Studied On Screening And Characteristics Of The Degradation Bacteria Of Residues OPs On Fruits And Vegetables

With the extensive use of organophosphorus pesticides, Pesticide residues in fruits and vegetables more and more attention has been paid. Screening excellent organophosphorus-degrading bacteria and building genetically engineered bacteria gradually become the focus of researchers. In order to obtain strains with capable of degrading organophosphorus pesticide residues in fruits and vegetables. In this study, a strain with excellent resistance and ability to degrade organophosphorus was isolated by primary screening and rescreening from the arable soil which always spraying organophosphorus pesticide. Morphological, physiological and biochemical and molecular biological identification, combined with the phylogenetic tree made after Blast in the NCBI. Determined the strain was Burkholderia cepacia and numbered DOP-Ma3, which accession number of GenBank is KT993571. The strain can tolerate up to 10000 mg/L chlorpyrifos substrate. The degradation rate was 69.87% within 48 h for 250 mg/L of chlorpyrifos substrate. And it was proved that the strain has good performance in broad spectrum of degradating organophosphorus pesticide.The strain was inoculated with 2% and fermented for 48 hours. At regular intervals samples and measured the amount of bacteria and the residues of chlorpyrifos. It was determined the type of enzyme-producing was growth-coupled by making grown curve and degradation curve of strains. To determine the optimal medium components by a single factor experiment. Optimum carbon and nitrogen content optimization combined with response surface methodology. Analysis experimental with Box-Behnken central composite design principles of Design-expert 8.0 software. To determine the optimum medium:15.38 g/L glucose,9.71 g/L peptone,15.54 g/L ammonium sulfate,0.01 g/L zinc sulfate,0.01 g/L ferrous sulfate,0.01 g/L copper sulfate. In this case the maximum amount of cell was 1.277 and the degradation rate of chlorpyrifos substrate was 67.76%. The result of validated experiment was closed with the maximum amount of cell was 1.269 and the degradation rate of chlorpyrifos substrate was 67.86%. And it was increased by 17.59% and 9.84% than previous. Determined optimum pH of the medium was 7.5 and the optimum culture temperature is 37 ?through the single factor experimental.The strain DOP-Ma3 fermented 48 h in conditions optimized. It determined the degradation enzyme is extracellular enzymes by cell localization for organophosphorus degrading enzyme. And chlorpyrifos as inducer was unnecessary. It was determined with single factor experiment that the effect of the activity of the enzyme reaction optimal concentration of 10% and a temperature of 50 ?, pH 7.5.30 min. By ammonium sulfate fractionation, salting-degrading enzyme was obtained in the range of 60%-80%. Load in Sephadex G-75 column after dialysis and concentration. Sampled at flow rate of 1.30 mL/min. The enzymatic activity collected In 55-60 min was 275.80 U/L. Load in DEAE Sepharose Fast Flow column after dialysis and concentration. Enzyme Activities of 517.94 U/L.was collected when elution with the buffer of 0.2-0.5 mol/L NaCl concentration. The molecular weight of the enzyme was determined about 30 kDa.by SDS-PAGE denaturing gel electrophoresis.By querying GenBank already included OPH gene, Analyzed and designed 8 pairs of specific primers and two pairs of degenerate primers with Vector NTI 10 software. OPH-BF amplified fragment was obtained after enzyme producing gene of the strain PCR. To determined OPH gene with a variety of OPH gene sequence homology reaches 99% by Blast in the NCBI after sending GeneBank sequenced. OPH gene translated and studied on physicochemical properties with software and website. Determined that the protein molecular formula is C1354H2137N365O402S4, molecular weight of 30 kDa, a length of 284 aa, isoelectric point of 7.26, extinction coefficient of 17420, fat index 89.01. Protein is a hydrophilic protein. And the total number of negatively charged residues (Asp+Glu) was the same as the total number of positively charged residues (Arg+Lys) with 28. There was a helix (Hh) 22.89%, extending chain (Ee) 26.41%, ? fold (Tt) 16.2%, random coil (Cc) 34.51% and no signal peptide in its secondary structure. Provide a theoretical basis for building genetically engineered bacteria in future.