesis optimization, highlights the potential of lesser-known biomimetic nanozymes and underscores the importance of refining the synthesis parameters.