Properties of fluorophore-labeled derivatives of class A beta-lactamases

vi, 142 leaves : ill. (some col.) ; 30 cm.PolyU Library Call No.: [THS] LG51 .H577M ABCT 2007 AuThe intensive use of beta-lactam antibiotics, such as penicillins and cephalosporins, has led to food contamination and the emergence of antibiotic-resistant bacteria. This is a very serious problem as it may reduce the efficacy of antibiotics and make them no longer able to kill the resistant infectious bacteria. This situation keeps worsening due to the evolution process of the bacteria. As a result, an effective detection method for any residual antibiotics and strategies to discover new effective beta-lactam drugs and beta-lactamase inhibitors are urgently required. Previously, the novel fluorescence biosensor (PenPC_E166Cf), derived from beta-lactamase I of Bacillus cereus 569/H, demonstrated its success in detecting beta-lactam antibiotics and beta-lactamase inhibitors. Its development opened up a new arena for antibiotic detection and novel drug screening. In order to evaluate the feasibility of using other class A beta-lactamases for the biosensor construction, four fluorescence biosensors (PenP_E166Cf, PenP_E166Cb, PenP_E166Cf/N170Q and PenP_E166Cb/N170Q) were developed in this study by a similar approach, using the PenP beta-lactamase of Bacillus Licheniformis 749/C as the basis. The PenP beta-lactamase is 58% identical to PenPC in the amino acid sequence but is more thermostable. Two PenP beta-lactamase mutants, E166C and E166C/N170Q, were constructed in the project. Mutations of E166C and N170Q were found to cause a dramatic decrease in the activity of the PenP beta-lactamase, in which the catalytic efficiencies of the E166C and E166C/N170Q were approximately 4 x 10⁴-fold and 1.2 x 10⁵-fold lower than that of the wild-type, respectively. The PenP biosensors showed the ability in detecting penicillins such as penicillin G and penicillin V, and cephalosporins such as cefotaxime and moxalactam. The detection limits of the biosensors for these beta-lactam antibiotics were approximately 10 nM. Biosensors developed from the double mutants gave longer detectable signals than those developed from the single mutants, because of the further suppressed deacylation of enzyme by the N170Q mutation. Apart from the detection of antibiotics, the capability of the PenP biosensors in signal generation in the presence of beta-lactamase inhibitors, such as sulbactam and clavulanate, may allow them to find their applications in drug discovery. However, in terms of developing a sensing system for detecting beta-lactams and screening beta-lactamase inhibitors, PenP biosensors labeled with fluorescein-5-maleimide seem to be more useful than the badan-labeled counterparts, because the formers confer a higher quantum yield with a lower incidence of false results. In comparison with the PenPC-based biosensors developed previously, the PenPC_E166Cb seems to be the best biosensor in detecting penicillin-type antibiotics because of its ability to generate significant and long detectable signals with a good detection limit. PenP_E166Cf seems to be the best in detecting cephalosporin-type antibiotics as it gave significant stable signals with a fast response time and a low incidence of false results. The PenP-based biosensors, which functioned well at temperature as high as 50 ℃, also demonstrated their high thermostability.Department of Applied Biology and Chemical TechnologyM.Phil., Dept. of Applied Biology & Chemical Technology, The Hong Kong Polytechnic University, 200