title: Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate.
abstract: A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691-699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7M; IC50 (M6): 4.6M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2min; t 1/2 (M6): 15min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ∼1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1-2M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substrate binding.
authors.affiliation: RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Li
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Danilo
authors.initials: D
authors.lastname: Roccatano
authors.affiliation: Molzym GmbH & Co. KG, Mary-Astell-Strasse 10, 28359 Bremen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Lorenz
authors.affiliation: RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.affiliation: RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany. Electronic address: u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001407:Bacillus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012967:Sodium Dodecyl Sulfate; D013381:Subtilisins; D019791:Guanidine
keywords: Chaotolerance; Directed evolution; Guanidinium chloride; Protease; SDS