Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate

Li, Zhenwei and Roccatano, Danilo and Lorenz, Michael and Martinez, Ronny and Schwaneberg, Ulrich (2014) Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate. Journal of Biotechnology, 169 . pp. 87-94. ISSN 0168-1656

Documents
Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate

Request a copy
[img] PDF
1-s2.0-S0168165613004835-main.pdf - Whole Document
Restricted to Repository staff only

1MB
Item Type:Article
Item Status:Live Archive

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): 2.7 M; IC50 (M6): 4.6 M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5 M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2 min; t 1/2 (M6): 15 min). 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 1–2 M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3 M 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.

Keywords:Chaotolerance, Directed evolution, Guanidinium chloride, Protease, Sodium dodecylsulfate (SDS), JCNotOpen
Subjects:C Biological Sciences > C560 Biotechnology
Divisions:College of Science > School of Mathematics and Physics
Related URLs:
ID Code:19352
Deposited On:27 Oct 2015 14:25

Repository Staff Only: item control page