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

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Woodyer, Ryan David

Contributors dc:contributor

• van der Donk, Wilfred A.
• Zhao, Huimin

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(MiAaPQ)AAI3199177

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84206