Rational Design of a Novel Fluorescent Biosensor for β-Lactam Antibiotics from a Class A β-Lactamase

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Abstract
A rational design strategy was used to construct a sensitive “turn-on” biosensor for β-lactam antibiotics and β-lactamase inhibitors from a class A β-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Ω-loop of the E166C mutant close to the active site of the β-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Ω-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.