Abstract
Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by Large-scale production of the final mutant enzyme by fermentation resulted in ∼6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.
Keywords: Directed evolution, protein engineering, biocatalysis, NAD(P)H regeneration, cofactor regeneration, soluble expression
Combinatorial Chemistry & High Throughput Screening
Title: Optimizing a Biocatalyst for Improved NAD(P)H Regeneration: Directed Evolution of Phosphite Dehydrogenase
Volume: 9 Issue: 4
Author(s): Ryan Woodyer, Wilfred A. van der Donk and Huimin Zhao
Affiliation:
Keywords: Directed evolution, protein engineering, biocatalysis, NAD(P)H regeneration, cofactor regeneration, soluble expression
Abstract: Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by Large-scale production of the final mutant enzyme by fermentation resulted in ∼6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.
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Cite this article as:
Woodyer Ryan, van der Donk A. Wilfred and Zhao Huimin, Optimizing a Biocatalyst for Improved NAD(P)H Regeneration: Directed Evolution of Phosphite Dehydrogenase, Combinatorial Chemistry & High Throughput Screening 2006; 9 (4) . https://dx.doi.org/10.2174/138620706776843246
DOI https://dx.doi.org/10.2174/138620706776843246 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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