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

Ho Wah Au, Man Wah Tsang, Pui Kin So, Kwok Yin Wong (Corresponding Author), Yun Chung Leung (Corresponding Author)

Research output: Journal article publicationJournal articleAcademic researchpeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)20493-20502
Number of pages10
JournalACS Omega
Volume4
Issue number24
DOIs
Publication statusPublished - 10 Dec 2019

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)

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