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PEDS Advance Access originally published online on September 2, 2006
Protein Engineering Design and Selection 2006 19(11):483-489; doi:10.1093/protein/gzl034
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

Norio Hamamatsu1, Yukiko Nomiya1, Takuyo Aita2, Motowo Nakajima1, Yuzuru Husimi2 and Yasuhiko Shibanaka1,3

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

3To whom correspondence should be addressed.E-mail: yasuhiko.shibanaka{at}novartis.com

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

Received April 12, 2006; revised July 17, 2006; accepted July 24, 2006.


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