Thermal instability of the industrial enzymes such as nitrile hydratase is a major problem in biotechnology. Insolubility of hydrophobic substrate of such enzymes in aqueous solvents is another problem. Enzymatic reactions in hydrophobic non-aqueous solvents may be a solution for these problems. Here, using molecular dynamic simulation it is shown for the first time that hydrophobic ionic liquids can protect nitrile hydratase from thermal instability at high temperatures. Different structural properties of the enzyme were analyzed at 300, 335, and 370 K respectively. Although enzyme was partially denatured by enhancing thermal stress in water, in 1-ethyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide, as a model hydrophobic ionic liquid, enzyme conformation remained native. Ionic liquid molecules interact strongly with the surface residues of the enzyme and rigidify the structure at high temperatures. Although the spatial arrangement of the catalytic residues in the active site was destructed in hot water, the active site remained native and therefore enzyme preserved its activity in high temperature ionic liquid. In contrast to the hydrophilic enzyme products, the substrates of nitrile hydratase are commonly hydrophobic. Therefore it is noteworthy that the application of hydrophobic ionic liquids not only is recommended for their thermal stability effects on the enzyme structure, but also is a very useful way for disposability of hydrophobic substrates to the active center of the enzyme and simple isolation of the hydrophilic products from solvent. The results of this paper can propose new promising technologies for nitrile hydratase catalyzed reactions and the industrial production of important materials such as acrylamide and nicotinamide.

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