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Converting a {beta}-glycosidase into a {beta}-transglycosidase by directed evolution
Hui-Yong Feng1, Jullien Drone, Lionel Hoffmann
1Biotechnologie, Biocatalyse, Biorégulation (UMR CNRS 6204), Université de Nantes Faculté des Sciences et des Techniques, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3, France.
The Journal of Biological Chemistry
|August 9, 2005
Summary
Directed evolution enhanced Thermus thermophilus beta-glycosidase for oligosaccharide synthesis via transglycosylation. Engineered enzymes significantly improved transferase activity, reducing hydrolysis and increasing yields for valuable oligosaccharides.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Beta-glycosidases are crucial enzymes in carbohydrate metabolism.
- Thermus thermophilus beta-glycosidase exhibits limited transglycosylation activity.
- Oligosaccharide synthesis requires efficient glycosyl transfer mechanisms.
Purpose of the Study:
- To engineer Thermus thermophilus beta-glycosidase for enhanced oligosaccharide synthesis.
- To improve the transglycosylation capabilities of the enzyme.
- To develop novel biocatalysts for oligosaccharide production.
Main Methods:
- Directed evolution involving random mutagenesis and in vitro recombination.
- Development of a screening procedure for high transferase activity.
- Molecular modeling to elucidate mutation effects.
Main Results:
- Mutant enzymes showed significantly reduced hydrolysis of substrates and products.
- Self-condensation synthesis yields approached quantitative levels.
- Transglycosylation yields on maltose and cellobiose reached 60% and 75%, respectively.
- Identified key mutations (F401S, N282T) influencing catalytic activity.
Conclusions:
- Directed evolution successfully created highly active transglycosidases.
- Mutations enhance glycosyl transfer by optimizing substrate binding and acceptor interaction.
- Engineered enzymes offer a promising alternative for efficient oligosaccharide synthesis.