Enhancing The Sensitivity Of Thermostable Esterase To The Organophosphorus Compounds By Rational Design

Title:Enhancing the Sensitivity of Thermostable Esterase to the Organophosphorus Compounds by Rational DesignAuthor: Yuexi WangAdvisor: Yan Feng, ProfessorMajor:Biochemistry and Molecular BiologyOrganophosphorus compounds (OPs) are widely used as pesticides in agriculture, which kill pests by inhibiting the activities of acetylcholinesterase (AChE) in the nervous system. The overuse of these compounds may lead to serious environmental pollution and cause problems to the public health. Therefore, it is important to detect the residual of pesticides in the environment. The common techniques for detecting pesticides are gas-liquid chromatography, thin-layer chromatography and so on. These techniques have high accuracy, but are difficult to operate, which is not suitable for detecting in spot and often requires complex pre-processing by liquid-liquid extraction or solid-liquid extraction. The biosensor could offer the real-time quantitative information by the smallest doses of the sample, which may replace the techniques mentioned before. The advantage of esterase using as OPs biosensor is convenience and sensitive, which are important for the environmental protection and food safety. The application of AChE as the biosensor for OPs, is not staying in the experimental stage. There are many pesticides detection kits in the market, that were developed from AChE. However, some properties of AChE limit its application. Thus, sensitive enzymes for detecting organophosphorus compounds are highly demanded. In this paper, thermophilic esterase of high thermostability was employed to study the inhibition kinetics, the half-inhibitory concentration, and so on.Here, a thermostable carboxylesterase from the hyperthermophilic archaeon Archaeoglobus fulgidus (AFEST) was employed in the inhibition study, which could hydrolyze P-nitrophenyl esters (pNP) with different acyl chain length and showed the highest activity towards pNPC6. The optimal temperature of AFEST was80℃, which indicated the high thermostability. In our research, we compared the IC50of AFEST (measured at50℃) and the AChE from the Rapid Aaaay Kit of Pesticide Residues(measured at37℃) towards seven OPs. Unfortunately, AFEST only showed smaller IC50than AChE towards dichlorovs, which indicated better sensitivity. Then we studied the inhibition kinetics of AFEST and OPs (paraoxon, dichlorvos, profenofos, and phoxim) through the continuous monitoring of the enzymatic reaction in the presence of inhibitor. The result indicated that AFEST showed the highest affinity with paraoxon, which was consisted with the results of the IC50. Through the analysis of the structure of AFEST, we constructed the mutant N44S/S48V. The mutant enhanced the catalytic activity for larger ester substrates (pNPC12), and the half-life of AFEST and the mutant N44S/S48V at37℃was16d and14d, respectively, which indicated that the stability of N44S/S48V is almost the same as that of the wild type AFEST. Compared with the wild-type AFEST, the mutant N44S/S48V exhibited1-3.6fold higher IC50for OPs. Before the mutagenesis, the wild type AFEST was more sensitive than AChE toward only one compound (dichlorvos); But after the mutagenesis, the N44S/S48V was more sensitive than AChE toward four compounds (paraoxon, dichlorvos, profenofos, and diazinon). The mutant N44S/S48V enhanced the sensitivity toward the OPs, and expanded the kinds of OPs that could be detected sensitively. These advantages are the important basis of AFEST as the biosensor for detecting OPs. Through the docking modules, we obtained more information about the residuals, which played important roles in the interaction of AFEST and OPs, and we also gained a clearer understanding of the structure of OPs, which was preferred mostly by AFEST. In this paper, we got deep understanding of structure and function of enzyme through the mutagenesis, inhibition kinetics, and computational biology, and these results offered us important information for the further study.Then, we immobilized AFEST on the nitrocellulose membrane to improve the availability of the enzyme. The immobilized AFEST was remarkable stable at37℃for reaction and at4℃for storage. The immobilized AFEST could be used over100times, which was an important basis for its application in industrial. Keyword:...