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Colorimetric High-Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase
Feng Liu1, Qiang Geng1, Chen Zhao1
1State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China.
Chembiochem : a European Journal of Chemical Biology
|May 31, 2022
Summary
A new colorimetric method aids directed evolution of Baeyer-Villiger monooxygenases (BVMOs). This resulted in an engineered enzyme variant with enhanced activity and stability for synthesizing chiral sulfoxides, crucial for pharmaceuticals.
Area of Science:
- Biocatalysis
- Enzyme Engineering
Background:
- Baeyer-Villiger monooxygenases (BVMOs) are vital biocatalysts for chiral sulfoxide synthesis, including pharmaceuticals for gastrointestinal disorders.
- Native BVMOs often exhibit insufficient activity and thermostability for industrial applications.
- Protein engineering strategies are hindered by a lack of high-throughput screening methods for BVMOs.
Purpose of the Study:
- To develop a novel colorimetric detection method for high-throughput screening of prazole sulfide monooxygenases.
- To engineer improved BVMO variants with enhanced catalytic activity and thermostability.
- To demonstrate the utility of the developed method in directed evolution for pharmaceutical applications.
Main Methods:
- Development of a colorimetric assay to differentiate sulfoxides from sulfides and sulfones.
- Application of directed evolution using the developed colorimetric method for prazole sulfide monooxygenases.
- Characterization of the engineered BVMO variant's activity and thermostability.
Main Results:
- A novel colorimetric detection method was successfully established for prazole sulfide monooxygenases.
- Directed evolution identified a CbBVMO variant with significantly improved performance.
- The engineered variant exhibited a 71.3% increase in conversion and a 6°C higher melting point compared to the wild-type enzyme.
- The variant efficiently catalyzes the asymmetric oxidation of lansoprazole sulfide.
Conclusions:
- The developed colorimetric method is effective for high-throughput screening of BVMO variants.
- Engineered BVMOs show promise for the practical synthesis of chiral sulfoxides, including pharmaceutical compounds.
- This approach facilitates the development of more robust and efficient biocatalysts for industrial applications.
