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Enhanced Thermostability and Anticancer Activity in Breast Cancer Cells of Laccase Immobilized on Pluronic-Stabilized Nanoparticles

Laccases are mainly found in fungi and plants known for catalyzing oxidation reactions. They are often named as multi-copper enzymes having a wide range of applications across a multitude of biotechnological processes.  Though widely used the major challenge is the low stability of the free enzyme which often restricts its usability for industrial purposes.  The development of new methods that can enhance the enzymatic activity is a need of time. In the present study, immobilization of trametes versicolor laccase on pH-responsive, pluronic stabilized silver nanoparticles (AgNPsTrp) was carried out. The results showed enhanced activity if enzyme upon stabilization of AgNPsTrp with amphiphilic copolymer Pluronic F127, this helps in changing the microenvironment of active site facilitating the improved activity. The results were confirmed using circular dichroism (CD) and fluorescence spectroscopy. Further analysis showed that lowering the activation energy and expanding the temperature window for substrate hydrolysis can play a crucial role in improving the enzyme activity. No change in the behavior of the nanocomposite indicated the stability of the enzyme as normal with the absence of any aggregation after immobilization. The efficiency of the nanocomposite in breast cancer cells- MCF-7 was observed which showed inhibition of cell proliferation by promoting cell apoptosis and beta-estradiol degradation. Further, qRT- PCR experiments were conducted to understand the underlying molecular mechanism hindering cell proliferation. The analysis showed a reduced level of mRNA levels of the anti-apoptotic genes and enhancement in level of pro-apoptotic genes. For example, BCL-2 and NF-kβ, and increase in levels of pro-apoptotic genes like p53. The entire study provides a new way of enhancing the enzymatic activity of Laccases and extending its subsequent applications.