Thermostable beta-lactamase mutant with its active site conjugated with fluorescein for efficient beta-lactam antibiotic detection

Abstract

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