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Title: Protein engineering of class A and C b-lactamases for b-lactam antibiotic detection
Authors: Tsang, Man-wah
Degree: Ph.D.
Issue Date: 2007
Abstract: With respect to the increasing demands for efficient screening systems for b-lactam compounds, a fluorescent b-lactamase E166Cf has been previously exploited for the purpose of generic detection of b-lactam antibiotics and b-lactamase inhibitors. Fabrication of this novel E166Cf sensor has been regarded as an innovative technology as it started a great milestone in the biosensor development by opening up the possibility of turning a non-allosteric b-lactam hydrolytic enzyme into a turn-on sensor for its substrates and inhibitors. E166Cf showed excellent properties as a sensing tool, including high sensitivity and amenability for high-throughput screening. However, owing to the differential substrate preference of the various classes of b-lactamases, E166Cf, based on a class A b-lactamase, demonstrated better responses to its preferred penicillin-type substrates than the cephalosporins. In order to have a biosensor specific for sensing cephalosporins, in this project, we aimed at constructing a class C b-lactamase, which shows a substrate preference on cephalosporins, into a fluorescent biosensor. In addition, it was postulated that this class C b-lactamase-based sensor would also be important for identifying potent class C b-lactamase inhibitors. In our study, a class C b-lactamase from Enterobacter cloacae P99 was engineered by rational design into a fluorescent protein designated as V211Cf for sensing b-lactam antibiotics and b-lactamase inhibitors. This novel V211Cf was a fluorescein-labeled b-lactamase prepared by labeling the V211C mutant of P99 b-lactamase with a thiol-reactive fluorescein-5-maleimide. The fluorescein molecule was tethered to this unique cysteine-containing mutant at the 211 position that is in close proximity to the active site via a maleimide linker. Kinetic analysis of V211Cf indicated that there was no loss of binding efficiency and hydrolytic capability of V211Cf with a fluorophore attached in vicinity to the active site. We had successfully tethered a fluorescent probe at the proximity of the active site with restoring enzyme activity. The fluorescein molecule located in close proximity of the active site in V211Cf could report the local environment changes at the enzyme active site. As revealed by fluorometric studies, addition of the two main classes of b-lactam antibiotics, including penicillins and cephalosporins, triggered fluorescence enhancements of V211Cf. Such increased signals then disappeared according to the rate of the hydrolysis of the substrates by V211Cf. In addition, V211Cf displayed distinct signal patterns for the b-lactamase inhibitors, including clavulanic acid, sulbactam, tazobactam, and transition-state analogs. These results indicated that V211Cf can probe for the presence of the b-lactam antibiotics and b-lactamase inhibitors. In addition, time-resolved fluorescence measurement of V211Cf allows real-time studies on the enzyme-substrate/-inhibitor interactions. With the aim of optimizing the performance of V211Cf in the detection for b-lactam antibiotics, Y150S/V211Cf, a derivative of V211Cf, was also constructed. As a catalytically-impaired derivative, Y150S/V211Cf did not hydrolyse the substrates, thus improving the signal stability. Furthermore, Y150S/V211Cf only showed fluorescence signals in the presence of cephalosporins but not penicillins, thus it was specific in sensing cephalosporin-type antibiotics. Comparison of performance was made among the class C labeled-b-lactamases, V211Cf and Y150S/V211Cf, as well as the previously reported class A b-lactamase-based E166Cf in the detection of b-lactam antibiotics. While E166Cf was superior to the fluorescent class C b-lactamases in sensitivity, the fluorescein-modified class C b-lactamases demonstrated a more rapid response time for sensing cephalosporins than E166Cf. To conclude, our results demonstrated for the first time the feasibility of the application of fluorescein-labeled class C b-lactamases for sensing b-lactam antibiotics and b-lactamase inhibitors. Furthermore, because of the conserved enzyme activity of V211Cf as the wild-type enzyme, V211Cf may serve as an attractive tool for mechanistic studies of the class C b-lactamase. Last but not least, our study further supported the applicability of utilizing a non-allosteric protein modified with a fluorophore close to the active site as a sensing system for its ligands. This opens up an innovative approach for the development of enzymes into reagentless biosensors.
Subjects: Hong Kong Polytechnic University -- Dissertations.
Protein engineering.
Beta lactamases.
Beta lactam antibiotics.
Pages: xv, 217 leaves : ill. (some col.) ; 30 cm.
Appears in Collections:Thesis

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