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Stabilization of substilisin E in organic solvents by site-directed mutagenesis

Martinez, Pascal and Van Dam, Mariana E. and Robinson, Amy C. and Chen, Keqin and Arnold, Frances H. (1992) Stabilization of substilisin E in organic solvents by site-directed mutagenesis. Biotechnology and Bioengineering, 39 (2). pp. 141-147. ISSN 0006-3592. https://resolver.caltech.edu/CaltechAUTHORS:20180822-140907258

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Abstract

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 → Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface‐charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 → Asn + Asn218 → Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1002/bit.260390204DOIArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1992 John Wiley & Sons. Manuscript received: 26 April 1991. Manuscript accepted: 14 June 1991.
Subject Keywords:protein engineering; enzymes in organic solvents; protein stabilization; subtilisin E
Issue or Number:2
Record Number:CaltechAUTHORS:20180822-140907258
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180822-140907258
Official Citation:Martinez, P. , Van Dam, M. E., Robinson, A. C., Chen, K. and Arnold, F. H. (1992), Stabilization of substilisin E in organic solvents by site‐directed mutagenesis. Biotechnol. Bioeng., 39: 141-147. doi:10.1002/bit.260390204
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89052
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:22 Aug 2018 21:54
Last Modified:03 Oct 2019 20:12

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