Ionic liquids (ILs) are utilized as the green solvents in the biocatalytic reactions. In this study, the effect of 1-Butyl- 3-methylimidazolium bromide ([BMIM][Br]) and 1-Butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]) ionic liquids on the structure and function of Chloroperoxidase (CPO) is investigated by molecular dynamics (MD) simulation. The results confirm that low and high concentrations of the liquids alter the enzyme structure. At low concentrations of the ionic liquids, the enzyme active site is maintained, or even, it is facilitated the substrate access to the site. Whereas, the high concentrations of the ionic liquids open the enzyme structure and reduce the flexibility of its residues. The high concentrations of the liquids also enhance the order of water molecules. Accurately, [BMIM][MeSO4] possesses greater influence on the enzyme structure, because of the special structure of the corresponding anion group. Besides, the number of cavities interprets the activation of the enzyme at the low concentrations and its inactivation at the high concentrations. The penetration of the anions into the enzyme structure is confirmed at the high concentrations of the ionic liquids. The role of ionic liquids at low concentrations is related to their binding to the enzyme structure. Their role at the high concentrations depends on the changes in the solvent arrangement as well as the attachment to the enzyme. Some binding sites of the anions and cations of the liquids are introduced precisely. Studies have shown that in the presence of ionic liquid [BMIM][Br] a new channel has been created which facilitate access of both active site and “Hem” to the surface and could lead to higher enzyme activity and more efficiency. Therefore, low concentration of ionic liquid play an important role and cause higher chloroproxidase activity. Whereas in high concentration due to surface coating of chloroproxidase by ionic liquid, despite the access of the substrate to the active site, CPO activity decreases.

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