有机磷及氨基甲酸酯类农药残留检测用豇豆酯酶的初步研究
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摘要
植物酯酶由于其来源丰富、价格低廉、检测灵敏性好等优点,在现代农药残留检测中有着良好的应用前景。本试验基于酶抑制原理,以常用植物酯酶酶源小麦为对照,以比活力和农药敏感性为测定指标,对10种植物酶源进行筛选。并对筛选出的适用于农药残留检测用的豇豆酯酶的提取、分离、固定化条件、酶学性质以及对11种有机磷及氨基甲酸酯类农药敏感性进行了研究。其主要研究结果如下:
1.在小麦(Triticum aestivuml.L)、杜仲(Eucommia ulmoides Oliv.)、刺槐(Robinia pseudacacia L.)、国槐(Sophora japonica L.)、腊梅(Chimonanthus praecox (L.))、洒金柏( Cv.aurea.nana )、侧柏( Platycladus orientalis (L.) Franco )、黄豆( Giycine max(L)Merrill)、绿豆(Vigna radiata)、菜豆(Phaseolus vulgaris Linn.)、豇豆(Vigna unguiculata (Linn.))和家蝇等12种材料中,豇豆酯酶比活力极显著高于其它酯酶,豇豆酯酶比活力是小麦的4.71倍,是敏感性家蝇乙酰胆碱酯酶的3.84倍。对敌敌畏、辛硫磷、敌百虫、速灭威、克百威和灭幼脲等6种有机磷及氨基甲酸酯类农药的敏感性测定显示:除小麦酯酶对辛硫磷和灭幼脲的灵敏度略高于豇豆外,豇豆酯酶对其他4种农药的灵敏度明显优于小麦,其对敌百虫灵敏度最好,达33.625,对敌敌畏等3种农药的灵敏度约是小麦酯酶的3-7倍。
2.豇豆酯酶最佳提取条件为:豇豆种子粉碎后过100目筛,按照料液比1:5加入0.3 mol/L pH 7.5的磷酸盐缓冲液,于4℃条件下浸提14h,在10000r/min冷冻离心机下离心15min,上清液即为粗酶液。
3.采用Box-Behnken设计法对其酶促反应条件进行优化,结果显示,酶液稀释1977倍后,在0.324mol/L pH 7.57的磷酸缓冲液中,30℃时酯酶的催化活性最高;三个因素对豇豆酯酶活力影响程度为稀释倍数>pH值>缓冲液摩尔浓度。
4.确定了两次硫酸铵盐析饱和度分别为50%和55%,豇豆酯酶经两次硫酸铵盐析,透析浓缩后上RESOURCE Q离子交换柱进行交换层析后的比活力比粗酶液比活力明显提高。
5.豇豆酯酶在20℃-40℃时热稳定性较好,20℃保温60min,酶残留活力为95.69%;30℃保温60min,残留活力为92.21%;40℃保温60min,残留活力为83.65%。豇豆酯酶米氏常数Km为1.303μmol/L。
6.豇豆酯酶对11种有机磷及氨基甲酸酯类农药敏感性测定结果显示:豇豆酯酶除对久效磷外,对其他10种农药的灵敏性都较好,最低检测限均低于其MRL值。通过纯化前后的豇豆酯酶对6种农药灵敏性测定结果表明,纯化前后的酯酶对农药的灵敏性无显著差异。
7.豇豆酯酶固定化的酶用量、固定化温度及固定化时间分别以5U/mL的酶液8mL、4℃和12h为最佳。
Because of its rich resources, low price, good detecting sensitivity and other merits, phytoesterases has a wider applicable prospect in detecting residues of modern pesticides. Based on Enzyme Inhibition Principle and compared with Triticum aestivuml.L, a commonly- used phytoesterases resource, by the detecting index of specific activity and pesticide sensitivity, ten kinds of phytoesterases resources are screened.Then further researches are made on the terms of extraction, separation, immobilization and the enzymatic properties of Vigna unguiculata (Linn.) esterase suitable for detecting pesticides residues.The major results are as follows:
I. Among twelve materials like Eucommia ulmoides Oliv.、Robinia pseudacacia L.、Sophora japonica L.、Chimonanthus praecox (L.)、Cv.aurea.nana、Platycladus orientalis (L.) Franco、Giycine max(L)Merrill、Vigna radiata (Linn.) Wilczek、Phaseolus vulgaris Linn.、Vigna unguiculata (Linn.)、Triticum aestivuml.L and Housefly.the specific activity of Vigna unguiculata (Linn.)is evidently higher than that of other esterases.The specific activity of Vigna unguiculata (Linn.) esterase is 4.71 times of Triticum aestivuml.L and 3.84 times of Acetylcholinesterase from sensitive housefly.Through detecting its sensitivity to six kinds of Organophosphorus and Carbamate pesticides like Dichlorvos, Phoxim, Trichlorfon, Metolcarb, Carbofuran and Mieyouniao, the result shows that Vigna unguiculata (Linn.) esterase’s sensi- vity to the rest four pesticides are evidently better than Triticum aestivuml.L’s .except that Triticum aestivuml.L esterases’s sensivity to Phoxim and Mieyouniao are a little higher than Vigna unguiculata(Linn.) esterases’s.The best sensitivity is to Trichlorfon, reaching 33.625, Its sensivity to the rest 3 pesticides like Dichlorvos is about 3 to 7 times of Triticum aestivuml. L’s.
II. The best extracting conditions of Vigna unguiculata(Linn.) esterase are:Screen the grinded seeds with 100 mesh sieve, then add 0.3 mol/L pH 7.5 of Phosphate Buffer according to the solid-liquid ratio,1:5,and extract for 14h at 4℃. Centrifuge it for 15 min at the rate 10000r/min with refrigerated centrifuge,the supernatant is crude enzyme.
III. Optimize the conditions of enzymatic reaction with Box-Behnken designing-method, the result shows that after diluting the enzyme 1977 times, the catalytic activity of esterase is the highest at 30℃in the 0.324mol/L pH 7.57 Phosphate Buffer.The affecting degree of the three factors to esterase activity of Vigna unguiculata (Linn. )is as follows:diluting times>PH value>molar concentration of buffer.
IV. The saturation degree of two ammonium sulfate salting-outs are 50% and 55% respectively.After two ammonium sulfate salting-outs, dialysis and concentration of Vigna unguiculata (Linn.) , put it in the RESOURCE Q ion exchange column to Chromatograph.The specific activity of purified esterase is evidently improved than that of crude enzyme.
V. The thermal stability of Vigna unguiculata (Linn.) esterase is better at 20℃-40℃。Keeping at 20℃for 60min, the remaining activity of esterase is 95.69%, Keeping at 30℃for 60min, the remaining activity is 92.21%,Keep at 40℃for 60min,the remaining activity is 83.65%.Michaelis constant Km of Vigna unguiculata(Linn.) esterase is 1.303umol/L.
VI. The detecting results on the sensitivity of Vigna unguiculata(Linn.) esterase to eleven kinds of Organophosphorus and Carbamate pesticides show that its sensitivity to the rest 10 kinds of pesticides is better except for Monocrotophos.The limits of detection of the ten pesticides are lower than their respective MRL value, Through the detecting results of its sensitivity to 6 kinds of pesticides before and after purification, it shows that Vigna unguiculata(Linn.)’s sensivitiy to pesticides is of no evident difference before and after purification.
VII. The amount of enzyme, temperature and time for immobilizing Vigna unguiculata (Linn.) Esterase is best at the condition of 8ml of 5U/mL enzyme、4℃and 12h.
1.在小麦(Triticum aestivuml.L)、杜仲(Eucommia ulmoides Oliv.)、刺槐(Robinia pseudacacia L.)、国槐(Sophora japonica L.)、腊梅(Chimonanthus praecox (L.))、洒金柏( Cv.aurea.nana )、侧柏( Platycladus orientalis (L.) Franco )、黄豆( Giycine max(L)Merrill)、绿豆(Vigna radiata)、菜豆(Phaseolus vulgaris Linn.)、豇豆(Vigna unguiculata (Linn.))和家蝇等12种材料中,豇豆酯酶比活力极显著高于其它酯酶,豇豆酯酶比活力是小麦的4.71倍,是敏感性家蝇乙酰胆碱酯酶的3.84倍。对敌敌畏、辛硫磷、敌百虫、速灭威、克百威和灭幼脲等6种有机磷及氨基甲酸酯类农药的敏感性测定显示:除小麦酯酶对辛硫磷和灭幼脲的灵敏度略高于豇豆外,豇豆酯酶对其他4种农药的灵敏度明显优于小麦,其对敌百虫灵敏度最好,达33.625,对敌敌畏等3种农药的灵敏度约是小麦酯酶的3-7倍。
2.豇豆酯酶最佳提取条件为:豇豆种子粉碎后过100目筛,按照料液比1:5加入0.3 mol/L pH 7.5的磷酸盐缓冲液,于4℃条件下浸提14h,在10000r/min冷冻离心机下离心15min,上清液即为粗酶液。
3.采用Box-Behnken设计法对其酶促反应条件进行优化,结果显示,酶液稀释1977倍后,在0.324mol/L pH 7.57的磷酸缓冲液中,30℃时酯酶的催化活性最高;三个因素对豇豆酯酶活力影响程度为稀释倍数>pH值>缓冲液摩尔浓度。
4.确定了两次硫酸铵盐析饱和度分别为50%和55%,豇豆酯酶经两次硫酸铵盐析,透析浓缩后上RESOURCE Q离子交换柱进行交换层析后的比活力比粗酶液比活力明显提高。
5.豇豆酯酶在20℃-40℃时热稳定性较好,20℃保温60min,酶残留活力为95.69%;30℃保温60min,残留活力为92.21%;40℃保温60min,残留活力为83.65%。豇豆酯酶米氏常数Km为1.303μmol/L。
6.豇豆酯酶对11种有机磷及氨基甲酸酯类农药敏感性测定结果显示:豇豆酯酶除对久效磷外,对其他10种农药的灵敏性都较好,最低检测限均低于其MRL值。通过纯化前后的豇豆酯酶对6种农药灵敏性测定结果表明,纯化前后的酯酶对农药的灵敏性无显著差异。
7.豇豆酯酶固定化的酶用量、固定化温度及固定化时间分别以5U/mL的酶液8mL、4℃和12h为最佳。
Because of its rich resources, low price, good detecting sensitivity and other merits, phytoesterases has a wider applicable prospect in detecting residues of modern pesticides. Based on Enzyme Inhibition Principle and compared with Triticum aestivuml.L, a commonly- used phytoesterases resource, by the detecting index of specific activity and pesticide sensitivity, ten kinds of phytoesterases resources are screened.Then further researches are made on the terms of extraction, separation, immobilization and the enzymatic properties of Vigna unguiculata (Linn.) esterase suitable for detecting pesticides residues.The major results are as follows:
I. Among twelve materials like Eucommia ulmoides Oliv.、Robinia pseudacacia L.、Sophora japonica L.、Chimonanthus praecox (L.)、Cv.aurea.nana、Platycladus orientalis (L.) Franco、Giycine max(L)Merrill、Vigna radiata (Linn.) Wilczek、Phaseolus vulgaris Linn.、Vigna unguiculata (Linn.)、Triticum aestivuml.L and Housefly.the specific activity of Vigna unguiculata (Linn.)is evidently higher than that of other esterases.The specific activity of Vigna unguiculata (Linn.) esterase is 4.71 times of Triticum aestivuml.L and 3.84 times of Acetylcholinesterase from sensitive housefly.Through detecting its sensitivity to six kinds of Organophosphorus and Carbamate pesticides like Dichlorvos, Phoxim, Trichlorfon, Metolcarb, Carbofuran and Mieyouniao, the result shows that Vigna unguiculata (Linn.) esterase’s sensi- vity to the rest four pesticides are evidently better than Triticum aestivuml.L’s .except that Triticum aestivuml.L esterases’s sensivity to Phoxim and Mieyouniao are a little higher than Vigna unguiculata(Linn.) esterases’s.The best sensitivity is to Trichlorfon, reaching 33.625, Its sensivity to the rest 3 pesticides like Dichlorvos is about 3 to 7 times of Triticum aestivuml. L’s.
II. The best extracting conditions of Vigna unguiculata(Linn.) esterase are:Screen the grinded seeds with 100 mesh sieve, then add 0.3 mol/L pH 7.5 of Phosphate Buffer according to the solid-liquid ratio,1:5,and extract for 14h at 4℃. Centrifuge it for 15 min at the rate 10000r/min with refrigerated centrifuge,the supernatant is crude enzyme.
III. Optimize the conditions of enzymatic reaction with Box-Behnken designing-method, the result shows that after diluting the enzyme 1977 times, the catalytic activity of esterase is the highest at 30℃in the 0.324mol/L pH 7.57 Phosphate Buffer.The affecting degree of the three factors to esterase activity of Vigna unguiculata (Linn. )is as follows:diluting times>PH value>molar concentration of buffer.
IV. The saturation degree of two ammonium sulfate salting-outs are 50% and 55% respectively.After two ammonium sulfate salting-outs, dialysis and concentration of Vigna unguiculata (Linn.) , put it in the RESOURCE Q ion exchange column to Chromatograph.The specific activity of purified esterase is evidently improved than that of crude enzyme.
V. The thermal stability of Vigna unguiculata (Linn.) esterase is better at 20℃-40℃。Keeping at 20℃for 60min, the remaining activity of esterase is 95.69%, Keeping at 30℃for 60min, the remaining activity is 92.21%,Keep at 40℃for 60min,the remaining activity is 83.65%.Michaelis constant Km of Vigna unguiculata(Linn.) esterase is 1.303umol/L.
VI. The detecting results on the sensitivity of Vigna unguiculata(Linn.) esterase to eleven kinds of Organophosphorus and Carbamate pesticides show that its sensitivity to the rest 10 kinds of pesticides is better except for Monocrotophos.The limits of detection of the ten pesticides are lower than their respective MRL value, Through the detecting results of its sensitivity to 6 kinds of pesticides before and after purification, it shows that Vigna unguiculata(Linn.)’s sensivitiy to pesticides is of no evident difference before and after purification.
VII. The amount of enzyme, temperature and time for immobilizing Vigna unguiculata (Linn.) Esterase is best at the condition of 8ml of 5U/mL enzyme、4℃and 12h.
引文
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