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PEDS Advance Access published online on January 11, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzl048
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vivo selection for the enhancement of Thermotoga maritima exopolygalacturonase activity at neutral pH and low temperature

Jullien Drone, Michel Dion, Charles Tellier, Claude Rabiller1 and Hugues Bedouelle

Université de Nantes, Nantes Atlantique Universités, UMR CNRS 6204, Biotechnologie, Biocatalyse, Biorégulation, Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, F-44322 Nantes, France

1 To whom correspondence should be addressed. E-mail: claude.rabiller{at}univ-nantes.fr

The aim of this study was to develop an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enabled the entry of oligogalacturonates into the cytoplasm of E. coli thus providing a modified strain usable for this purpose. This tool was used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In only one round of error-prone PCR and selection, mutants of TmGalU with a 4-fold increased turnover at pH 7.0 and 2-fold more active at 37°C than wild-type enzyme were isolated. These results show the versatility of this strain for the evolution of oligogalacturonate-active enzymes.

Keywords: directed evolution/exopolygalacturonase/metabolic selection/oligogalacturonates/Thermotoga maritima

Received August 1, 2006; revised October 30, 2006; accepted October 2, 2006.


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