Degradation Characteristics Of Crude Enzyme And Immobilized Enzyme To Insecticides Chlorpyrifos

In this dissertation, taking chlorpyrifos as a model pollutant, its GCanalytical method, the extract of its degrading enzyme, condition ofimmobilized enzyme and degradation characteristics of its crude enzyme andimmobilized enzyme, were deeply studied. And application of chlorpyrifosdegrading enzyme (WZ-Ⅰ) to degrade chlorpyrifos residues on the surface ofvegetable was studied systemically. The results could be summarized asfollows:1. The situation of application, pollution and mobility in environment,ecological toxicological characteristics and pollutant management ofchlorpyrifos was introduced and it has been applied extensively all over theworld and recently in china, accordingly author presents the issues that wouldbe investigated.2. The extraction and analytical methods of chlorpyrifos in water and cabbagesamples were set up. Chlorpyrifos residues in water samples were extractedwith petroleum ether; for extraction of chlorpyrifos residues from vegetablesamples, the samples were transferred to 250 mL conical flask with 60mL ofacetone and shaken for 30min on an orbital shaker, the extract was cleaned upby liquid-liquid separation and then analyzed by gas chromatography.Chlorpyrifos was detected by GC–14C gas chromatography equipped with aflame Photometric detector (FPD) and a capillary column (30m×0.53mm i.d)packed with 100%Dimethyl polysiloxane OV-101. The operating conditionswere described below: flow rate of Carrier gas (99.99% nitrogen), hydrogen,and air were 50ml·min-1, 60ml·min-1and 60 ml·min-1, respectively. Temperatureof column, injector and detector were 230,260 and 260℃, respectively. Underthese conditions , the retention times for Chlorpyrifos was 1.76min, and limitof detection was 2.0×10-12g, and the linearity range is 5.0×10-11～10-8g. Therecovery rates from water with concentration of 0.01128, 0.1128, 1.128,5.0mg·L-1 were 102.25±3.21,　104.44±3.16,　98.05±1.30　and　104.21±1.43,　respectively, and from cabbage samples with concentration of0.01,0.03,0.05,0.5,and 5.0 mg·kg-1were 83.21±2.00,　80.81±3.56,　86.68±2.47,　87.68±1.15,　86.78±1.63, respectively, The results showed thatthis method could meet the requirement for analysis of chlorpyrifos residues.Chlorpyrifos in buffer was determined by Ultra-violet spectrophotometry.3. Degradation characteristics of the crude enzyme, which was extracted fromthe isolated strain WZ-Ⅰ(Fusarium LK.ex Fx), to degrade chlorpyrifosinsecticides were researched. The objective of this study was to initiallyascertain the best separating conditions and the degrading characteristics ofchlorpyrifos. The content of this study included the form and orientation of thedegrading enzyme for chlorpyrifos; rate of degradation for chlorpyrifos by itsintracellular enzyme, extracellular enzyme and cell fragment was 60.8%,11.3% and 48%, respectively. The enzymic activity of the degrading enzymeextracted from the fungus incubated for 8 generations in the condition ofnon-inducement lost less, the results showed that this enzyme was anintracellular and connatural enzyme. The solubility protein of the crudeenzyme was 3.36mg·mL-1 which was determined with Albumin (bovine scrum)as standard protein. The pH optimum for crude enzyme was 6.8 for enzymaticdegradation of chlorpyrifos,It had comparatively high activity in the rage ofpH 6.0～9.0. And the optimum temperature for enzymatic activity was at 40℃.It still had comparatively high activity in the rage of temperature 20～50℃,but the activity of enzyme rapidly reduced at 55℃, only 41% of the maximalactivity. The crude enzyme showed Km value for chlorpyrifos of1.04926mmol·L-1, and the maximal enzymatic degradation rate was0.2535μmol·(mg·min)-1. Additional experimental evidence suggests that thecrude enzyme of fungus WZ-Ⅰhad good stability and endurance fortemperature and pH which could effectively degrade chlorpyrifos.4. Fungus WZ-Ⅱwas incubated in the induced and non-induced conditionsrespectively, from which degrading enzyme of chlorpyrifos was extracted. Theresults showed that this crude enzyme was induced one. By the orientatingexperiment of enzyme, it indicated that this enzyme was intracellular one. Thesolubility protein of the crude enzyme was 1.99mg·mL-1. The crude enzymeshowed Km value for chlorpyrifos of 0.691mmol·L-1, and the maximalenzymatic degradation rate of 0.2777μmol·(mg·min)-1. The pH optimum forcrude enzyme was 7.0 for enzymatic degradation of chlorpyrifos,It hadcomparatively high activity in the rage of pH 6.8～7.2. And the optimumtemperature for enzymatic activity was at 25℃. Additional experimentalevidence suggests that the enzyme had the relatively stronger sensitivity fortemperature and pHvalue.5. The immobilized methods of free enzyme were studied. The free enzyme,extracted from the strain identified as Fusarium LK.ex Fx (fungus WZ-Ⅰ),which could degrade chlorpyrifos effectively, was immobilized with thesodium alginate and calcium chloride. The results indicated that the freeenzyme was immobilized for 4h under conditions of 30g·L-1 sodium alginateand 3% protein at the 4℃.6. Degradation characteristic of the immobilized enzyme and the free enzymeto chlorpyrifos were compared. The immobilized enzyme showed maximalactivity at pH 8.0 and also comparatively high activity in the range of pH6.5～9.0., the optimum temperature for enzyme activity was at 45℃, besidesin the range of temperature 40～55℃, degradation activity to chlorpyrifos wasabove 80%. The immobilized enzyme showed Km and Vmax value forchlorpyrifos of 0.1672mmol·L-1 and 50.246nmol·min-1, respectively, less thanthe free enzyme. Additional experimental evidence suggests that theimmobilized enzyme showed good stability and endurance for temperature and...