BADAN-conjugated β-lactamases as biosensors for β-lactam antibiotic detection.

β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for β-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP β-lactamase E166C and E166C/N170Q mutants.

Thermostable β-Lactamase Mutant with Its Active Site Conjugated with Fluorescein for Efficient β-Lactam Antibiotic Detection.

Monitoring the β-lactam antibiotic level has been an important task in food industry and clinical practice. Here, we report the development of a fluorescent PenP β-lactamase, PenP-E166Cf/N170Q, for efficient β-lactam antibiotic detection. It was constructed by covalently attaching fluorescein onto the active-site entrance of a thermostable E166Cf/N170Q mutant of a PenP β-lactamase.

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase.

Background: β-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood.

Results: Here we report the crystal structure of a Class A β-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf) and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime), to illustrate its biosensing mechanism.