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Updated: Oct 22, 2025
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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
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Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics
C J Markin1, D A Mokhtari1, F Sunden1
1Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Science (New York, N.Y.)
|August 26, 2021
Summary
High-Throughput Microfluidic Enzyme Kinetics (HT-MEK) enables rapid characterization of enzyme variants. This platform maps enzyme function to specific residue regions, advancing molecular understanding for medicine and engineering.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Kinetics
Background:
- Enzyme efficiency is crucial for medicine and engineering.
- Systematic investigation of enzyme variants is needed.
- Existing methods limit high-throughput characterization.
Purpose of the Study:
- Introduce High-Throughput Microfluidic Enzyme Kinetics (HT-MEK) platform.
- Enable high-throughput expression, purification, and characterization of enzyme variants.
- Map enzyme function to specific residue regions.
Main Methods:
- Developed a microfluidic platform (HT-MEK).
- Characterized 1036 mutants of alkaline phosphatase PafA.
- Performed over 670,000 reactions to determine kinetic and physical constants.
Main Results:
- Uncovered extensive kinetic partitioning to a misfolded state.
- Isolated catalytic effects and identified functional residue regions.
- Mapped enzyme architecture from active site to surface, revealing structure-function relationships.
Conclusions:
- HT-MEK facilitates large-scale enzyme variant characterization.
- Identified specific residue regions critical for enzyme function.
- Provides a novel approach for understanding enzyme mechanisms and design.
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