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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Directed evolution of transketolase activity on non-phosphorylated substrates
Edward G Hibbert1, Tarik Senussi, Sean J Costelloe
1Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK.
Journal of Biotechnology
|September 11, 2007
Summary
Directed evolution enhanced E. coli transketolase activity on non-phosphorylated substrates. Mutants showed up to 5-fold improvement by targeting conserved or phylogenetically varied active-site residues.
Area of Science:
- Biocatalysis
- Protein Engineering
- Enzyme Activity
Background:
- E. coli transketolase naturally acts on phosphorylated substrates.
- Improving activity on non-phosphorylated substrates is crucial for novel biocatalytic applications.
- Directed evolution offers a powerful approach to enzyme engineering.
Purpose of the Study:
- To enhance the activity of E. coli transketolase towards non-phosphorylated substrates.
- To identify key active-site residues for improving substrate specificity.
- To explore the utility of saturation mutagenesis in enzyme optimization.
Main Methods:
- Active-site targeted directed evolution using saturation mutagenesis.
- Selection of residues based on structural proximity or phylogenetic variation.
- Screening of mutant libraries for activity on hydroxypyruvate and glycolaldehyde.
Main Results:
- Mutants from phylogenetically defined libraries showed up to 3-fold increased specific activity.
- Mutants targeting conserved residues interacting with phosphate groups yielded up to 5-fold improvement.
- Improved mutants often had substitutions differing from naturally occurring residues.
Conclusions:
- Phylogenetically variant residues can modulate activity on homologous substrates.
- Conserved residues, when no longer interacting with natural substrates, are valuable targets for mutagenesis.
- This study provides insights into engineering enzyme specificity for non-natural substrates.
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