Please use this identifier to cite or link to this item:
Title: Structural and functional studies on class A β-lactamase-derived biosensors
Authors: Wong, Wai-ting
Degree: Ph.D.
Issue Date: 2011
Abstract: The extensive use of beta-lactam antibiotics in veterinary and clinical applications has led to food contamination and reduced efficacy of β-lactam antibiotics as bacteria become antibiotic-resistant due to selective pressure. The major mechanism of antibiotic resistance in bacteria is β-lactamase that inactivates the β-lactam antibiotics. Recently, our group has constructed a "switch-on" fluorescent biosensor from a β-lactamase (Chan P.-H. et al.; J. Am Chem. Soc., 2004, 126, 4074) that can successfully detect trace amount of β-lactam antibiotics. In this project, the β-lactamase from Bacillus licheniformis 749/C (PenP) was engineered by site-directed mutagenesis to form the mutant E166C, in which the Glu166 residue in the Ω-loop is replaced with a cysteine residue. It was separately labeled with two thiol-reactive fluorophores, badan (b) and fluorescein-5-maleimide (f) to form two biosensors, PenP_E166Cb and PenP_E166Cf, respectively. This project concentrates on the study of the biosensing mechanisms of both biosensors in order to explain how they generate fluorescence changes upon β-lactam binding. The fluorescence studies of both PenP_E166Cb and PenP_E166Cf with various β-lactams (mainly cephalosporins) reveal that the formation of enzyme-substrate complex enhances the fluorescence of the biosensors significantly. For PenP_E166Cb, the response is selective, and only the addition of oxyimino-cephalosporins gave significant enhancement in fluorescence intensity. Structural studies of PenP_E166Cb and PenP_E166Cf, as well as their antibiotic-bound intermediates show that the biosensing mechanisms in E166Cb and E166Cf are different. The results show that the E166Cb only detects oxyimino-cephalosporins because of the steric clashes between the side chain of oxyimino-cephalosporins and the Ω-loop residues, which induces a change in the local environment around the fluorophore. But E166Cf detects all cephalosporins as the fluorescein label is likely to share a common space with the incoming β-lactam in the active site so that the fluorescein label is displaced from the active site to a more solvent exposed environment. Binding kinetics was studied by ESI-MS to investigate the binding rate of cefotaxime for PenP_E166Cb in comparison with the unlabeled enzyme E166C. We found that the flexibility of the Ω-loop in PenP_E166Cb is significantly increased and the Ω-loop changed to a new conformation after incorporation of the badan label. The new conformation results in the improved binding of cephalosporins with large side chains. Furthermore, based on the crystal structure of PenP_E166Cb, some residues that are in close proximity to the badan label were changed and the biosensing properties of the β-lactamase-based biosensor were improved.
Subjects: Biosensors.
Beta lactamases.
Beta lactam antibiotics.
Hong Kong Polytechnic University -- Dissertations
Pages: xxi, 274 p. : ill. (some col.) ; 30 cm.
Appears in Collections:Thesis

Show full item record

Page views

Last Week
Last month
Citations as of Jun 4, 2023

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.