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Improving Activity and Stability of Candida boidinii Formate Dehydrogenase Through Rational Active Site Engineering
Marisa Bickmann1,2, Andrea Rodil1, Jan Deska1
1Department of Chemistry, University of Helsinki, Helsinki, Finland.
Chembiochem : a European Journal of Chemical Biology
|August 14, 2026
Summary
Engineered formate dehydrogenases (FDHs) show enhanced activity and stability. This research improves FDH biocatalysis using specific amino acid substitutions for better cofactor regeneration in enzymatic reactions.
Area of Science:
- Biocatalysis and Enzyme Engineering
- Protein Engineering
- Industrial Biotechnology
Background:
- Formate dehydrogenases (FDHs) are crucial for regenerating NADH cofactors, essential for many enzymatic reductions and oxygenations.
- Native FDHs often exhibit low specific activity, limiting their industrial application despite the advantages of using formate as a reductant.
- The active site vicinity of FDHs presents opportunities for protein engineering to enhance enzyme performance.
Purpose of the Study:
- To engineer Candida boidinii FDH variants with improved catalytic activity, stability, and operational profiles.
- To investigate the impact of incorporating nonconsensus amino acids from Saccharomyces cerevisiae FDH into the Candida boidinii FDH active site.
- To identify specific mutations that enhance FDH performance for cofactor regeneration applications.
Main Methods:
- Rational design approach utilizing knowledge of FDH active site architecture.
- Site-directed mutagenesis to introduce specific amino acid substitutions.
- Biochemical characterization of engineered FDH variants, including specific activity, pH/temperature profiles, and stability assays.
Main Results:
- A double variant (C23S/F285D) demonstrated significantly higher specific activity compared to the wild-type enzyme.
- The engineered variant exhibited an improved pH and temperature profile, making it more suitable for industrial conditions.
- Enhanced stability was observed in the double variant, indicating greater robustness for biocatalytic processes.
Conclusions:
- The C23S/F285D double variant of Candida boidinii FDH represents a significant improvement over the native enzyme.
- This engineered FDH offers a more efficient and stable solution for NADH cofactor regeneration in biocatalysis.
- The findings highlight the potential of targeted protein engineering in optimizing FDHs for biotechnological applications.
