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Directed evolution of a thermostable esterase

Giver, Lori and Gershenson, Anne and Freskgard, Per-Ola and Arnold, Frances H. (1998) Directed evolution of a thermostable esterase. Proceedings of the National Academy of Sciences of the United States of America, 95 (22). pp. 12809-12813. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:GIVpnas98

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Abstract

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14°C increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.


Item Type:Article
Additional Information:© 1998 by The National Academy of Sciences Edited by Norman R. Pace, University of California, Berkeley, CA, and approved September 5, 1998 (received for review April 30, 1998) We thank Dr. Litian Fu for helpful discussions and Candace Chang for excellent technical assistance. This research is supported by the Department of Energy and by the Army Research Office. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Record Number:CaltechAUTHORS:GIVpnas98
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:GIVpnas98
Alternative URL:http://www.pnas.org/cgi/content/abstract/95/22/12809
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:638
Collection:CaltechAUTHORS
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Deposited On:09 Sep 2005
Last Modified:26 Dec 2012 08:40

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