A fluorescein-modified β-lactamase as biosensor for β-lactam antibiotics

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A fluorescein-modified β-lactamase as biosensor for β-lactam antibiotics

 

Author: Chan, Pak-ho
Title: A fluorescein-modified β-lactamase as biosensor for β-lactam antibiotics
Degree: M.Phil.
Year: 2002
Subject: Hong Kong Polytechnic University -- Dissertations.
Beta lactamases.
Antibiotics.
Department: Dept. of Applied Biology and Chemical Technology
Pages: ix, 191 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1657656
URI: http://theses.lib.polyu.edu.hk/handle/200/345
Abstract: b-Lactam antibiotics (e.g. cephalosporins and penicillins) and b-lactamase inhibitors (e.g. sulbactam and clavulanate) are clinically important antibacterial agents widely used in the clinical treatment of bacterial infections. Because of their effective antibacterial activities, a great variety of b-lactam antibiotics and b-lactamase inhibitors are produced in the pharmaceutical industry and they are used routinely in the clinical treatment of human beings and livestock. Therefore, a new sensor that can effectively detect these drugs is highly desired. Such a sensor will be of importance in drug screening, discovery of new b-lactamase inhibitors and routine measurement of antibiotic concentration in, for example, contaminated food samples. In our study, a new biosensor for b-lactam antibiotics and b-lactamase inhibitors has been prepared by labeling a mutant of b-lactamase I (E166C) with the environment-sensitive fluorescent probe fluorescein-5-maleimide. The mutant was constructed by site-directed mutagenesis in which a particular amino acid (Glu-166) on the O-loop of the enzyme was replaced with a thiol-containing cysteine residue. The cysteine residue was then labeled with the thiol-reactive fluoresein-5-maleimide via the formation of thioether to give a fluorescein-labeled b-lactamase I (E166Cf). As the wild-type b-lactamase I contains no cysteine residue, this single-point mutation allows the attachment of the thiol-reactive fluorophore to a specific site on the enzyme. The Glu-166 residue was chosen as the labeling site not only of its closeness to the active site, but also of the flexibility of the O-loop, which allows the fluorophore to move when the antibiotic enters the active site. Spectrophotometric assays showed that the activity of the E166Cf enzyme is conserved after the labeling reaction. Fluorescence measurements indicated that the fluorescence intensity of the E166Cf enzyme increases with the concentration of penicillins (penicillin G, penicillin V and ampicillin) and cephalosporins (cefuroxime, cefoxitin and moxalactam). The fluorescence enhancement is attributed to the conformational change in the active site upon binding of the antibiotic. Such a conformational change causes the fluorescein label to depart from the active site, thus enhancing the fluorescence quantum yield of the fluorescein molecule. Time-resolved fluorescence measurements revealed that the E166Cf enzyme can detect b-lactam antibiotics at trace level (10-7 M). Moreover, the E166Cf enzyme is capable of distinguishing penicillin antibiotics from cephalosporin antibiotics. With penicillin G, penicillin V and ampicillin as substrates, the fluorescence intensity of the E166Cf enzyme increases and then declines slowly. In the presence of cefuroxime, cefoxitin and moxalactam, the fluorescence signal of the labeled enzyme increases slowly but does not decline. The E166Cf enzyme also exhibits different fluorescence signals when incubating with the b-lactamase inhibitors (sulbactam and clavulanate). The use of the E166Cf enzyme in screening bacteria for b-lactamases against a panel of b-lactam antibiotics has been investigated. b-Lactamase II, penPC b-lactamase, penP b-lactamase and TEM-1 b-lactamase secreted by Bacillus subtilis and Escherichia coli were screened against penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin and moxalactam using the E166Cf enzyme as a reporting tool. The fluorescence signals from the E166Cf enzyme allow one to distinguish antibiotics that are resistant to the hydrolytic activities of the bacterial b-lactamases from those that are unstable towards the bacterial enzymes.

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