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Abstract
Laccase is a highly versatile biocatalyst with broad applications across bioremediation, biofuel production, organic synthesis and in industries such as food and paper manufacturing. However, the industrial use of laccase is often limited by its inactivation under harsh conditions, including the presence of non-aqueous solvents, extreme pH and high temperatures. To overcome these challenges, various solvent engineering strategies have been explored, including the use of natural deep eutectic solvents (NADESs) as environmentally friendly alternatives to traditional organic solvents. In this study, the effects of NADESs composed of betaine, choline chloride, L-proline, and sorbitol were evaluated. Sorbitol was consistently used as the hydrogen bond donor to assess the impact of different hydrogen bond acceptors. Laccase activity, structural stability and interactions with the NADES components were investigated using enzyme activity assays, intrinsic fluorescence, circular dichroism (CD) spectroscopy and molecular docking studies. The results revealed a significant enhancement in laccase activity when Betaine:Sorbitol NADES was used, suggesting its potential as a sustainable solvent system for industrial applications. In contrast, the NADESs containing choline chloride and L-proline as the acceptors showed an inhibitory effect on laccase activity, highlighting the importance of carefully selecting NADES components. CD and intrinsic fluorescence measurements indicated that the reduced activity of laccase in the L-proline:sorbitol system was due to a substantial structural change in the enzyme. In contrast, the enhanced activity observed in the Betaine:Sorbitol NADES could be attributed to the retention of laccase structural stability and favorable interactions between the NADES components and the enzyme's active site residues. These findings provide valuable insights into the design and selection of NADESs for various industrial applications, with the potential for improved enzyme stability and activity.
