Molecular insights on the effects of anions towards hydrolases in [BMIM]-based ionic liquids

The use of non-aqueous solvents in biocatalysis has shown improvements in enzyme performance. A new type of non-aqueous solvent has attracted a lot of interests in biocatalysis, called the Room Temperature Ionic Liquid (RTIL). A lot of biocatalysis experiments have showed that RTILs can further increase the reaction rates and yields when used instead of conventional organic solvents. However, since there are many RTIL combinations available, selecting a specific RTIL for use in biocatalysis have proven to be quite challenging. A detailed understanding on the effects that different RTIL combination imposed on enzymes is therefore important. Here, the behavior of enzymes in RTILs was characterized by their effects when different RTIL anions were used. A series of molecular-level investigations were conducted using molecular dynamics (MD) and stochastic dynamics (SD) simulations in order to gain more information on the structural and dynamics properties of enzymes in RTILs. Four hydrolases, consisted of α-Chymotrypsin, thermolysin, Candida Antarctica Lipase B (CALB) and Candida rugosa Lipase (CRL) were studied. These hydrolases were solvated in aqueous and five, 1-butyl-3- methylimidazolium ([BMIM])-based RTILs with different anions such as hexafluorophosphate ([PF6]-), tetrafluoroborate ([BF4]-), chloride ([Cl]-), trifluoromethanesulfonate ([TfO]-) and bis-trifluoromethylsulfonylimide ([Tf2N]-). The effects of water molecules in the systems were studied at 5%, 10%, 15%, 20% and 50% of water, based on the weight/weight percentages of the protein mass (w/w protein). All RTIL solvent models produced a liquid ordering at room temperature and an average density that was close to experimental data with a percentage error of below than 5%. The structural stability of all hydrolases studied showed a dependency towards the water content, in which the minimum atomic displacements were observed around 10 to 20% of water. Around this water percentage region, [TfO]- anion rendered the most stable conformation for α-Chymotrypsin, CALB and CRL. The smallest [Cl]- anion was found to produce the least stable conformations compared to other RTILs studied. In the case of thermolysin, the order of structural stability between the RTIL anions at 15% of water was [PF6]- > [TfO]- ~ [Tf2N]- > [Cl]-~ [BF4]- which was different from other hydrolases studied. Further investigations revealed that in [BMIM][PF6], thermolysin showed better structural stability than in aqueous, even when simulated at 90 °C. The effect of changing the RTIL anions towards the enzyme flexibility was only clearly visible at higher water content (20% and 50% w/w protein), especially for [PF6]-and [Tf2N]- anions. The analysis on local flexibility showed that only the surface of the protein was affected. For the lipases, the local flexibility was found significantly reduced in certain regions which were highly flexible in aqueous solution, particularly for the lid of the CRL. MD simulations revealed a structured ordering of RTIL anions around the enzymes while the water molecules were found localized at certain region of the protein surface. Hydrophobic anions such as [PF6]- covered more areas and were more organized at low water content while [Cl]-anion behave otherwise. Meanwhile, a number of water molecules were stripped off from the surface of α-Chymotrypsin, CALB and CRL. RTILs with [PF6]- and [TfO]- anions retained more water on the surface as compared to [BF4]- and [Cl]- anions, consistently for the three hydrolases. [Tf2N]- anion was found stripping the most number of water for the case ofα-Chymotrypsin and CALB while the least was found for CRL. The solvation thermodynamics of amino acid side chain analogues in water and five [BMIM]-based RTILs was investigated using SD simulations. The solvation free energy was calculated using Bennett’s Acceptance Ratio method. Results from the simulations in water were in agreement with published experimental and simulation data. RTILs showed better solvation capabilities when compared with water. Nonpolar analogues produced lower solvation free energy in hydrophobic anions such as [PF6]- and [Tf2N]- while the polar ones showed better solvation in hydrophilic anions such as [BF4]-, [Cl]- and [TfO]-. The solvation properties in [BMIM][Cl] also explained why the enzymes experienced more conformational distortions in this RTIL at low water content. Overall, computer simulations were able to explain several effects of RTIL anions on the structure and dynamics of enzymes at molecular level. The structural stability and flexibility of the enzymes were found affected by the water content, more than the types of the RTIL anions studied. MD simulation results were correlated with experimental reports. It was found that the behavior of anions and water at the protein surface played a major role towards the properties of enzymes in RTILs. The results also suggested that the surface properties of the biocatalyst and the physicochemical properties of the substrate should be taken into consideration when choosing a particular RTIL as the solvent system