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A generic, whole-cell-based screening method for Baeyer-Villiger monooxygenases
Hanna M Dudek1, Petra Popken, Edwin van Bloois
1Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands.
Journal of Biomolecular Screening
|March 29, 2013
Summary
A new whole-cell screening method enables rapid engineering of Baeyer-Villiger monooxygenases (BVMOs). This method efficiently identifies beneficial enzyme mutants for biocatalysis by screening Escherichia coli cell libraries.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Molecular Biology
Background:
- Baeyer-Villiger monooxygenases (BVMOs) are crucial biocatalysts with growing industrial demand for enzyme redesign.
- Screening large libraries of enzyme mutants is essential for efficient enzyme engineering.
Purpose of the Study:
- To develop a generic, high-throughput screening method for Baeyer-Villiger monooxygenases (BVMOs).
- To facilitate the rapid engineering of BVMOs for diverse biocatalytic applications.
Main Methods:
- Established an Escherichia coli expression system for periplasmic production of phenylacetone monooxygenase (PAMO).
- Implemented extracellular coenzyme (NADPH) regeneration using phosphite dehydrogenase and a chromogenic phosphate assay.
- Incorporated a detection method to exclude false-positive NADPH oxidase activity.
Main Results:
- Successfully developed and validated a 96-well plate-based whole-cell screening method for BVMOs.
- Identified PAMO mutants with altered catalytic properties through screening of site-saturation libraries.
- Demonstrated the method's utility for screening BVMO libraries with various substrates.
Conclusions:
- The developed screening method is a powerful tool for engineering BVMOs.
- This approach accelerates the discovery of novel biocatalysts for industrial applications.
- Enables efficient screening of enzyme libraries for desired catalytic activities.
