| Sulfonamides(SAs)are a group of synthetic broad-spectrum antimicrobial agents.Illegal use of sulfonamides may leave residues in animal-derived foods,thus endangering the health of consumers.Accordingly,there is a crying demand for cost-effective,high-throughput,time-and labor-saving detection methods for SAs residue monitoring.In this study,two dihydropteroate synthases(DHPS)were obtained and subjected to directional evolution respectively.Then four detection methods were developed for the multi-detection of sulfonamides in animal-derived food by using DHPS as recognition reagents.First,the dihydropteroate synthase of Staphylococcus aureus(SaDHPS)was expressed,and it was used as a recognition reagent to develop a fluorescence polarization assay(FPA)for detection of the 31 SAs in chicken muscle samples.Results demonstrated that a single assay could be completed within 5 minutes,and the limits of detection(LODs)for the 31 SAs were in the range of 2.0-38.5 ng/g.In addition,the change trends of half of the inhibition concentrations(IC50)for the 31 drugs were identical with the SaDHPS-SAs affinities.Results of molecular docking showed that the core structures of 31 SAs matched well with the SaDHPS binding pocket,and their side chains were out of the binding pocket.Hydrogen bonds and hydrophobic interactions were the main intermolecular forces,with Gly171 and Lys203 as the key amino acids.The binding sites in SAs molecules were mainly around the para-aminobenzenesulfonamide part.Second,the Arg204 and Pro216 in the SaDHPS binding pocket were mutated to His and Arg respectively to produce a mutant.Results of molecular docking showed that the affinities of the mutant for 35 SAs were generally increased due to the increase of hydrogen bonds and cation-Pi interactions.Then this SaDHPS mutant was used as a recognition reagent to establish an enhanced FPA for the detection of the 35 SAs in pork.Results demonstrated that the LODs for the 35 SAs were in the range of 0.15-5.58 ng/g,and the sensitivities were improved for 2.8-8.6 folds compared with the conventional FPA.Third,a type of magnetic photoaffinity-labeled activity-based protein profiling probe was synthesized,and a natural DHPS(EcDHPS)was "hooked" directly from Escherichia coli.Then this natural EcDHPS was used as a recognition reagent to develop a direct competitive assay for the determination of 40 SAs in pork.Results demonstrated that the LODs for the 40 SAs were in the range of 0.001-0.016 ng/g,and the sensitivities were improved for 32-88 folds compared with the conventional direct competitive assay.In addition,the change trends of IC50 for the 40 drugs were identical with the EcDHPS-SAs affinities.Results of molecular docking showed that the key amino acids of EcDHPS-SAs were Lys221,Ser222,Arg63 and Pro64.Fourth,the Ser222 in the binding pocket of natural EcDHPS was mutated to Arg to produce a mutant.The binding energies and affinities of the mutant for 44 SAs were significantly increased.Then this EcDHPS mutant was used as a recognition reagent to develop a direct competitive fluorescence method for the detection of the 44 SAs in milk.Results showed that the LODs for the 44 SAs were in the range of 0.025-0.65 ng/g,and the sensitivities were improved for 6-86 folds compared with the conventional direct competitive fluorescence method.In conclusion,in this study,two bacteria-derived DHPS were prepared by the in vitro expression method and the magnetic probe capture method,and two mutants with higher affinity were obtained through directional evolution.The interaction mechanism between the four DHPS and SAs was explored by molecular docking techniques and surface plasmon resonance experiments.Then these four DHPS were respectively used as recognition reagents for the development of four rapid multi-detection methods.The present four methods could be used as practical tools for multi-screening the sulfonamide residues in animal-derived foods,which is extremely important for food safety and consumer health. |
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