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

Abstract

Laccases are multi-copper oxidase enzymes having widespread applications in various biotechnological fields. However, low stability of free enzymes restricts their industrial use. Development of effective methods to preserve and even increase the enzymatic activity is critical to maximize their use, though this remains a challenge. In the present study we immobilized Trametes versicolor laccase on pH-responsive (and charge-switchable) Pluronic-stabilized silver nanoparticles (AgNPsTrp). Our results demonstrate that colloidal stabilization of AgNPsTrp with the amphiphilic copolymer Pluronic F127 enhances enzyme activity (AgNPsTrpF1 + Lac6) by changing the active site microenvironment, which is confirmed by circular dichroism (CD) and fluorescence spectroscopy. Detailed kinetic and thermodynamic studies reveal a facile strategy to improve the protein quality by lowering the activation energy and expanding the temperature window for substrate hydrolysis. The immobilized nanocomposite did not show any change in flow behavior which indirectly suggests that the enzyme stability is maintained, and the enzyme did not aggregate or unfold upon immobilization. Finally, assessing the anticancer efficacy of this nanocomposite in breast cancer MCF-7 cells shows the inhibition of cell proliferation through β-estradiol degradation and cells apoptosis. To understand the molecular mechanism involved in this process, semi qRT-PCR experiments were performed, which indicated significant decrease in the mRNA levels of anti-apoptotic genes, for example, BCL-2 and NF-kβ, and increase in the mRNA level of pro-apoptotic genes like p53 in treated cells, compared to control. Overall, this study offers a completely new strategy for tailoring nano-bio-interfaces with improved activity and stability of laccase.

• Authors: Prakram Singh Chauhan, Murali Kumarasamy, Alejandro Sosnik, Dganit Danino
• Journal: ACS applied materials & interfaces (ACS Appl Mater Interfaces) Vol. 11 Issue 43 Pg. 39436-39448 (Oct 30 2019) ISSN: 1944-8252 [Electronic] United States
• PMID: 31580644 (Publication Type: Journal Article)

Chemical References

• Enzymes, Immobilized
• Fungal Proteins
• Neoplasm Proteins
• Poloxamer
• Laccase

Topics

• Breast Neoplasms (drug therapy, metabolism, pathology)
• Enzyme Stability
• Enzymes, Immobilized (chemistry, pharmacology)
• Female
• Fungal Proteins (chemistry, pharmacology)
• Gene Expression Regulation, Neoplastic (drug effects)
• Humans
• Laccase (chemistry, pharmacology)
• MCF-7 Cells
• Neoplasm Proteins (biosynthesis)
• Poloxamer (chemistry, pharmacology)
• Polyporaceae (enzymology)