Tailoring Oxidoreductase Function Through Development of Mutant Enzyme Libraries
Major
Biology
Submission Type
Poster
Area of Study or Work
Biochemistry
Faculty Advisor
Noah Haskin
Location
CNS Atrium
Start Date
4-12-2025 8:30 AM
End Date
4-12-2025 9:30 AM
Abstract
Aldehydes and carboxylic acids are commonly synthesized in cellular reactions catalyzed by oxidoreductases. These enzymes utilize redox-active cofactors for both energy capture and biosynthesis. Dependence on expensive cofactors and narrow substrate tolerance limit the applications of these enzymes outside of their native pathways. Enzyme engineering through guided mutagenesis is a promising technique for generating novel biological catalysts and selecting those with desirable functions. Here, we demonstrate the cloning, expression, and purification of two E. coli oxidoreductases. Successful cloning and overexpression are verified by plasmid sequencing, SDS-PAGE and activity assays. Mutant libraries will be developed from these wild type expression platforms, with the goal of altering cofactor dependence in NADP-dependent aldB and expanding the substrate tolerance of NAD-dependent fucO. Identifying mutations circumventing these limitations may offer broadly applicable strategies to engineering useful oxidoreductases.
Tailoring Oxidoreductase Function Through Development of Mutant Enzyme Libraries
CNS Atrium
Aldehydes and carboxylic acids are commonly synthesized in cellular reactions catalyzed by oxidoreductases. These enzymes utilize redox-active cofactors for both energy capture and biosynthesis. Dependence on expensive cofactors and narrow substrate tolerance limit the applications of these enzymes outside of their native pathways. Enzyme engineering through guided mutagenesis is a promising technique for generating novel biological catalysts and selecting those with desirable functions. Here, we demonstrate the cloning, expression, and purification of two E. coli oxidoreductases. Successful cloning and overexpression are verified by plasmid sequencing, SDS-PAGE and activity assays. Mutant libraries will be developed from these wild type expression platforms, with the goal of altering cofactor dependence in NADP-dependent aldB and expanding the substrate tolerance of NAD-dependent fucO. Identifying mutations circumventing these limitations may offer broadly applicable strategies to engineering useful oxidoreductases.