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Applied and Environmental Microbiology, October 2005, p. 6390-6393, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6390-6393.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

Barbara Petschacher and Bernd Nidetzky*

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria

Received 9 March 2005/ Accepted 28 April 2005

Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2'-phosphate binding pocket were tested for NADPH versus NADH selectivity (Rsel) in the presence of physiological reactant concentrations. The experimental Rsel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274->Arg and Arg-280->His substitutions, which individually improved wild-type Rsel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

* Corresponding author. Mailing address: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. Phone: 43-316-873-8400. Fax: 43-316-873-8434. E-mail: bernd.nidetzky{at}tugraz.at.

Applied and Environmental Microbiology, October 2005, p. 6390-6393, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6390-6393.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Watanabe, S., Abu Saleh, A., Pack, S. P., Annaluru, N., Kodaki, T., Makino, K. (2007). Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis. Microbiology 153: 3044-3054 [Abstract] [Full Text]  


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