Droplet microfluidic screening to engineer angiotensin-converting enzyme 2 (ACE2) catalytic activity

Area of Science:

• Biochemistry
• Enzyme Engineering
• Microfluidics

Background:

• Angiotensin-Converting Enzyme 2 (ACE2) is vital for peptide hormone signaling, regulating blood pressure and inflammation.
• ACE2's therapeutic potential for viral infections, ARDS, and diabetes is hindered by inefficient enzyme variants.
• Traditional screening methods using surrogate substrates lack biological relevance for enzyme optimization.

Purpose of the Study:

• To develop an ultra-high-throughput droplet microfluidic platform for screening peptidase activity on native substrates.
• To engineer enhanced ACE2 variants with improved therapeutic potential.

Main Methods:

• Developed an ultra-high-throughput droplet microfluidic platform for screening peptidases.
• Assay detects substrate cleavage by measuring free amino acid release, reflecting native activity.
• Screened a large library of ACE2 variants to identify key residues for activity enhancement.

Main Results:

• Identified position 187 as a critical site for enhancing ACE2 activity.
• Discovered the K187T ACE2 variant with a fourfold increase in catalytic efficiency (kcat/KM) compared to wild-type.
• Demonstrated the platform's ability to identify highly active enzyme variants.

Conclusions:

• Droplet microfluidics offers a powerful tool for therapeutic peptidase engineering.
• The developed platform provides a robust and accessible method for optimizing enzyme properties.
• This approach can accelerate the development of enzymes for clinical applications.