|Author:||Liu, Sze Yan|
|Title:||Biosensor development based on class C beta-lactamase|
Hong Kong Polytechnic University -- Dissertations
|Pages:||1 online resource (xii, 214 pages) : illustrations|
xii, 214 pages : illustrations
|Abstract:||Since the first discovery in the 20th century, antibiotics have been prescribed for combating bacterial infections extravagantly. Owing to comparatively high effectiveness, low cost, ease of delivery and minimal side effects, β-lactams are not only extensively used by clinical doctors, but also in veterinary applications. This led to food allergy and reduced the efficacy of β-lactam antibiotics as the bacteria become drug resistant under selective pressure. Recently, our group successfully developed an innovative screening tool for class C β-lactamase inhibitors. An engineered class C β-lactamase mutant from Enterobacter cloacae P99, V211C, was prepared by replacing valine 211 with cysteine by site-directed mutagenesis (Tsang et al., 2011). The purified V211C was labeled by attaching maleimide-5-fluorescein which is a thio-reactive fluorophore and named as V211Cf. The fluorescent sensor enzyme generated has minimum activity loss and acts as a "natural" fluorescent drug target for screening active site binding molecules or inhibitors. However, the AmpC P99 β-lactamase from Enterobacter cloacae is a narrow-spectrum β-lactamase and therefore, this labeled sensor enzyme V211Cf can only be used to screen for inhibitors against the AmpC P99 β-lactamase. Extended-spectrum cephalosporins are poor substrates for both class A and class C β-lactamases. The AmpC P99 Y221G mutant is a well characterized class C extended-spectrum β-lactamase. In this project, V211C expressed in E. coli, was prepared and named as AmpC_P99_V211C. The AmpC P99 β-lactamase was also engineered to form a double mutant AmpC_P99_Y221G/V211C by rational design. This purified mutant was labeled with fluorescein-5-maleimide to form a new biosensor, AmpC_P99_Y221G/V211Cf. The fluorescence studies, kinetics studies and mass spectrometry studies revealed that AmpC_P99_Y221G/V211Cf has a similar binding efficiency with AmpC_P99_V211Cf. However, the rates of hydrolysis of penicillin G, cephalothin and cefoxitin by AmpC_P99_Y221G/V211Cf are much lower than AmpC_P99_V211Cf, indicating that Tyr221 should have an important role in the deacylation process during hydrolysis of penicillins and cephalosporins. The new AmpC_P99_Y221G/V211Cf not only has properties that allow it to act as a better tool to study the active site kinetic features, detecting β-lactam antibiotic residues in milk, more important is that it gives meaningful signals in the presence of inhibitors and it could be useful in discovering potential inhibitors against extended-spectrum β-lactamases.|
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