Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase
Alejandro Gran-Scheuch1, Friso Aalbers2, Yannick Woudstra3
1Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands; Department of Chemical and Bioprocesses Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile.
Methods in Enzymology
|January 23, 2021
Summary
Optimizing enzyme fusions for industrial organic synthesis is key. This study demonstrates that linker length significantly impacts the performance of bifunctional enzymes, crucial for cofactor-dependent biocatalysis.
Area of Science:
- Biocatalysis and Enzyme Engineering
- Organic Synthesis
- Protein Engineering
Background:
- Enzymes offer high selectivity in organic synthesis.
- Cofactor-dependent enzymes require self-sufficient systems for industrial application.
- Enzyme fusion can create bifunctional systems with enhanced stability and product channeling.
Purpose of the Study:
- To investigate the impact of linker length on the performance of fused enzymes.
- To optimize bifunctional enzyme systems for industrial biocatalysis.
- To establish protocols for evaluating protein-protein fusions.
Main Methods:
- Cloning of various glycine-rich linker variants.
- Expression analysis of fused enzymes.
- Determination of enzyme thermostability, activity, and conversion levels.
Main Results:
- Fifteen linker variants of varying lengths were created and analyzed.
- Linker length was found to significantly affect enzyme expression, stability, and performance.
- Optimal linker design is critical for successful enzyme fusion.
Conclusions:
- Linker characteristics are crucial for the efficacy of fused enzymes.
- Tailoring linker length can enhance the industrial applicability of biocatalytic systems.
- The developed protocols are applicable to other protein-protein fusion studies.
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