The Rational Design Of Recombinant Butylcholineserase For The Detection Of Pesticide Residue

Organophosphorus pesticides have been commonly used for its high efficiency, species diversity and wide application. Due to the excessive use of organophosphorus pesticides, serious environmental pollution and non-targeted organisms toxicity. Therefore, there is an urgent need for development of organophosphorus pesticides detection.The main measuring methods of pesticide residues is enzyme inhibition, the main enzyme sorce is AChE. But the following deficiencies still exist as we use it for detection: The sensitivity needs to be improved; secondly because of the large number of organophosphorus pesticides, A single AChE can not meet detect all. Therefore, we hope through biotechnology to transform the BChE which has the similar structure to AChE. In order to find the enzyme source which has a better sensitivity and wide detection range.BChE shares many common properties with AChE in in structure and properties. Scientist, previously, tried to use it for organophosphorus pesticides detection. By studying the structure of BChE, we transform the important site of human BChE and use the method of site-directed mutagenesis, protein expression and purification, and enzyme kinetics detection. Finally,40 mutants of hBChE are produced. And then we successfully establish a screening model to select the most favorite one. By measuring the bimolecular rate, we find that hAChE don’t recognize Profenofos. hBChE compared with hAChE,has better identify to dichlorvos, profenofos, phoxim.Through mutant screening, we found F227, S287, N68, P285 and other sites for identifying influence. The mutant A328W has improved 17.9-folded on detecting ethyl paraoxon.