RESUMO
If natural proteases are used in organic synthesis, they are often inactivated or give a low rate of reaction in non-aqueous or aqueous-organic media. Therefore, to reveal the molecular mechanism governing the stability of proteases in organic solvents and increase protease stability in those systems is of intriguing interest. In the present study, the activity and conformational changes of an organic solvent-stable protease (OSP) from Bacillus sphaericus DS11 in different concentrations of methanol were investigated by measuring fluorescence, UV-Vis spectra, circular dichroism (CD), and conducting molecular dynamics (MD) simulations. The OSP expanded with increasing methanol concentration. The methanol molecules were able to enter into the OSP, leading to microenvironmental changes around the aromatic amino acids. More hydrophobic groups were exposed to the solvents at high methanol concentrations, and the original hydrophobic interaction in the protein decreased, thus resulting in the secondary and tertiary structure change in the OSP. Our results provide helpful insight into the molecular mechanism of the OSP tolerance to organic solvent and indicate directions for future work to design and engineer proteases that are stable at high organic solvent concentrations.
