Biocatalysis in presence of organic solvents has numerous industrially attractive advantages in comparison to traditional aqueous solvents. In some cases, the presence of organic molecules such as methanol in the processes such as enzymatic production of biodiesel is inevitable. However, enzyme inactivation and/or instability in organic solvents limits such biotechnological processes. Although it was found that some enzymes are more and others are less tolerant against organic solvents, the structural basis of such differences is relatively unknown. In this work using molecular dynamics simulations, we have investigated the structural behavior of enzymes with completely different structural architecture including lipase, laccase and lysozyme in presence of methanol as polar and hexane as non-polar organic solvents. In agreement with the previous experimental observations, simulations showed that lipase is more tolerant against both polar and non-polar organic solvents. It is found that lipase has high stability in pure hexane even higher than that obtained in aqueous solvent. In contrast, laccase shows better stability in aqueous conditions. To obtain general mechanism of enzyme inactivation in presence of methanol and hexane, we have treated lysozyme as model enzyme in different percentages of these solvent in long MD simulations. It is found that lysozyme is completely denatured in high concentrations of methanol, but it remains native at low concentration of this solvent. Interestingly, the concentration-dependence structural behavior of enzyme was completely different in presence of hexane. It was obtained that low concentrations of hexane may impose more instability on the enzyme conformation than higher percentages. Results also showed that presence of water is determining factor in the enzyme stability in high concentrations of hexane. Pure hexane may also lead to the surface denaturation of the enzymes. Both methanol and hexane denaturation mechanisms were initiated by diffusion of organic solvent in hydrophobic core. However, enzyme denaturation in hexane was continued by a collapse of hydrophobic core and entering hexane molecules to the core, but in methanol it was completed by decomposition of the secondary structures. In both cases it was found that beta structures are more prone to destabilize than helix structures. This may be a reason for obtained results about lower stability of laccase with β-barrel architecture than lipase with multiple helixes at it surface. In total, by our extensive structural data, it was found that the forces which stabilize tertiary structure have pivotal role in enzyme tolerance against both polar and non-polar organic solvents

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