Screening Of New Plant Esterase Sources And Application In The Pesticide Residues Detection

The rapid detection technology of pesticide residue by plant esterase is widely employed for its simplicity, rapidity, sensitivity, practicality and low cost, but the plant esterase used presently is extracted mostly from grain and crops . The total esterase activity and specific activity in 7 food crops and 6 pasture were detected, the results showed that the specific activity of alfalfa esterase is the maximum, so alfalfa was determined to be the appropriate source of plant esteras.In this thesis, we select alfalfa- the king of forages as the source of plant esterase, and design a series of single factor tests to study the effect factors such as the type of extraction, pH , time, temperature, the ratio of liquid to material, etc. The optimum extraction conditions were determined by orthogonal test, and the alfalfa esterase was also preliminarily purified . A rapid detection method of organophosphorus and carbamates pesticide residues in vegetables by alfalfa esterase was developed , The lowest detection limit of 5 organophosphorus and 2 carbamate pesticides in chinese cabbage was detemined.1.The plant esterase were extracted from rice, millet, coixseed, corn, oat, wheat flour, soybean, alfalfa, caragana sinica, guinea grass, shamrock, setaria viridis, calamagrostis angustifolia, and the total esterase activity and specific activity were detected for the plant esterase resources. The results showed that alfalfa esterase had the highest specific activity, which was 1.34 times as high as the soybean esterase, 1. 43 times as the wheat esterase. So the alfalfa is the best plant esterase resources.2.The influence factors involved the type of extracting agents, pH, temperature, time and the ratio of liquid to material, and the process is carried out according to L16(45)orthogonal table. The best extraction conditions of alfalfa was as follows: the fresh alfalfa is well grounded at 4℃with the ratio of liquid to material being 1:10, pH =7.0, in phosphate buffer solution (PBS). The solution was shocked for 10 min at 4℃, and then filtered with 4-layer gauze, centrifuged for 15 min with the speed of 4000⁶000r·min-1.3.The sensitivity experiments of alfalfa, wheat and soybean esterase were carried out to detect 5 organophosphorus pesticides (omethoate, monocroto-phos, phorate,methamidophos, methyl parathion) and 2 carbamates pesticides (carboiuran, methomyl) in cabbage.The results showed that the alfalfa esterase had the best sensitivity to the above 7 pesticides. When the concentration of pesticides was in 0.001μg·mL^-1 and 0.100μg·mL^-1, the inhibition rate was in linear relation with the logarithm of the pesticide concentration. The detection limit of the alfalfa esterase to omethoate, monocroto-phos, phorate, methamidophos, methyl parathion was 0.024, 0.050, 0.009, 0.003, 0.002, 0.013, 0.250μg·mL^-1 respectively. The recovery is as high as 90.2%¹18.3%.4. The crude extract of alfalfa esterase was purified by ammonium sulfate precipitation. The result showed that the specific activity of the purified plant esterase was 2.6 times as the crude esterase, the recovery of activity is 31.53% times as the crude testerase.5.A rapid detection method of organophosphorus and carbamates pesticide residues in vegetables by alfalfa esterase was developed. The organophosphorus and carbamates pesticide residues in vegetables(tomato, cucumber, celery, leeks, cabbage, brassica, eggplant, pepper, boy choy and cut green beans) sown in summer earth and in winter plastic tents were deteted. The result showed that the vegetables sown in plastic tents had the highest pesticide residue beyond the standard value. 6. The characteristics of enzymology was studied.The loss of enzyme activity is little when theoptimum temperature is 40℃,pH 7.0; the thermal stability is not satisfactory at the condition of alkalescence, and it is practical to preserve the plant esterase at the low temperature of -20℃. At 4℃for 120 days, only 30% of activity remains; while at -20℃for 360 days, nearly 36% of activity still remains, so it is wise to preserve the alfalfa plant esterase at low temperature for a long period without the lose of enzyme activity. It is edible after defrosting, but it is not wisely to frost and defrost repeatedly.