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Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution

Wintrode, Patrick L. and Miyazaki, Kentaro and Arnold, Frances H. (2000) Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution. Journal of Biological Chemistry, 275 (41). pp. 31635-31640. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:WINjbc00

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Abstract

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (kcat) at 10 °C 6.6 times and a catalytic efficiency (kcat/KM) 9.6 times that of wild type. Its half-life at 70 °C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in kcat is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77.4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.


Item Type:Article
Additional Information:Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, May 24, 2000, and in revised form, July 14, 2000. Originally published In Press as doi:10.1074/jbc.M004503200 on July 21, 2000 We are grateful to Professor Alan Porter (National University of Singapore) for providing the gene for wild-type SSII. We also thank Francisco Valles for technical assistance. This work was supported by Procter & Gamble.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Record Number:CaltechAUTHORS:WINjbc00
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:WINjbc00
Alternative URL:http://dx.doi.org/10.1074/jbc.M004503200
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6206
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:28 Nov 2006
Last Modified:26 Dec 2012 09:18

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