Understanding, Optimization, and Application of Phosphite Dehydrogenase: Advancing NAD(P)H Regeneration
type: CreativeWork
description: 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.
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.
author: Woodyer, Ryan David
language: eng
keywords: Chemistry, Biochemistry
date published: 2015-09-25T22:13:29Z
date modified: 2026-07-22T22:26:22Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/84206