In this paper, new findings about enzyme microenvironment in supercritical CO2/ionic liquid biphasic system were obtained. All-atom molecular dynamic simulation of three systems composed of enzyme, ionic liquid, and CO2 molecules was performed in 313 K and 100 bar. Percentages of ILs in the solvent phase are about 50, 10, and 0 % v/v in systems 1-3 respectively. Simulation clearly showed that enzyme and ionic liquid molecules form a supramolecular-like structure in supercritical CO2. Ionic liquid molecules function as a coating layer and protect enzyme from denaturing condition in supercritical CO2. Ionic liquid molecules function as a coating layer and protect enzyme from denaturing condition in supercritical CO2. By analysis of root mean square deviation it was found that the enzyme has more native and stable conformation in supercritical CO2/ionic liquid system than in supercritical CO2. Moreover, based on the radius of gyration values, it is concluded that enzyme has a more compact and active conformation in supercritical CO2/ionic liquid system than supercritical CO2. Root mean square fluctuations of enzyme flexible residues including α5 and α10 helixes are highly reduced by adding Ionic liquid molecules to supercritical CO2. Although α-helix and β-sheet content of the enzyme in supercritical CO2 reduced to some extent, they almost remained unchanged in supercritical CO2/ionic liquid. At the molecular level, the results of this research reasonably confirmed that the use of ionic liquid molecules is an efficient method for stabilizing enzymes in supercritical CO2.

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