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pHluorin-based in vivo assay for hydrolase screening
Sascha Schuster1, Markus Enzelberger, Harald Trauthwein
1Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Analytical Chemistry
|April 30, 2005
Summary
A novel pHluorin-based assay enables in vivo hydrolase screening by detecting intracellular pH changes in Escherichia coli (E. coli). This method efficiently identifies enzyme activity using flow cytometry for rapid analysis.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Microbial Assays
Background:
- pHluorin, a pH-sensitive green fluorescent protein (GFP) mutant, responds to intracellular pH variations.
- Hydrolytic enzymes induce pH shifts, making them potential targets for biosensor development.
Purpose of the Study:
- To develop and validate a pHluorin-based in vivo assay for screening hydrolase activity.
- To demonstrate the assay's utility in Escherichia coli (E. coli) for detecting heterologous enzyme expression and function.
Main Methods:
- Utilized pHluorin as a biosensor for intracellular pH changes in E. coli.
- Expressed model hydrolases (esterase, hydantoinase, amidase) in E. coli.
- Evaluated assay performance using flow cytometry to detect population shifts.
Main Results:
- Successfully established a pHluorin-based in vivo assay for hydrolase screening in E. coli.
- Demonstrated detection of esterase, hydantoinase, and amidase activity through pH-induced fluorescence changes.
- Showcased the assay's compatibility with flow cytometric analysis for population-level readout.
Conclusions:
- The pHluorin-based in vivo assay is a robust tool for screening hydrolase activity in microbial systems.
- This method offers a sensitive and efficient approach for enzyme discovery and characterization.
- Flow cytometry provides a high-throughput readout for this pHluorin-based detection system.