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. 2019 Nov 27;4(24):20493-20502.
doi: 10.1021/acsomega.9b02211. eCollection 2019 Dec 10.

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

Ho-Wah Au  1 , Man-Wah Tsang  1 , Pui-Kin So  1 , Kwok-Yin Wong  1 , Yun-Chung Leung  1
Affiliations

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

Ho-Wah Au et al. ACS Omega. .

Abstract

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 Bacillus licheniformis PenP β-lactamase. It gave a fluorescence turn-on signal toward various β-lactam antibiotics, where the fluorescence enhancement was attributed to the acyl-enzyme complex formed between PenP-E166Cf/N170Q and the β-lactam antibiotic. It demonstrated enhanced signal stability over its parental PenP-E166Cf because of the suppressed hydrolytic activity by the N170Q mutation. Compared with our previously constructed PenPC-E166Cf biosensor, PenP-E166Cf/N170Q was more thermostable and advanced in detecting β-lactams in terms of response time, signal stability, and detection limit. Positive fluorescence signals generated by E166Cf/N170Q in response to the penicillin-containing milk and mouse serum illustrated the feasibility of the biosensor for antibiotic detection in real samples. Taken together, our findings suggest the potential application of PenP-E166Cf/N170Q in biosensing β-lactam antibiotics.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Hydrolytic Reaction of β-Lactam Antibiotics by a Class A β-Lactamase
Binding of β-lactamase (E) with the antibiotic substrate (S) leads to the formation of the Michaelis complex (ES). ES then undergoes an irreversible step of acylation to form an acyl–enzyme complex (ES*). Deacylation of ES* eventually takes place, leading to a release of the hydrolyzed product (P) from the active site of the enzyme.
Figure 1
Figure 1
Formation of the acyl–enzyme complex between PenP-E166Cf/N170Q and penicillin G was monitored by (a) stopped-flow fluorescence measurement and (b) MS. (a) Initial fluorescence change of the PenP-E166Cf/N170Q after the addition of penicillin G was recorded for 100 s. (b) Reaction between PenP-E166Cf/N170Q and penicillin G was quenched at different time intervals and subjected to mass spectrometric analysis. Peak A refers to the free enzyme of PenP-E166Cf/N170Q (E), whereas peak B refers to the covalent acyl–enzyme complex formed between PenP-E166Cf/N170Q and penicillin G (ES*).
Figure 2
Figure 2
Fluorescence spectra of 0.1 μM PenP-E166Cf and PenP-E166Cf/N170Q biosensors in 50 mM potassium phosphate (pH 7.0) with various concentrations of (a) ampicillin, (b) penicillin G, and (c) penicillin V: 0 (orange); 0.01 μM (red); 0.1 μM (green); 0.5 μM (brown); 1 μM (purple); and 10 μM (blue).
Figure 3
Figure 3
Fluorescence spectra of 0.1 μM PenP-E166Cf and PenP-E166Cf/N170Q biosensors in 50 mM potassium phosphate (pH 7.0) with various concentrations of (a) cefotaxime, (b) cefoxitin, and (c) moxalactam: 0 (orange); 0.01 μM (red); 0.05 μM (cyan); 0.1 μM (green); 1 μM (purple); and 10 μM (blue). The figure of detection of cefotaxime by PenP-E166Cf (a; left panel) was adapted from our previously published data and reprinted in part with permission from Wong et al. Copyright 2011 BMC.
Figure 4
Figure 4
Thermostability of the PenP and PenPC biosensors. (a) Thermal denaturation curves of the biosensors determined by CD. Temperature at which the relative ellipticity at 222 nm is reduced to 50% is regarded as the melting temperature (Tm) of the protein. (b) Residual activities of PenP- and PenPC-based biosensors at 50 °C.
Figure 5
Figure 5
Detection of penicillin G in the (a) mouse serum and (b) milk sample using PenP-E166Cf/N170Q. Upper panels: Time-resolved fluorescence traces of PenP-E166Cf/N170Q with mouse serum or milk containing various concentrations of penicillin G: 0 (orange); 0.01 μM (red); 0.1 μM (green); 1 μM (purple); and 10 μM (blue). Each point of the trace was the average value collected from three independent measurements. Lower panels: The corresponding calibration curve for the detection of penicillin G. Each point shows the mean ± S.E.M. of three measurements.
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