title: Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase.
abstract: A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.
authors.affiliation: Department of Applied Biology and Chemical Technology and Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hunghom, Kowloon, Hong Kong, P. R. China.
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.forename: Hong-Bing
authors.initials: HB
authors.lastname: Liu
authors.forename: Yu Wai
authors.initials: YW
authors.lastname: Chen
authors.forename: Kwok-Chu
authors.initials: KC
authors.lastname: Chan
authors.forename: Chun-Wai
authors.initials: CW
authors.lastname: Tsang
authors.forename: Yun-Chung
authors.initials: YC
authors.lastname: Leung
authors.forename: Kwok-Yin
authors.initials: KY
authors.lastname: Wong
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000737:chemistry; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005506:Food Contamination; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000032:analysis* / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D047090:beta-Lactams; D001618:beta-Lactamases