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PEDS Advance Access published online on September 2, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl034
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© 2006 The Author(s).
Received April 12, 2006
Revised July 17, 2006
Accepted July 24, 2006

Article

Directed evolution by accumulating tailored mutations: Thermostabilization of lactate oxidase with less trade-off with catalytic activity

Norio Hamamatsu 1, Yukiko Nomiya 1, Takuyo Aita 2, Motowo Nakajima 1, Yuzuru Husimi 2, and Yasuhiko Shibanaka 1 *

1 Tsukuba Research Institute, Novartis Pharma K.K., Ohkubo 8, Tsukuba 300-2611, Japan
2 Department of Functional Materials Science, Saitama University, Saitama 338-8570, Japan

* To whom correspondence should be addressed.
Yasuhiko Shibanaka, E-mail: yasuhiko.shibanaka{at}novartis.com


  Abstract

We assumed that adverse effects posed by introducing multiple mutations could be decomposed into those of each of the component mutations and that the risk could be reduced by the accumulation of mutations that were finely tuned for directed improvement of a specific property. We propose here a directed evolution strategy for improving a specific property with less effect on other ones. This strategy is composed of fine-tuning of mutations and their accumulation by our original mutation-assembling method. In this study, we selected lactate oxidase (LOX) as a model enzyme, because its directed evolution had showed a trade-off between thermostability and catalytic activity. Mutation profiling at each of the sites found by error-prone PCR revealed a strong inverse relationship between the two properties. Thermostable mutations with less effect on catalytic activity were selected at each site and accumulated with ideal combinations by our method. The resultant multiple mutants exhibited 5- to 10-fold superior catalytic activity and comparable thermostability with those created by accumulating thermostable mutations, which were not tuned for catalytic activity. This result demonstrates that the accumulation of fine-tuned mutations is an advantageous approach to reduce the risk of adverse effects posed by accumulating multiple mutations.

Keywords: additivity principles; directed evolution; lactate oxidase; local fitness landscape; thermostability.
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