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Directed evolution converts subtilisin E into a functional equivalent of thermitase

Zhao, Huimin and Arnold, Frances H. (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Engineering, 12 (1). pp. 47-53. ISSN 0269-2139. doi:10.1093/protein/12.1.47.

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We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83°C (3.5 min) and temperature optimum for activity (Topt, 76°C) are identical with those of thermitase. The Topt of the evolved enzyme is 17°C higher and its half-life at 65°C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90°C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity.

Item Type:Article
Related URLs:
URLURL TypeDescription
Zhao, Huimin0000-0002-9069-6739
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1999 Oxford University Press Received June 26, 1998; accepted September 24, 1998. This work was supported by the US Department of Energy's program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables.
Funding AgencyGrant Number
Department of Energy (DOE)UNSPECIFIED
Subject Keywords:in vitro evolution, StEP recombination, subtilisin E, thermitase, thermostability
Issue or Number:1
Record Number:CaltechAUTHORS:ZHApeds99
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2205
Deposited By: Archive Administrator
Deposited On:15 Mar 2006
Last Modified:08 Nov 2021 19:46

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