A Preliminary Study On The Development Of Enzyme Preparation And Genetically Engineered Microorganism Used For Pesticides Degradation

Pesticides are synthetic materials that have been widely used by people for insect control on both agricultural and home insecticides. Continuous high yield of agriculture products can be obtained due to such a long time and extensive usage of synthetic pesticides. However, we have to admit that the usage of pesticides lead us to face the compound pesticides pollution and health risk problem. Bioremediation, as a security and cost-effectiveness means for environmental control, gets increasing concern and plays a crucial role in pesticides degradation.On one hand, pesticides pollution can be seen from that excessive pesticide been tested from agriculture products. Currently, there are many studies on microbiological degradation, but most of them focus on isolation of pesticides-degrading bacteria and metabolic pathway. These methods play a little role in pesticides degradation in the environment. In this study, PytH was successfully over-expressed and 20mg protein could be purified from 1 liter of culture. We were willing to degrade the fenpropathrin on the surface of lettuce with the crude enzyme preparation and pure enzyme preparation detecting by gas chromatography. We used two methods to prepare the research. One is spraying the crude enzyme preparation and pure enzyme preparation on the "surface of lettuce. The other is immersing the lettuce into PBS with the crude enzyme preparation and pure enzyme preparation. On the two methods above, the degradation rates of fenpropathrin with the crude enzyme preparation are 61.81% and 50.97%. The degradation rates of fenpropathrin with pure enzyme preparation are 76.78% and 76.41%. From the results, we can conclude that not only the pure enzyme preparation but also the crude enzyme preparation can obtain a degradation rates over than 50%, which indicates a promising application of enzyme preparation.On the other hand, pesticides pollution can be seen from soil and water pollution with multiple pesticides, in which pyrethroids、parathion-methyl、chlorothalonil and chloroacidamide play a major role in pesticides pollution. Nowadays, no reported pesticides-degrading bacteria have broad substrate spectrum toward pesticides degradation. The ideal strain for bioremediation of contaminated environments needs to degrade more kinds of Pesticides. Thus, we constructed a multifunctional pesticides degrading genetically engineered microorganism called P. putida KT2440-pmca by homologous recombination. PytH、mpd、chd、amp A were integrated into the chromosome of P. putida KT2440 using the method of homologous recombination. PytH that can degrade pyrethroids was cloned from Sphingobium faniae JZ-2; mpd that can degrade parathion-methyl was cloned from Plesiomonas sp. M6; chd that can degrade chlorothalonil was cloned from Pseudomonas sp. CTN-3; ampA that can degrade chloroacidamide was cloned from Paracoccus sp. FLN-7. Then we verified the degradation function of P. putida KT2440-pmca by experiment of transparent circle、UV dictation and HPLC dictation which means that P. putida KT-pmca truly can degrade four kinds of pesticides.With the increasing use of pesticides, compound pesticides pollution and health risk problem could be getting worse and worse. We studied an enzyme preparation that can degrade many kinds of pyrethroids. And we then constructed a multifunctional pesticides degrading genetically engineered microorganism P. putida KT2440-pmca that can degrade four kinds of pesticides, which brought a well foundation for multifunctional pesticides degradation.