Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase.
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.
Chan
Pak-Ho
PH
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.
Liu
Hong-Bing
HB
Chen
Yu Wai
YW
Chan
Kwok-Chu
KC
Tsang
Chun-Wai
CW
Leung
Yun-Chung
YC
Wong
Kwok-Yin
KY
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
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D047090:beta-Lactams; D001618:beta-Lactamases