A New Method For Detecting The Chloramphenicol With The Mutanted Chloramphenicol Acetyltransferase

Small Molecular Trapping (SMT) is one novel methods which use mutanted enzyme to detect small molecular. By gene engineering the enzymes' activity is changed with lower catalytic activity and steady binding activity. Bacteria containing chloramphenicol acetyltransferase (CAT) when exposed to CM convert the antibiotic to acetylated products and is resistant to the growth inhibition normally observed upon exposure to CM. Chloramphenicol acetyltransferase is a kind of prokaryotic enzyme and is abundantly expressed in E.coli. With the characteristics, chloramphenicol acetyltransferase gene is a ideal reporter gene.Variants of chloramphenicol acetyltransferase come from a variety of bacterial species .The more attention is paid on CAT I , IK III ,among which CAT I has the highest substrate affinity.Structure study indicates His in the catalytic center plays an essential part in catalytic activity. Replacement of His by alanine results in apparent decreases in kcat of 9X 10⁵-fold, whereas Km value for chloramphenicol is similar to those of wild-type CAT .In this thesis, the site—directed mutant of CAT I is gained by PCR, which has a dramatic fall in catalytic activity and steady chloramphenicol binding activity. The mutant is suitable for detecting the remnant CM in the food. Coat wells of 96-well plates with the mutant, add optimum concentration of HRP-conjugated CM and free CM diluted to the serial concentration then plot standards against absorbance .The free CM concentration of above 1 μg/ml against absorbance (OD_490nm) has a linear relation. The method of detecting CM with the mutanted chloramphenicol acetyltransferase is feasible and provides a new way for detecting CM.