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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23341 43433 2014 8 PDF Available
Title
Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate
Abstract

•We increased the activity and resistance of a subtilisin E variant in the presence of chaotropic agents.•Circular dichroism analysis revealed that that the secondary structure in GdmCl or SDS were not significantly perturbed.•Structural analysis suggests there is an inactivation step prior denaturation.•This study suggests that GdCl or SDS “stabilizing” a substitutions should be focused in the active site of the enzyme.

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; SDS
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Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 169, 10 January 2014, Pages 87–94
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us