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A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Published on: October 2, 2012
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A thermostable transketolase evolved for aliphatic aldehyde acceptors.
Dong Yi1, Thangavelu Saravanan, Titu Devamani
1Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany. fessner@tu-darmstadt.de.
Chemical Communications (Cambridge, England)
|November 22, 2014
Summary
Directed evolution enhanced transketolase activity for aliphatic aldehydes by up to 16-fold. The engineered enzyme also demonstrated high enantioselectivity (>95% ee) in asymmetric carboligation.
Area of Science:
- Biocatalysis and enzyme engineering
- Protein evolution and directed evolution
- Synthetic organic chemistry
Background:
- Transketolase enzymes are crucial for carbon-carbon bond formation.
- Improving transketolase activity and selectivity is essential for biocatalytic applications.
- Directed evolution offers a powerful approach for enzyme optimization.
Purpose of the Study:
- To enhance the activity and enantioselectivity of thermostable transketolase from Geobacillus stearothermophilus.
- To develop a pH-based colorimetric screening method for identifying improved transketolase variants.
- To investigate the potential of engineered transketolase in asymmetric carboligation reactions.
Main Methods:
- Directed evolution of Geobacillus stearothermophilus transketolase using smart libraries.
- Implementation of a pH-based colorimetric screening assay for high-throughput selection.
- Characterization of mutant enzymes for activity towards aliphatic aldehydes and enantioselectivity in carboligation.
Main Results:
- Several transketolase mutants with significantly enhanced activity for aliphatic aldehydes were identified (up to 16-fold increase).
- The engineered mutants exhibited high enantioselectivity (>95% ee) in the asymmetric carboligation step.
- The pH-based screening method proved effective for isolating superior enzyme variants.
Conclusions:
- Directed evolution is a successful strategy for improving transketolase performance.
- Engineered transketolase variants show great promise for efficient and selective biocatalytic synthesis.
- This work provides valuable tools for advancing asymmetric carboligation reactions.
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