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Optimisation and evaluation of a generic microplate-based HPLC screen for transketolase activity
Oliver J Miller1, Edward G Hibbert, Christine U Ingram
1Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.
Biotechnology Letters
|June 29, 2007
Summary
A new microplate assay speeds up transketolase research by quickly measuring enzyme activity. This method helps optimize biocatalysis and find better enzyme variants for creating chiral compounds.
Area of Science:
- Biochemistry
- Enzymology
- Biocatalysis
Background:
- Transketolase is a key enzyme for enantioselective carbon-carbon bond formation.
- Optimizing biocatalytic processes and screening enzyme variants requires efficient assays.
- Existing methods may not be suitable for high-throughput screening or process optimization.
Purpose of the Study:
- To develop a rapid, microplate-based HPLC assay for transketolase.
- To enable efficient determination of substrate and product concentrations.
- To facilitate optimization of biocatalytic conditions and screening of directed evolution libraries.
Main Methods:
- Development of a microplate-based High-Performance Liquid Chromatography (HPLC) assay.
- Optimization of sample preparation including microplate-based fermentation, cell lysis, and cofactor/substrate addition.
- Determination of dissociation constants for transketolase cofactors.
Main Results:
- The assay accurately determines substrate and product concentrations for transketolase.
- Dissociation constants for two essential cofactors were determined with 5-11% error.
- Optimized sample preparation led to increased transketolase activity.
- The assay enabled identification of enzyme variants with 3-fold improved activity.
Conclusions:
- The developed microplate-HPLC assay is a valuable tool for transketolase research.
- It significantly enhances the efficiency of biocatalytic process optimization.
- The assay facilitates rapid screening of directed evolution libraries for improved enzyme variants.
