Development and optimization of fluorescent biosensors from various class A beta-lactamases

Pao Yue-kong Library Electronic Theses Database

Development and optimization of fluorescent biosensors from various class A beta-lactamases

 

Author: Chung, Wai-hong
Title: Development and optimization of fluorescent biosensors from various class A beta-lactamases
Degree: Ph.D.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Beta lactam antibiotics.
Beta lactamases.
Biosensors.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xvi, 224 p. : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2285820
URI: http://theses.lib.polyu.edu.hk/handle/200/5289
Abstract: Abuse of antibiotics results in the emergence of antibiotic-resistance pathogenic bacteria which cause undesirable effect on human health. Because of the effectiveness of b-lactam antibiotics, they are commonly used in food producing animals and can thus cause contamination in food. Monitoring and avoiding contamination of b-lactams in food is thus extremely important. Although several tests and protocols for detecting b-lactam residues in food are available, they are either time consuming or semi-quantitative. To solve this problem, several promising fluorescent biosensors based on fluorophore-modified b-lactamase from B. cereus (PenPC), B. licheniformis (PenP) and E. cloacae (AmpC P99) have been developed. To further improve the sensitivity and detection limit of modified b-lactamase biosensors, different modifications were applied to three class A b-lactamases, namely PenP, PenPC and TEM-1. Different amino acid residues in these enzymes were chosen to be mutated to cysteine, and these mutants were separately labeled with 3 different fluorophores (6-bromoacetyl-2-dimethylaminonaphthalene (badan), tetramethylrhodamine-5-maleimide (TMRM), and fluorescein-5-maleimide (FM)). The badan-labeled PenP N170C mutant (PenP N170Cb) has the best performance among all prepared PenP-based biosensors and gives the largest improvement in sensitivity with a 3 folds increase in fluorescence intensity upon penicillin V binding. The FM-labeled TEM-1 V216C (TEM-1 V216Cf), with 2 folds fluorescence increase upon penicillin G binding, has a better sensitivity than the other prepared TEM-1 based FM-labeled biosensor, TEM-1 E166Cf. The sensitivity of TEM-1 V216Cf was further improved by 3 additional mutations (E104K, M182T and G238S) which resulted in 3.6 folds increase in fluorescence intensity upon penicillin G binding, and the improved biosensor was denoted as TEM-52 V216Cf. El66 is the best residue for cysteine mutation and fluorophore labeling in the PenPC biosensor. The FM-labeled PenPC E166Cf has 2 folds increase in fluorescence intensity upon addition of penicillin G and the fluorescence increase is raised to 3 folds by introducing the Y105W mutation. Labeling TMRM to PenPC Y105W/E166C (PenPC Y105W/E166Cr) produced an even better biosensor, with 4 folds increase in fluorescence intensity upon penicillin G addition. Molecular model of TEM-52 V216Cf showed that the binding of penicillin G caused the departure of the FM label from the active site and increased the distance between the FM label and tyrosine 105 (Tyr-105), which is a well known fluorescence quencher. The Increase in fluorescence lifetime of TEM-52 V216Cf upon penicillin G binding supported that the FM-label was quenched by Tyr-105 in the active site. In PenPC Y105W/E166Cr, replacing Tyr-105 by tryptophan (trp), which is a stronger fluorescence quencher than tyrosine, caused reduction in quantum yield of the free enzyme PenPC Y105W/E166Cr but not the substrate bound PenPC Y105W/E166Cr. The background fluorescence intensity of PenPC Y105W/E166Cr was therefore suppressed and the sensitivity of the biosensor was improved. The information obtained thus provides direction for the future design of more efficient biosensors.

Files in this item

Files Size Format
b22858209.pdf 5.325Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. 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.
  3. 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.

     

Quick Search

Browse

More Information