|Title:||Development and optimization of fluorescent biosensors from various class A beta-lactamases|
|Subject:||Hong Kong Polytechnic University -- Dissertations.|
Beta lactam antibiotics.
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xvi, 224 p. : ill. (some col.) ; 30 cm.|
|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.|
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