University of Illinois Urbana-Champaign

Understanding, Optimization, and Application of Phosphite Dehydrogenase: Advancing NAD(P)H Regeneration

Woodyer, Ryan David

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Description

Title
Understanding, Optimization, and Application of Phosphite Dehydrogenase: Advancing NAD(P)H Regeneration
Author(s)
Woodyer, Ryan David
Issue Date
2005
Doctoral Committee Chair(s)
  • van der Donk, Wilfred A.
  • Zhao, Huimin
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
NAD(P)H regeneration is an industrially important process that supports biocatalytic reactions that utilize these cofactors. PTDH shows promise for NAD(P)H regeneration and was therefore optimized for industrial application. The cofactor specificity of PTDH was relaxed by rational design using the homology model. Cofactor specificity was changed from 100-fold in favor of NAD to 3-fold in favor of NADP, with improvements in the catalytic efficiency (1000-fold for NADP). Utilizing directed evolution, the activity, expression, and thermostability of PTDH were also improved. A 3-fold increase in heterologous expression, 2-fold improvement in kcat, and 7000-fold increase in thermostability were achieved. The best mutants from each type of improvement were characterized in detail. Combined mutants with relaxed cofactor specificity, improved activity, stability, and expression level were tested in small-scale regeneration reactions for the production of several industrially interesting compounds such as xylitol and L- tert-leucine. The results obtained show that the combined mutant PTDH enzymes allow high productivity and conversion, representing one of the best NAD(P)H regeneration methods currently available.
Graduation Semester
2005
Type of Resource
text
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http://hdl.handle.net/2142/84206

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