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Patterns of adaptation in a laboratory evolved thermophilic enzyme

Wintrode, Patrick L. and Miyazaki, Kentaro and Arnold, Frances H. (2001) Patterns of adaptation in a laboratory evolved thermophilic enzyme. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1549 (1). pp. 1-8. ISSN 0167-4838. https://resolver.caltech.edu/CaltechAUTHORS:20180822-085420199

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Abstract

The heat sensitive psychrophilic protease subtilisin S41 was previously subjected to three rounds of mutagenesis/recombination and screening, resulting in variant 3-2G7, whose half-life at 60°C is approx. 500 times that of wild-type. Here we report the results of five additional generations of laboratory evolution starting from 3-2G7. The half-life of 8th generation enzyme 8-4A9 at 60°C is 1200 times that of wild-type, and slightly more than twice that of 3-2G7. This half-life is >20-fold greater than those of homologous mesophilic subtilisins SSII and BPN′. Circular dichroism melting curves indicate that subtilisin 8-4A9 unfolds at temperatures approx. 25°C higher than wild-type. It is also substantially more resistant to proteolysis at 30°C. Nearly half of the 13 amino acid substitutions accumulated in 8-4A9 involve the mutation of serine residues. This mirrors a pattern observed in natural proteins, where serines are statistically less prevalent in thermophilic enzymes compared to mesophilic ones.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/S0167-4838(01)00226-6DOIArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2001 Elsevier Science B.V. Received 23 February 2001, Revised 17 May 2001, Accepted 17 May 2001, Available online 18 September 2001. This research was supported in part by Procter and Gamble, the US Army Research Office and by Maxygen, Inc.
Funders:
Funding AgencyGrant Number
Procter and GambleUNSPECIFIED
Army Research Office (ARO)UNSPECIFIED
Maxygen, Inc.UNSPECIFIED
Subject Keywords:Thermophile; Psychrophile; Directed evolution; Subtilisin; Protein engineering
Issue or Number:1
Record Number:CaltechAUTHORS:20180822-085420199
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180822-085420199
Official Citation:Patrick L Wintrode, Kentaro Miyazaki, Frances H Arnold, Patterns of adaptation in a laboratory evolved thermophilic enzyme, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Volume 1549, Issue 1, 2001, Pages 1-8, ISSN 0167-4838, https://doi.org/10.1016/S0167-4838(01)00226-6. (http://www.sciencedirect.com/science/article/pii/S0167483801002266)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89011
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:22 Aug 2018 16:22
Last Modified:03 Oct 2019 20:12

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