Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease

Abstract

Proteases 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 "chaophilic" protease that would be resistance to GdmCl and SDS, for application in diagnostic kits. In three iterative rounds of directed evolution, variant SeSaM1-5 (S62I/A153V/G166S/I205V) was generated, with improved activity (330 %) and increased half life in 1 M GdmCl (<2 min to 4.7 h) or in 0.5 % SDS (<2 min to 2.7 h). Saturation mutagenesis at each site in the wild-type subtilisin E revealed that positions 62 and 166 were mainly responsible for increased activity and stability. A double mutant, M2 (S62I/G166M), generated by combination of the best single mutations showed significantly improved kinetic constants; in 2 M GdmCl the K(m) value decreased (29-fold) from 7.31 to 0.25 mM, and the k(cat) values increased (fourfold) from 15 to 61 s(-1) . The catalytic efficiency, k(cat)/K(m), improved dramatically (GdmCl: 247 mM(-1)s(-1) (118-fold); SDS, 179 mM(-1)s(-1) (13-fold)). In addition, the SeSaM1-5 variant showed higher stability in 2.0 % SDS when compared to the wild-type (t(1/2) 54.8 min (>27-fold)). Finally, molecular dynamics simulations of the wild-type subtilisin E showed that Gdm(+) ions could directly interact with active site residues, thereby probably limiting access of the substrate to the catalytic centre.