Isolation And The Basic Relative Study On Biochemical, Molecular Biology Of Pyrethroid-degrading Strain

In recent years, pyrethroid pesticides have showed many environmental deficiencies and toxic effects against men. Especially in the procession of export trade, some products such as tea, vegetable etc., have encountered "green barrier" because of pyrethroid residues, It is the residues of pyrethroid pesticides that have led to great economic losses. And then the research on pyrethroid-degrading mediated by microorganism should be payed much attention by researcher and government.In this experiment, a bacterial strain which can use pyrethroid pesticides as sole carbon and energy sources, was isolated from the polluted soil samples collected from the pesticide factory in Changzhou of Jiangsu provience. By Biolog microstation system and 16S rDNA sequence analysis, we identified the strain. And that of broad spectrum about pyrethroid and organphosphorus pesticides-degrading were studied. All of those as follows:The strain named as 4-D temporarily, by Biolog microstation system and 16S rDNA sequence analysis, has been identified as Acinetobacter sp. It was gram-negative unmotile rod and broad-spectrum, which is able to use pyrethroid and organphosphorus pesticide as a sole source of carbon. The optimum temperature and pH are 28℃ and 8.0. Additionally, It's growth temperature and pH are much wider. When the temperature are 4℃ or 50℃, the strain doesn't lose the degrading ability on pyrethroid. When pH are 7~9, the degrading rates on pyrethroid don't have significant difference .The enzyme of pyrethroid-degrading was purified from 4-D strain by acetone precipitation, Sephadex G-100 gel filtration and DEAE-52 ion-exchange column chromatography. When the volumn was 50~70 milliliter, the enzyme of pyrethroid-degrading was washed from Sephadex G-100. And it's tightly combined with DEAE-52 ion-exchange column chromatography. When the concentration of sodium chloride in washing buffer is 500mmol/L, it was washed down. The purified enzyme shows a single band on SDS-PAGE gel with an approximate molecular weight of 55 X 103Da. The optimum temperature and pH for the enzyme are 30℃ and 9.0. The degrading rate of coarse enzyme on pyrethroid was 0.312 nmol min~-1 per milligram protein, when the enzyme was purified, the rate was improved highly. It was 10.8 nmol. min~-1' per milligram protein.By the molecular biology methods, constituted the gene library of 4-D strain, and attained the recombined plasmid 1-2, which can use beta_cypermethrin and cyfluthrin as sole carbon and energy sources. Partial sequence of the fragment is homoeologous with that of Rhs family proteins, and the data is 59%. But he relationship of Rhs family proteins and pyrethroid-degrading should be further studied.