Molecular level insight into stability, activity, and structure of Laccase in aqueous ionic liquid and organic solvents: An experimental and computational research

Abstract

Herein, we performed meticulous experimental and computational studies to explore the stability, activity, and dynamics of laccase in the presence of various organic and inorganic solvents. It is well known that laccases are eco-friendly enzymes, which can quickly eliminate recalcitrant chemicals from contaminated media. We determined the Asp96 (COO−)---Arg43 (N-H2), Asp131 (COO−)---Arg197(N-H1), Asp138(COO−)---Arg195 (N-H2), Asp140(COO−)---Arg199(N-H2), Asp214(COO−)---Arg260(N-H2), Asp224(COO−)---Arg423(N-H2), Asp424(COO−)---Arg243(N-H1), Asp424(COO−)---Arg243(N-H2), Glu288(COO−)---Arg176(N-H1) and Glu288(COO−)---Arg176(N-H2) salt bridges as the crucial ones in maintaining the structural integrity of laccase. Furthermore, the fluorescence, circular dichroism (CD) spectroscopies, and molecular dynamics simulation outcomes highlighted that the secondary structure elements and tertiary structure of the enzyme did not change significantly in the presence of both selected organic (ethanol and hexane) and inorganic (1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) co-solvents. The obtained experimental results demonstrated that the enzyme was more stable in the hexane and aqueous ionic liquid solutions than in the ethanol solution. Additionally, laccase exhibited more activity in the presence of the hexane and aqueous ionic liquid solutions. Probably due to more accessibility of the substrate to the active site of laccase. Interestingly, the activity of laccase in the presence of the co-solvents was in decreasing order of hexane<[BMIM][PF6] < ethanol and the same order was observed for the number of water molecules in the enzyme's hydration shell. The results also indicated that the biocatalyst may keep a balance between the interactions with both water molecules and co-solvents to maintain its native conformation. The results also showed that there were some co-solvent molecules in the first hydration shell of the enzyme, but they were not enough to make a considerable change in the enzyme structure.