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

With the improvement of living standards,food safety issues are increasingly conce rned by people.Among them,the problem of residues pesticides in fruits and vegetable s is becoming more and more serious,which poses a huge threat to human health,in or der to solve this problem,through the microbial fermentation method to degrade pes tici des.In this paper,we mainly studied the screening of organophosphorus pesticide d egrading bacteria,the optimization of culture medium,the isolation and purification of organ ophosphorus pesticide degrading enzyme,then studied its enzymological properties and its gene.In order to reduce the amount of pesticide residues in fruits and vegetables,96-we 11 plates were screened from long-term spraying of pesticides in arable soils one can ef fectively degrade organophosphorus pesticides strain,Morphology analysis,physiological and biochemical properties and 26S rDNA sequences in GenBank indicated that the st rain belonged to Rhodotorula genus.Therefore,to determine the strain of Rhodotorula mucilaginosa and numbered RM-DY21.The content of organic phosphorus pesticide wa s determined by UV Spectrophotometry.The degradation rate of RM-DY21 was 70.04%for 50 mg/L chlorpyrifos in 32 h.Strains with chlorpyrifos as sole carbon source in t he medium grew better,followed by oxygen dimethoate,trichlorfon the lesser,It is wid ely described strain may degrade organophosphorus pesticides.To improve the degradation rate of Rhodotorula mucilaginosa RM-DY21 to organo phosphorus pesticides,The culture medium and culture conditions were optimized by si ngle factor and Box-Behnken analysis.The optimum culture medium is lactose 20.0 g/L,ammonium sulfate 20.0 g/L,The addition of Ca2+,Mg2+ and Zn2+ were 0.8,0.6,a nd 0.8 mg/20mL,respectively,yeast extract 10.0 g/L and analysis experimental with Bo x-Behnken central composite design principles of Design-expert 8.0 software.To determ ine the optimal culture condition is initial pH 5.0,fermentation temperature 30?,fer mentation time 36 h.In the optimized medium,organic phosphorus pesticide degradatio n rate of Rhodotorula mucilaginosa RM-DY21 can be increased to 83.86%and enhanc ed 10.62%.It will establish a good foundation for the enzyme production of strain in f uture.Experiments were conducted to determine the organophosphorus degrading enzyme produced by strain Rhodotorula mucilaginosa RM-DY21.And by using the method of s alting out and column chromatography on organic phosphorus degrading enzyme was p urified.The experimental results show that the organic phosphorus degrading enzyme R hodotorula mucilaginosa RM-DY21 strain generated by extracellular enzyme.By ammon ium sulfate fractionation,salting-degrading enzyme was obtained in the range of 60%?80%(w/v);Load in Sephadex G-100 column after dialysis and concentration,The enz ymatic activity was 350.63 U/mg.Load in DEAE Sepharose Fast Flow column after di alysis and concentration,Enzyme Activities of 693.94 U/mg was collected when elution with the buffer of 0.2-0.5 mol/L NaCl concentration.The molecular weight of the enz yme was determined about 40 kDa by SDS-PAGE denaturing gel electrophoresis.The characterization of enzyme reaction activity:The reaction temperature of organi c phosphorus degrading enzyme is 30?50 ?,the optimum reaction temperature is 40?;the best save temperature is at-20 ?,the reaction pH is 7?9,the optimum p H is 8.The optimum reaction time was 30 min,the optimum enzyme dosage was 20?L.Different metal ions have different effects on the activity of organophosphorus degr ading enzymes.The concentration of metal ions 0.2 mmol/L,Na+ and Zn2+ have strong activation effect on degrading enzymes,Ca2+,Mg2+ activation was weak,Li+,K+,Mn2?,Ag+ has a strong inhibitory effect on degrading enzymes,Cu2+,Fe3+ and Fe2+ had in hibitory effect on the enzyme degradation of small.Isopropyl alcohol and acetone could inhibit the activity of enzyme,Ethanol increases with increasing concentration.High c oncentrations of methanol have a slight increase in enzymatic activity.And the kinetic parameters Km = 24.213 ?mol L-1,Vmax = 18.553 ?mol(mg min)-1.According to the sequence of organic phosphorus degrading enzymes in GenBank,Analyzed and designed 2 pairs of specific primers by Vector NTI 10 software.The ge ne of organic phosphorus degrading enzymes in the experimental strain was amplified b y PCR and sequencing,and the amino acid sequences were analyzed by the software.Sequencing results shows,the sequence of the organophosphorus-degrading enzyme gene.of the experimental strain had a higher consistency with other strains.The opd gene co ntains 1155 nucleotides,encoding 384 amino acids,the protein molecular formula is C1830H2944N530O540S11 its molecular weight is 41363.39 Da,is hydrophobic and stable pr otein the isoelectric point is 9.05,the atoms total number is 5855,and has Signal pepti de,belongs to secreted proteinsecreted protein.There was a helix(Hh)38.54%,exten ding chain(Ee)19.53%,? fold(Tt)8.33%,random coil(Cc)33.59%in its second ary structure.The enzyme has a consistency of 99.39%with 2r11.1.A(organophosphoru s hydrolase)as a template for modeling,coverage can be 86%,the three level structure of OPD protein contains 2 copper atoms.Provide a theoretical basis for building gene tically engineered bacteria in future.