Methods For Determination Of β-lactam Residues Based On Genetically Encoding Unnatural Amino Acid Technology

Food safety is important for public health and the foundation of national stability. The ratio of animal derived food is increasing in residential diet in China, and antibiotic residues within have become one of the main issues of food safety. P-lactam antibiotics are widely used in veterinary. To develop a facile, reliable and sensitive method to detect and monitor their residues in food is important for safeguarding food.In this dissertation we site-specifically incorporated a fluorescent amino acid, （7-hydroxycoumarin -4-yl）)ethylglycine （7HC）, into different sites of the binding domain of the penicillin binding protein PBP2x by using the genetically encoding Unnatural amino acid Technology （UaaTech）. After multi-criteria optimization, the recombinant protein PBP2x₇HC （W374U and Y561U） were successfully obtained from E. coli in the yields of mg per liter, and 7HC incorporation was confirmed by mass spectrometry.After systematic analyses of fluorescence spectra of W374U before and after binding 15 β-lactams, we found that, upon binding Penams, the fluorescence emission intensity of W374U dramatically increased when excited at 325 nm, but decreased significantly when excited at 366nm. In contrast, binding of Cephems decreased W374U emission intensity regardless of excitation wavelength. To be compatible with both sub-group, we chose 366nm and 450 nm as the excitation and emission wavelengths, respectively, and immobilized W374U in microplates for developing the fluorescence assay. The LODs of our assay are in the range of 0.49-2.44 ng/mL for penicillin G, ampicillin, amoxicillin, cloxacillin, oxacillin, dichloxacillin, ceftiofur, cefquinome, ceflonium, cefazolin, cefoperazone, cephapirin and cephacetrile, all lower than the maximum residue limit （MRL） of European Union. The detection range is broad with excellent regression coefficients （R2>0.99）. Milk spiked with antibiotics could be detected after simple pre-processing; the recovery rate was 78%-102% in raw milk and 76%-112% in processed milk. We also developed a multi-residue screening methods using amoxicillin as the standard:setting threshold at 3.6 ng/mL, penicillin G and other 13 p-lactams were detected as positive when they were added in milk in MRL concentrations. Six raw milk samples and nine processed milk samples purchased from supermarkets were measured, the results are consistent with the results measured by the β-lactam strip on the market.To improve the yield of W374U further, we studied the effect of UaaTech to the host organism on the proteomic level for the first time. We discovered that, upon strong suppression of the amber stop codon, E. coli immediately tries to inactivate the responsible gene using transposon insertion. If transposons miss the target and amber codon suppression persists, the expression levels of 51 endogenous proteins were changed more than 2 fold. These results reveal novel avenues for increasing the yield of W374U and for overcome the intrinsic limitation of the UaaTech.In summary, using the UaaTech we have generated a reagent that innovatively served as both capture and signal functions, and developed a sensitive fluorescence method for the determination of β-lactam residues. This work represents a non-competitive receptor assay to detect small molecule analytes, and should be a valuable new addition to the technological arsenal for the detection and control of veterinary drug residues.