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Proteases are industrially important enzymes and have niche applications in diagnostic kits that use cell lysis and thereby require high resistance towards chaotropic salts and detergents, such as guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS). Subtilisin E, a well-studied serine protease, was selected to be re-engineered by directed evolution into a “chaotolerant” protease that would be resistant towards GdmCl and SDS, for application in diagnostic kits. In three iterative rounds of directed evolution, the variant SeSaM1-5 (S62I/A153V/G166S/I205V), designated as M4, was generated, with improved activity (330% on Suc-AAPF-AMC) and increased half life in 1 M GdmCl (27 fold)). Two other variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/ A153V/G166S/I205V/N218S/T224A) were constructed by combination of additional beneficial substitutions for further improvement of activity and resistance towards GdmCl and SDS. Inactivation curves showed that the activity of M6 towards Suc-AAPF-pNA as substrate was much improved compared to M4 in the presence of up to 5 M GdmCl (IC50 (M4): 2.7 M; IC50 (M6): 4.6 M) and up to 4% SDS (IC50 (M4): 1.5% ; IC50 (M6): 4.0%). Half life value of M6 in the presence of GdmCl was also increased (3 M GdmCl: t1/2 68 min; 2% SDS: t1/2 66 min). M5 was slightly more active in GdmCl but less active in SDS compared to M4. Additionally M5 was more active towards complex protein substrate (Azocasein) when compared to both M4 and M6. However, M5 was not stable in GdmCl or SDS in terms of its half life. Circular dichroism (CD) spectra analysis showed that secondary structures of subtilisin E wild type and three mutants (M4, M5 and M6) were almost intact at lower concentrations of GdmCl (1-2 M) and SDS (0.5-2.0%), indicating inactivation precedes conformation changes in subtilisin E in the presence of chaotropic agents. Finally, model building, molecular docking and molecular dynamics (MD) simulations of subtilisin E wild type showed that the guanidinium cation (Gdm+) or the monomeric dodecylsulfate anion (DS-) are likely to directly interact with the active site or substrate binding pocket residues, probably limiting the access of the substrate to the catalytic centre. Simulations of M4 indicated that the residues forming part of the active site or substrate binding pocket of the mutant are less affected or accessible by Gdm+ or DS-. |
