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

ABSTRACT: Monitoring the ?-lactam antibiotic level has been an important task in food industry and clinical practice. Here, we report the development of a fluorescent PenP ?-lactamase, PenP-E166Cf/N170Q, for efficient ?-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 ?-lactamase. It gave a fluorescence turn-on signal toward various ?-lactam antibiotics, where the fluorescence enhancement was attributed to the acyl-enzyme complex formed between PenP-E166Cf/N170Q and the ?-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 ?-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 ?-lactam antibiotics.

SUBMITTER: Au HW 

PROVIDER: S-EPMC6906784 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature