Related Experiment Video
Updated: Sep 15, 2025
10:28
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
9.8K
A novel P450 enzyme assay utilizing an NADP+-based biosensor
Sifan Shangguan1,2, Taichang Wang1,2, Di Zhao1,2
1State Key Laboratory of Green Papermaking and Resource Recycling, Qilu University of Technology, Jinan, 250353, Shandong, Republic of China.
Biotechnology Letters
|July 16, 2025
Summary
A novel biosensor enables real-time monitoring of cytochrome P450 (P450) enzyme activity by tracking intracellular NADP+ levels. This tool offers a robust solution for high-throughput screening of P450s.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Traditional high-throughput screening methods for cytochrome P450 enzymes (P450s) have limitations in throughput, real-time monitoring, and versatility.
- Existing assays like colorimetric, mass spectrometric, and fluorescence-based methods do not fully meet the demands of modern biochemical research.
Purpose of the Study:
- To develop a novel biosensor for real-time monitoring of intracellular NADP+ levels to enable P450 activity detection.
- To overcome the limitations of current P450 screening techniques by providing a more versatile and precise method.
Main Methods:
- Developed a biosensor using glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation to monitor intracellular NADP+ dynamics.
- Applied the sensor to track P450 activity by measuring the conversion of NADPH to NADP+.
- Reduced background fluorescence by knocking down NADPH-dependent aldehyde reductase (YqhD) to enhance detection precision.
Main Results:
- The biosensor demonstrated a linear NADP+ detection range from 1 μM to 10 mM, suitable for P450 assays.
- Validated sensor performance by comparing P450 activities in engineered strains with traditional gas chromatography.
- Successfully monitored intracellular NADP+ dynamics to infer P450 enzyme activity.
Conclusions:
- The developed biosensor is a robust tool for real-time screening of P450 enzyme activity.
- This novel approach holds significant potential for advancing P450 research and drug discovery.
