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Microbial sensor for new-generation cephalosporins based in a protein-engineered beta-lactamase
J L García1, C J Nuñez, E G González
1Department of Bioengineering, National University of Mexico, Cuernavaca, Morelos, México.
Applied Biochemistry and Biotechnology
|October 21, 1998
Summary
Researchers engineered a beta-lactamase enzyme to create a whole-cell biosensor for detecting new-generation cephalosporins. This novel biosensor offers rapid and stable detection of important antibiotics.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Biosensor Development
Background:
- Beta-lactamase enzymes are crucial for antibiotic resistance.
- Developing sensitive and rapid detection methods for cephalosporins is essential for clinical and environmental monitoring.
- Protein engineering offers a powerful approach to enhance enzyme specificity and activity.
Purpose of the Study:
- To engineer a beta-lactamase with broadened specificity for cephalosporin degradation.
- To develop a whole-cell biosensor for detecting new-generation cephalosporins using engineered bacteria.
- To characterize the performance of the developed biosensor in terms of sensitivity, response time, and stability.
Main Methods:
- Site-directed mutagenesis was used to engineer a beta-lactamase (E104M/G238S) in Escherichia coli.
- Culture conditions were optimized to increase beta-lactamase specific activity.
- Whole bacterial cells were immobilized on agar membranes and coupled to a flat pH electrode to create a biosensor.
- The biosensor's ability to detect cephalosporins was evaluated by measuring pH changes.
Main Results:
- The engineered beta-lactamase demonstrated degradation of first-, second-, and third-generation cephalosporins.
- Beta-lactamase specific activity was increased up to twofold through culture condition optimization.
- The whole-cell biosensor successfully detected second- and third-generation cephalosporins, including cefamandole, cefotaxime, and cefoperazone, within specific concentration ranges (0.3-4 mM).
- Biosensor response times ranged from 3.5 to 11 minutes.
- The biosensor exhibited stability for at least 7 days, with capacity for up to 100 tests.
Conclusions:
- Protein engineering can yield beta-lactamases with broadened cephalosporin specificity.
- An immobilized whole-cell biosensor based on pH changes provides a viable method for detecting new-generation cephalosporins.
- The developed biosensor offers a stable, rapid, and sensitive platform for antibiotic detection.