How a protein can remain stable in a solvent with high content of urea: Insights from molecular dynamics simulation of Candida antarcetica lipase B in urea:choline chloride deep eutectic solvent

Abstract

Deep eutectic solvents (DESs) are utilized as green and inexpensive alternatives to the <br><br>classical ionic liquids. It was known that some of DESs can be used as solvent in the <br><br>enzymatic reactions to obtain very green chemical processes. DESs are almost poorly <br><br>understood at the molecular level. Moreover, we do not know much about the enzyme <br><br>microstructure in such systems. For example, how some hydrolase can remain active and <br><br>stable in a deep eutectic solvent including 9M of urea? In this study, molecular dynamic of <br><br>DESs as a liquid has been simulated at the molecular level. Urea: Choline chloride as a well-known eutectic mixture was chosen as model DESs. Behavior of the lipase as a biocatalyst <br><br>was studied in this system. For comparison, the enzyme structure was also simulated in urea <br><br>8M. Thermal stability of the enzyme was also evaluated in DESs, water, and urea 8M. The <br><br>enzyme showed very good conformational stability in urea:choline chloride mixture with <br><br>about 66% urea (9M) even at high temperatures. The results are in good agreement with <br><br>recent experimental observations. In contrast, complete enzyme denaturation occurred in urea <br><br>8M with only 12% urea in water. It was found that urea molecules denature the enzyme by <br><br>interrupting the intra-chain hydrogen bonds in a “direct denaturation mechanism”. However, <br><br>in urea:choline chloride deep eutectic solvent, as a result of hydrogen bonding with choline <br><br>and chloride ions, urea molecules have a low diffusion coefficient and cannot reach to the <br><br>protein domains. Interestingly, urea, choline, and chloride ions form hydrogen bonds with the <br><br>surface residues of the enzyme which, instead of lipase denaturation, leads to more enzyme <br><br>stability. To the best of our knowledge, this is the first study in which the microstructural <br><br>properties of a macromolecule are examined in a deep eutectic solvent.