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Directed evolution of a thermostable esterase

L Giver1, A Gershenson, P O Freskgard

  • 1Division of Chemistry and Chemical Engineering, Mail Code 210-41, California Institute of Technology, Pasadena, CA 91125, USA.

Proceedings of the National Academy of Sciences of the United States of America
|October 28, 1998
PubMed
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In vitro evolution stabilized an esterase enzyme for higher temperatures without losing low-temperature catalytic activity. This research challenges the idea that enzyme stability and activity are always inversely correlated.

Area of Science:

  • Enzyme engineering
  • Protein evolution
  • Biochemistry

Background:

  • Previous studies suggested a trade-off between high-temperature stability and low-temperature catalytic activity in enzymes.
  • This inverse correlation is attributed to differing flexibility requirements for optimal function at distinct temperatures.

Purpose of the Study:

  • To investigate the relationship between stability and activity in a mesophilic esterase using in vitro evolution.
  • To determine if enhanced thermostability necessitates a compromise in catalytic activity at lower temperatures.

Main Methods:

  • Employing six generations of random mutagenesis, recombination, and screening.
  • Utilizing Bacillus subtilis p-nitrobenzyl esterase as the model enzyme.
  • Analyzing the stability and activity of numerous random mutants.

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Main Results:

  • Significant stabilization of the esterase was achieved, with a >14°C increase in melting temperature (Tm).
  • Catalytic activity at lower temperatures was maintained despite enhanced thermostability.
  • Analysis indicated that stability and activity are not necessarily inversely correlated.

Conclusions:

  • Enhanced thermostability can be achieved without sacrificing low-temperature activity.
  • The evolutionary pathway and constraints are crucial; mutations for increased thermostability while retaining low-temperature activity are rare.
  • Without specific constraints, evolving one property often comes at the expense of the other.