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AbstractElectrostatic interactions of enzymes and their effects on enzyme activity and stability are poorly understood in non-aqueous conditions. Here, we investigate the contribution of the electrostatic interactions on the stability and activity of enzymes in the non-aqueous environment using molecular dynamics simulations. Lipase was selected as active and lysozyme as inactive model enzymes in non-aqueous media. Hexane was used as a common non-aqueous solvent model. In agreement with the previous experiments, simulations show that lysozyme has more structural instabilities than lipase in hexane. The number of hydrogen bonds and salt bridges of both enzymes is dramatically increased in hexane. In contrast to the other opinions, we show that the increase of the electrostatic interactions in non-aqueous media is not so favorable for enzymatic function and stability. In this condition, the newly formed hydrogen bonds and salt bridges can partially denature the local structure of the enzymes. For lysozyme, the changes in electrostatic interactions occur in all domains including the active site cleft, which leads to enzyme inactivation and destabilization. Interestingly, most of the changes in electrostatic interactions of lipase occur far from the active site regions. Therefore, the active site entrance regions remain functional in hexane. The results of this study reveal how the changes in electrostatic interactions can affect enzyme stability and activity in non-aqueous conditions. Moreover, we show for the first time how some enzymes, such as lipase, remain active in a non-aqueous environment.Communicated by Ramaswamy H. Sarma

Deep eutectic solvents (DESs) are one of the most interesting research subjects in green chemistry nowadays. Due to their low toxicity, simple synthesis, and lower prices, they have gradually taken the place of other green solvents such as ionic liquids (ILs) in sustainable processes. However, problems such as high viscosity and high polarity limit the applications of DESs in areas such as extraction, catalysis, and biocatalysis. In this work, we introduce and evaluate the potential application of scCO2/DES for the first time. Molecular dynamics simulations were used to examine the phase behavior, polarity, molecular mobilities, and microstructure of this system. Results show that CO2molecules can significantly diffuse to the DES phase, while DES components do not appear in the scCO2phase. The diffused CO2molecules significantly enhanced the molecular mobility of the DES components. The presence of CO2molecules changes the DES polarity so that hexane can be solubilized and dispersed in the DES phase. Radial distribution functions show that the solubilized CO2molecules have negligible effects on the microstructure of DES. It was shown that chloride and urea are the main interaction sites of CO2in DES. The results of this study show that scCO2/DES as a new class of green and versatile solvents can open a new promising window for research in sustainable chemistry and engineering.