|Author:||Chow, Ka Yan|
|Title:||Development of a biosensing system from chloramphenicol acetyltransferase|
Food -- Microbiology.
Food -- Analysis.
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xiii, 193 leaves : ill. (some col.) ; 30 cm.|
|Abstract:||Chloramphenicol is a low-cost and effective antimicrobial agent that binds to the bacterial ribosome and interferes with protein synthesis. It is commonly used in clinical treatments and veterinary practices. However, because of its harmful side effects such as bone marrow depression, the use of chloramphenicol in food production has been banned in many countries. Nevertheless, residual chloramphenicol is still found in some food products, such as in aqua organisms and honey in some countries. As a result, it is essential to develop rapid and sensitive methods for detecting and screening of chloramphenicol residues in food samples. In this project, we aimed to construct a fluorescent biosensor for chloramphenicol detection based on the enzyme chloramphenicol acetyltransferase (CAT). In the construction of chloramphenicol biosensor, a series of modified chloramphenicol were synthesized by connecting chloramphenicol base to fluorescein-5-isothiocyanate (FITC) through a short linker. The products were named as FITC-l-CM where l (l = 4, 5, 6, or 7) is the number of carbon atom present in the linker. In addition, the CAT gene was cloned and three amino acid residues of the enzyme (V28, F138 and E140) were mutated to tryptophan. These three sites were selected because they are close to the active site which can probably interact with the bound chloramphenicol. These two components, FITC-l-CM and CAT mutants were used together for chloramphenicol detection and determination.|
Spectrophotometric assays indicated that chloramphenicol and FITC-l-CM can bind to the active site of those CAT mutants. Fluorescence measurements showed that only mutants V28W, V28W/F138W, V28W/E140W and V28W/F138W/E140W can quench FITC-l-CM. Addition of chloramphenicol can subsequently restore the fluorescence intensity of the quenched FITC-l-CM. The fluorescence change is attributed to the quenching effect exerted by the tryptophan residues on CAT mutants and competitive binding with chloramphenicol. When FITC-l-CM binds to the active site of CAT, the fluorophore interacts with the nearby tryptophan residues provided that the linker is of appropriate chain length. Since tryptophan is an effective quencher of the fluorophore, the bound FITC-l-CM is quenched through photo-induced electron transfer (PET). When chloramphenicol is added, it competes for the binding site with FITC-l-CM. Once FITC-l-CM is displaced from the active site, it is freed from the quenching effect of tryptophan. As a result, the fluorescence intensity increases again. Among different combinations of FITC-l-CM/CAT mutant, FITC-5-CM with the V28W mutant and FITC-6-CM with the V28W/E140W mutant exhibited the strongest change in fluorescence signals and therefore they were selected as the best candidates for biosensor fabrication. The fluorescence intensity of FITC-5-CM decreased about 40% after the addition of the V28W mutant and increased about 60% after addition of chloramphenicol while those of FITC-6-CM with the V28W/E140W mutant are about 60% and 70% respectively. For both FITC-5-CM and FITC-6-CM, the fluorescence intensities are mostly restored to that before quenching. The detection limits of chloramphenicol for FITC-5-CM with the V28W mutant and the FITC-6-CM with the V28W/E140W mutant are 0.1μM and 1μM respectively. Our results demonstrated the applicability of mutated CAT and fluorophore-linked chloramphenicol as a biosensing system for chloramphenicol determination. It is fast, simple and specific, though the sensitivity needs to be further improved before its application to real samples.
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