Preliminary Research On Mungbean Esterase Used For Detecting Organophosphorus Pesticides Residue

Abstract: In this paper, mungbean was chosen as the resource of phytoesterase and astudy has been taken into mungbean esterase, which includes extraction, separation,purification and charaterization. The chromogenic reaction system’s conditions ofmungbean esterase hydrolyzes1-naphthyl acetate were also optimized. The methodof rapid detection of organophosphorous pesticides residue based on mungbeanesterase was established.The main results of this paper are as follows:(1) The optimal conditions and technical parameters of extractction are asfollows: mungbean germinated for48h, peeled and crushed under low temperature,add0.2mol/L pH7.1phosphate buffer solution, the proportion of mungbean andphosphate buffer solution is1:5(w/v),30℃water bath surging for4h, then to gainthe percolate by filtration using multilayer gauze, centrifuge the percolate at8000rpm under4℃for5min, gain the supernatant fluid, crude enzyme solution is got.(2) Mungbean esterase’s separation and purification: the crude enzyme solutionwas salt out by ammonium sulphate at the concentration of60%, dialysis andconcentrated to gain the enzyme, then followed by Sephadex G-100gel filtrationchromatography and collect the enzyme activity phase. After the steps of purification,the active unit per milliliter, active unit per milligram protease, purification multipleand activity recovery of mungbean esterase are0.964U/mL,0.410U/mg,3.90timesand13.5%respectively. Evaluated the mungbean esterase with SDS-polyacrylamidegel electrophoresis, the molecular weight of active component is between30kD and60kD.(3) Preliminary study on the enzymatic properties of mung bean esterase: theenzymatic reaction’s optimum temperature is40℃, optimum pH is6.7, the suitableconcentration of phosphate buffer solution is0.15mol/L. Mungbean esterase hasgood thermal stability when the temperature belows40℃,the enzyme activity almostlost when the temperature is70℃.Cryopreservation is conducive to preserve themungbean esterase’s activity. The freeze-dried powder of mungbean esterase stored at-18℃, the relative activities will remain82.7%,75%,69%respectively for about4weeks,8weeks and12weeks accordingly.(4) The chromogenic reaction system’s conditions of mungbean esterasehydrolyzes1-naphthyl acetate were optimized as follows: in the0.15mol/L pH6.7phosphate buffer solution’s reaction system, study the effect of0.057U/mLmungbean esterase’s addition, fast blue B color reagent’s addition and color time onthe chromogenic reaction system when the reaction temperatue is40℃. The resultsindicate that the1mL0.057U/L mungbean esterase,1mL0.25mg/mL fast blue Bcolor reagent and10min for color are suitable.(5) The rapid detection system for the organophosphorus pesticides residuebased on the mungbean esterase inhibition was established. The system’s compositionand specific operation are as follows:10.0mL test tube with stopper, add0.15mol/LpH6.7phosphate buffer solution2.0mL,0.057U/mL mungbean esterase solution1.0mL and test sample1.0mL,40℃water bath for3min, then add1mg/mL1-naphthyl acetate1.0mL and0.25mg/mL fast blue B color reagent1.0mL,40℃water bath for10min，adding5mol/L acetic acid2.0mL to terminate the reaction andvolume to10.0mL with distilled water, then measure the reaction solution’sabsorbance at520nm. Enzyme activity was measured with distilled water instead oftest sample according to the above conditions.(6) Six kinds of organophosphorus pesticides were detected by rapid detectionsystem which based on mungbean esterase inhibition method. The results show thatmungbean esterase represented highest sensitivity to trichlorfon, but the sensitivity tophoxim is relative poor. The minmum limit of detection of the six organophosphoruspesticides are all below the maximum residue limit value of national standard.Ultrasonic method rapidly extracting the cabbage which was added withorganophosphorus pesticides for10min, then use mungbean esterase inhibitionmethod to assay the six kinds of organophosphorus pesticides, the spiked recoveriesare between80%and95%, the results show good.The main innovations of this dissertation are listed as follows:(1) Extract and separate phytoesterase from mungbean, the mungbean esterasecan be used for detecting organophosphorus pesticides residue. (2) Based on the reseach of mungbean esterase’s enzymatic properties andreaction conditions, a rapid method for detecting organophosphorus pesticides residuewas established.