High-throughput selection system for assessing the activity of epoxide hydrolases

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Synthetic Organic Chemistry

Background:

• Directed evolution requires high-throughput assays to screen enantioselective enzymes for synthetic applications.
• Existing enantiopurity (ee) assays are time-consuming, necessitating pre-selection methods for enzyme activity.
• Efficient pre-selection systems are crucial for streamlining the identification of active enzyme mutants.

Purpose of the Study:

• To develop an efficient and simple pre-selection test for assessing epoxide hydrolase activity.
• To correlate bacterial growth on agar plates with epoxide hydrolase catalytic function.

Main Methods:

• Development of a pre-selection test using bacterial (E. coli) growth on agar plates.
• Utilizing epoxide substrates that are hydrolyzed by active epoxide hydrolases.
• Visual inspection of bacterial colonies on agar plates to identify active enzyme variants.

Main Results:

• Bacterial growth on agar plates directly correlates with the presence and activity of epoxide hydrolases.
• The test allows for the identification of active epoxide hydrolase mutants through simple visual inspection.
• This method effectively screens large libraries of mutant epoxide hydrolases for catalytic activity.

Conclusions:

• An efficient pre-selection test for epoxide hydrolase activity has been successfully developed.
• This assay simplifies the screening process by relying on visual assessment of bacterial growth.
• The developed method is valuable for directed evolution of enantioselective epoxide hydrolases.