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Applied and Environmental Microbiology, October 2005, p. 5728-5734, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5728-5734.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

Tyler W. Johannes,2 Ryan D. Woodyer,1 and Huimin Zhao1,2*

Departments of Chemistry,1 Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 618012

Received 17 January 2005/ Accepted 13 May 2005

NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to L-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.

* Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801. Phone: (217) 333-2631. Fax: (217) 333-5052. E-mail: zhao5{at}uiuc.edu.

Applied and Environmental Microbiology, October 2005, p. 5728-5734, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5728-5734.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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