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Updated: May 12, 2026
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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
A high-sensitivity strategy to screen NAD(P)H-dependent reductase activity by coupled enzyme cascade
Trisha Ghosh1, Jacob Sicheri1, David H Kwan2
1Department of Biology, Concordia University, Montreal, Quebec, Canada.
The Journal of Biological Chemistry
|May 10, 2026
Summary
Researchers developed a sensitive fluorescence assay to detect enzyme activity. This new method significantly improves upon traditional absorbance assays for screening and engineering biocatalysts, particularly oxidoreductases dependent on nicotinamide adenine dinucleotide phosphate (NAD(P)H).
Area of Science:
- Biocatalysis
- Green Chemistry
- Enzyme Engineering
Background:
- Oxidoreductase enzymes are crucial for green chemistry, enabling diverse chemical transformations via redox reactions.
- Detecting oxidoreductase activity often relies on monitoring nicotinamide adenine dinucleotide phosphate (NAD(P)H) absorbance changes at 340 nm.
- The limited sensitivity of absorbance assays hinders the screening and engineering of low-activity or novel biocatalysts.
Purpose of the Study:
- To develop a highly sensitive assay for detecting NAD(P)H-dependent oxidoreductase activity.
- To overcome the limitations of conventional absorbance-based detection methods.
- To facilitate the screening and directed evolution of biocatalysts.
Main Methods:
- Developed a fluorescence-based enzyme cascade-coupled system.
- The system recycles NAD(P)H from NAD(P)+.
- Cleavage of a fluorogenic probe by the coupled cascade releases a fluorescent signal.
Main Results:
- Achieved orders of magnitude greater sensitivity compared to absorbance-based assays.
- Enabled detection of very low levels of oxidoreductase activity.
- The assay is suitable for high-throughput screening.
Conclusions:
- The fluorescence-based assay provides a sensitive tool for biocatalyst discovery and engineering.
- This method enhances the ability to identify and improve enzymes for green chemistry applications.
- Facilitates directed evolution efforts by enabling detection of subtle enzymatic activities.
