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

Abstract

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. They gave fluorescence turn-on signals in response to β-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four β-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 μM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated β-lactamases as biosensing devices for β-lactam antibiotics.

Fig 1. Fluorescence scanning of BADAN-conjugated β-lactamases, (A) E166Cb and (B) E166Cb/N170Q.

Spectra were obtained by scanning 0.1 μM labeled enzyme in 50 mM potassium phosphate buffer (pH 7.0) with an excitation at 380 nm. Blue line: enzyme without penicillin G; Red line: enzyme with the addition of 100 μM penicillin G.

Fig 2. Models of E166Cb with and without penicillin G.

(A) In the apo-E166Cb model, BADAN (green) was slightly buried in the active site pocket and stayed close to Tyr-105 (blue). Its naphthalene core was stacking on the aromatic ring of the tyrosine. (B) In the presence of penicillin G (red), BADAN moved slightly towards the aqueous environment and it was spatially separated from Tyr-105.

Fig 3. Fluorescence detection of β-lactam antibiotics by BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q.

Fluorescence traces were acquired after adding 0.1 μM labeled enzyme with (A) penicillin G, (B) penicillin V, (C) cefotaxime and (D) moxalactam in 50 mM potassium phosphate buffer (pH 7.0). Antibiotic concentrations: 0 (orange), 10 nM (red), 50 nM (cyan), 0.1 μM (green), 0.5 μM (brown), 1μM (purple) and 10 μM (blue). Fluorescence measurements were conducted several times with similar results. Representative traces were illustrated.