| The aim of this study is to explore the possibilities to gain the sensitive phyto-enzyme to organophosphate and carbamate pesticides in order to replace animal-origin acetycholinesterase (AChE). Therefore, a systemic study has been taken from the probe into the sensitive enzyme, which includes extraction, separation, purification and its properties, to establish the new methods for rapid detection the pesticide residues based on the soybean esterase.The main results are as follows:1. The optimal conditions and technical parameters of aqueous liquid extraction are as follows: the proportion of flour of soybean seed and water is 1:5, surge or mix round for 30 min, laying tranquilly under 4℃in refrigerator, and then, gained crude enzyme liquid by filtration and centrifugal. The extraction, separation and purification of soybean esterase by the optimizing experimentation based on the independency factor and multi-factors orthogonal design, the critical factors selected for the enzyme recovery ratio were pH value of PBS buffer, PBS mole intensity, and time in file, the optimum ranges of proportion of flour of soybean seed and PBS buffer, surge or mix round time, and laying time are 1:5, 7.0, 30min and laying overnight under the 4℃in refrigerator respectively, then to get the percolate by filtration using multilayer gauze. The supernatant fluid were separated from the percolate after centrifuging at 6000 rpm, 4000 rpm, 2000 rpm, 1000 rpm for 15-20 min in every stage from higher speed to lower speed ordinal. The incorporative supernatant fluid was salt out for the crude protease precipitation by slowly added ammonium sulfate at the concentration of 60%, then, dialysis and concentrated to gain the crude extraction liquid, followed by DEAE-32 cellulose column chromatographic fractionating to purification the crude enzyme to obtain the soybean esterase by monitoring the enzyme activities, collecting the enzyme activity phase, concentrated it or freeze-dry. The enzyme products should been storage under -20℃. The active unit, active unit per milligram protease, purification multiple and activity recovery of soybean esterase are 62.48 U/mL, 6.455 U/mg.pro, 90.92 times and 6.32% respectively.2. The characteristic of the soybean esterase are as follows: Km is 2.97μmoL/L. Adaptation temperature ranges of the enzyme catalysis reaction from 25℃to 40℃, and optimum temperature is 30℃. Adaptation pH ranges of the enzyme catalysis reaction between 7.0 and 8.0, and optimum pH is 7.5. The enzyme activities will losing around 50% under the temperature of 50℃-60℃for 1h, quarter activities will remain under this temperature for over 12h. The enzyme activities will lose quickly in the acidity condition below pH≤6. It will keep higher activities in the 0.2 moL/L-0.3 moL/L PBS buffer system, and the optimal hydronium intensity of PBS buffer is 2.5moL/L. The soybean esterase activity was less altered by freezer storage at -20℃, the relative activities will remain 87.4%, 64.8% and 42.6% respectively for about 60 d, 120 d and 180 d accordingly. The enzyme has a dimeric structure with molecular weights of 44.67KD and 69.18KD. The soybean esterase activity was assayed using a-Naphthyl acetate as substrate, fast blue B salt as color development reagent; the liquid colour should be measured by colorimetry in 10min.3. The rapid detection system for the organophosphate and carbamate pesticide residues based on the soybean esterase inhibition coupled with colorimetry has been established in this study. The evaluation test was showed that the soybean esterase represented well sensitivity to 24 organophosphate and carbamate pesticide except fenthion, and the limit of detection (LOD) low far from their maximal residue limit (MRL). The LOD ranges of 18 organophosphate pesticides were 0.03125 mg.kg-1 - 0.0625 mg.kg-1, and the LOD ranges of 6 carbemate pesticides were 0.03125 mg.kg-1 -0.25 mg.kg-1. Thereinto, the LOD of chlorpyrifos, dimethoate and methidathion can reach its 1/32 MRL. So, we can conclude that the methods established in this study can meet the twenty-four kinds of these pesticides demand of thier MRL compared with national specified. The recoveries of apple sample for five organophosphate pesticide residues were 78.5%-102.1% with C.V.≤5.1% and the recoveries of apple sample for three carbamate pesticide residues was 87.4%-94.0% with C.V.≤3.8%, it is completely in conformity to the results of gas chromatography determination.4. The rapid detection system for the organophosphate and carbamate pesticide residues based on the soybean esterase inhibition coupled with potentiometry has been established also in this study. The sensitivity of twenty kinds of organophosphate pesticides and eight kinds of carbemate pesticides were determined by this method. The result shows that the LOD of 19 organophosphate pesticides for the water sample were 0.000625 mg.kg-1- 0.0625 mg.kg-1, and the LOD of 8 carbemate pesticides for the water sample were 0.015 mg/kg - 0.3125 mg/kg. The recoveries of apple sample for 27 organophosphate pesticide residues were 82.5%-92.3% with CV≤5.22%. So, twenty-seven kinds of these pesticides (except fenthion) can meet the demand of its MRL compared with national specified. Over 83% in conformity to the results of gas chromatography determination by detection the apples and some vegetables from market.5. Nitrocellulose membrane (NC) was used to immobilized soybean esterase, the optimal conditions and technical parameters of the immobilized soybean esterase, the characteristics of the immobilized enzyme, and the efficiency of the immobilization was studied in this paper.Six batch totalling sixty times of this immobilized enzyme membrane has tested shows it has well recurs performance in use. The enzyme activity recoveries of the NC immobilized enzyme are 15.37%, over 60% activities will remained after seven times use, and its optimal pH rise 0.5 pH unit compare with the dissociative enzyme, the optimum temperature is 35℃, rise the 5℃compare with the dissociative enzyme, and its stability has improved obviously.Ultrastructure and the chemical structure of the NC membrane and NC immobilized enzyme membrane was expressed by Quanta 200 Scanning Electron Microscope(SEM) and Fourier Transform Infrared Spectrometer to investigate the mechanism of immobilization. It shows that soybean esterase can immobilized on NC membrane effectively, and it is more and even distributed on the membrane compare with the enzyme simplely loaded on it by SEM observation. Infrared spectra photograph of NC membrane, before and after immobilized soybean esterase, indicated that amino on NC membrane was covalent bond with amino acide residues of the enzyme through glutaraldehyde interlinking arm crosslinked. PBS buffer treated on NC membrane can enlarge the hole on its surface, meanwhile, this treatment can make the dissociative group exposure fully, therefore it will advantage enzymatic immobilization.The rapid detection method has been established using the NC immobilized enzyme membrane. The application test was carded to investigate capabilities to detect the pesticides. The result shows that the LOD of twenty nine organophosphate and carbemate pesticides for the water sample was 0.4 mg.kg-1-5 mg.kg-1 by eyeballing method, and the LOD of eight pesticides for the water sample was 0.00625 mg.kg-1- 0.0625 mg.kg-1 by colorimetry method. The recoveries of apple sample for 8 pesticide residues were 85.3%-91.8% with CV≤5.21%. The result shows that this method is conformity with gas chromatography determination.6. The possibilities of KGM membrane immobilized enzyme (soybean esterase) was explored in this research, the optimal conditions and technical parameters were as follows: 1% KGM sol to adjust the pH to 8.0 by Na2CO3 solution to improve its property, and adding 1% glycerin to refine its plasticity, soybean esterase was added finaly, and then form the sol into membrane. The result indicates that the KGM enzyme membrane has some good properties such as superthin, transparency, fiexility and good plasticity as well as well capabilities which achieve 50% enzyme activities recoveries. The activities of KGM immobilized soybean esterase nearly have no loss compare with the same dissociative enzyme under the same condition, which indicate the soybean esterase can express activity highly in KGM membrane.The mechanism of KGM immobilized soybean esterase was investigated through the ultrastructure observation and the chemical functional group changing analysis by Quanta 200 SEM and Fourier Transform Infrared Spectrometer. It shows soybean esterase was well embedded and well-proportioned in the KGM membrane. Infrared spectra photograph of KGM membrane, before and after immobilized soybean esterase, indicated that carbonyl on KGM membrane was boned with amino acide residues of the enzyme by hydrogen bond or conjugated effect. This method of immobilization was more warmly to enzyme, and very advantages to maintain the acticities of the immobilized enzyme.KGM membrane as novel membranous material to immobilize enzyme has a good potential to be applied in biosensor for detection pesticides because of its above good properties, and meanwhile, it can be a reference for a methodology of making other enzyme biosensor membrane.
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