|Title:||Development of β-lactamase-based biosensor for anti-tuberculosis drug screening|
|Advisors:||Leung, Thomas (ABCT)|
Tsang, Edman (ABCT)
|Subject:||Hong Kong Polytechnic University -- Dissertations|
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xvi, 170 pages : color illustrations|
|Abstract:||Tuberculosis has been one of the deadliest disease in history. Although it became curable since the discovery of antibiotics, drug resistance was soon developed and the needs in new antituberculosis treatment escalated. In the past, our group has developed an effective β-lactamase-based fluorescent biosensor for β-lactam antibiotics detection. BlaC, a β-lactamase responsible for the β-lactam antibiotic resistance in Mycobacterium tuberculosis, was engineered to be a sensitive and efficient biosensor for screening potential inhibitors to be used in combination with currently available β-lactam antibiotics. Thr-216 was mutated to Cys by site-directed mutagenesis and was named T216C. Various fluorophores were labelled on the mutated residue and the prospective fluorescent sensor enzyme was screened. The functionality of the most promising fluorophore-labelled enzyme T216Cf against antibiotics and inhibitors was investigated with fluorescence spectrometry. Immediate surges in fluorescence intensity of T216Cf upon penicillins addition and positive signals induced by carbapenems and inhibitors were observed, but the response to cephalosporins was insignificant. The relationship between enzyme-inhibitor complex formation and fluorescence intensity change was also revealed by the combination of ESI-MS and fluorescence spectrometry. The kinetic parameters were measured and calculated to examine the impact of mutagenesis and labelling. A second mutation to Trp was introduced at Ile-105 and Thr-237 in an attempt to enhance the biosensor sensitivity. T216Cf/I105W showed a major improvement in sensitivity, as much as from +35% to +83% upon inhibitor addition, although further optimisation can be done on the biosensor efficiency. Molecular dynamics simulations were conducted to understand the mechanism behind the fluorescence signals, which is transferable to similar β-lactamase-based biosensor. The change in residues in proximity to the fluorescein, solvent exposure, and interaction with quenchers such as tryptophan all contribute to the fluorescence signals. The promising results suggested that the new T216Cf has great potential to be refined as a powerful biosensor for drug screening.|
|Rights:||All rights reserved|
Files in This Item:
|991022287145903411.pdf||For All Users||8.23 MB||Adobe PDF||View/Open|
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item: