大豆酯酶同工酶的分离纯化及其对有机磷和氨基甲酸酯类农药敏感性的研究
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摘要
自有机氯农药禁止使用后,作为替代品的有机磷、氨基甲酸酯类农药被广泛使用。但是由于我国制药技术落后和施药技术不规范,导致这两类农药中的高毒化学农药大量使用,不仅破坏农业生态环境,制约农业经济发展,同时因食用农药污染的食品所引起的食物中毒事件频频发生,带来了严重的食品安全问题。因此,急需研究和开发一些适合我国农产品产销特点的简便、快速、可靠、灵敏、实用的农药残留分析检测技术,将高农药残留的农产品杜绝于市场之外。
酶抑制法具有需时短,成本低,技术要求不高等优点,易于在农产品生产基地和批发市场推广,适于现场快速检测。目前市场上的速测卡、检测箱、pH测量、传感器法及酶催化动力学光度法等都是基于酶抑制所建立起来的方法。植物酯酶酶原丰富,取材方便,对农药的敏感性与动物乙酰胆碱酯酶相当,因而近年来备受关注。本研究选用本实验室筛选的大豆酯酶作为农药快速检测用生物识别元件,对大豆中酯酶同工酶进行了分离纯化,制得纯的大豆酯酶同工酶制品,并分别研究了各种大豆酯酶同工酶的酶学特性及对有机磷和氨基甲酸酯类农药的敏感性。研究内容及结果如下：
(1)确定了大豆酯酶同工酶的分离纯化工艺路线。即：大豆种子粉碎后,按1：5料液比加0.3mol／L,pH 7.0的磷酸盐缓冲液,搅拌30min,4℃冰箱中浸提过夜,2-3层纱布过滤后,上清液依次进行1000r／min、2000r／min、4000r／min、6000r／min的差速冷冻离心,合并有酶活的部分,用60%硫酸铵盐析1h,沉淀溶解并透析,真空冷冻干燥制成酶粉,然后依次进行纤维素DEAE-32离子交换层析和葡聚糖Sephadex G-100凝胶过滤层析,分别收集酶活力峰,最终得到三种纯的大豆酯酶同工酶(Soybean Esterase isozyme, SEI)制品,分别记为SEI1,SEI2和SEI3。
(2)对三种大豆酯酶同工酶的理化性质和动力学特性分别进行了研究。研究表明：SEI1的亚基相对分子量为40.7KDa；SEI2含有两种亚基,相对分子量分别为37.2KDa和21.4KDa；SEI3的亚基相对分子量为74.1KDa。三种酯酶催化底物α-乙酸奈酯的米氏常数Km分别为22.7μmol／L,58.6μmol／L和13.4μmol／L,最适温度分别为30℃,30℃和25℃,最适pH分别为6.5,6.0和6.5。三种大豆酯酶同工酶对α-乙酸萘酯和β-乙酸萘酯有催化能力,对乙酰胆碱和有机磷酸酯类化合物甲基对硫磷无催化能力。
(3)考察了三种大豆酯酶同工酶的热稳定性、酸碱稳定性和保存稳定性,并确定了液态大豆酯酶同工酶的有效稳定保护剂。结果表明：SEI2的热稳定性好,SEI1和SEI3较差；三种酶的pH适应范围均较广,且碱性条件下相对稳定；三种酶在4℃的保存效果比—20℃好,且BSA、蔗糖和甘油可以有效地保护酶的稳定性,保存效果BSA>蔗糖>甘油。
(4)研究了常见有机溶剂、金属离子、螯合剂及表面活性剂对三种大豆酯酶同工酶的影响。结果表明：乙腈、甲醇、丙酮、二氯甲烷、乙酸乙酯、苯、正己烷等对酶的活性影响很大,且极性溶剂的影响大于非极性溶剂；金属离子除CaCl2外对三种酯酶均有很大的影响；螯合剂EDTA和表面活性剂SDS对三种酯酶均有抑制作用。
(5)改进了分光光度法检测有机磷和氨基甲酸酯类农药的检测体系。即：1.45 mL的磷酸缓冲液中依次加入0.5 mL酶液和0.5 mL的农药,混匀后在30℃水浴中反应10 min,取出试管,加入50μLα-乙酸萘酯丙酮溶液,混匀,在30℃水浴中反应15 min,再加入0.5mL固兰B盐溶液,混匀放入30℃水浴中反应10min,然后在595 nm波长处测定吸光度,用缓冲液代替酶和农药作空白,调零。
(6)进行了三种酯酶同工酶对有机磷和氨基甲酸酯类农药的敏感性试验,结果表明：SEI2对有机磷和氨基甲酸酯类农药的敏感性最好,SEI3其次,而SEI1无敏感性。故而选择SEI2作为快速检测有机磷和氨基甲酸酯类农药的生物识别元件。
Since Organochlorine pesticide was forbidden, Organophosphate and Carbamate Pesticides as succedaneums have been used widely. However, being backward in making technique and being back of the specification in using pesticides led them abused, which not only destroyed agricultrural ecology environment and restricted agricultrural economic development, but also brought serious problems of food safety. At present, events about food poisoning for eating food polluted by pesticides had happened repeatedly. So, it is badly in need to study and develop a rapid detection technology for pesicides which is simple, convenient, fast, reliable, sensitive and practical. This technology is suitable for our country's producing and selling characteristics and can stop the agricultural products containing high pesiticides residues outside of the market.
Due to its less detection time, low cost and low technology demanding, Enzyme-inhibition methods are suitable for on-site rapid detection for pesiticids and can be spreaded in producing base and wholesale market of agricultural products.At present, the detection methods in market almost are on the base of Enzyme-inhibition principle. Due to its similar sensitivity to cholinesterase, easier gaining and abundant source, phytoesterase was paid close attention in the past few years. In this study, esterase from Soybean seeds was selected for a biomarker for rapid detection of pesticide residues. Then the separation and purification, the characteristics, the sensitivities to Organophosphate and Carbamate Pesticides of different Soybean Esterase isozymes were studied explicitly in this paper.
The results are as follows:
1. The technical process on purification of phytoesterase from soybeans is as follows:soybean flour was surged for 30 min with 5 times phosphate buffer(0.3mol/L, pH 7.0), then, put it at 4℃for more than 12 hours and filtrate by 2～3 layers gauze. Next, centrifugalize the filtrate at 1000r/min to 6000r/min by differential centrifugation. The supernatant fluid was collected, selt-out with 60% ammonium sulfate saturation 1h and dialysed. The active enzyme was further purified by DEAE-32 ion exchange column chromatography and Sephadex G-100 gel filtration column chromatography, with collecting the enzyme active parts. At last, Three Soybean Esterase isozymes (SEIs)of electrophoresis purity were obtained, marked with SEI1, SEI2 and SEI3.
2. The physico-chemical and kinetics characteristics were separately studied of three SEIs. It demonstrated that the molecular weight of SEI1 is 40.7 KDa, SEI2 has two kinds of subunits with the molecular weight 37.2 KDa and 21.4 KDa, and the molecular weight of SEI3 is 74.1 KDa.The Km of SEI1 is 22.7μmol/L, with the optimal temperature 30℃and pH 6.5.The Km of SEI2 is 58.6μmol/L, with the optimal temperature 30℃and pH 6.0.The Km of SEI3 is 13.4μmol/L, with the optimal temperature 25℃and pH 6.5. They all have catalyzine ability to a-Naphthyl acetate and P-Naphthyl acetate, and have no catalyzine ability to AChE and methyl paraoxon.
3. The storage stability, temperature stability, acid alkali stability and storage condition of Soybean Esterase for pesticide residues rapid detection were separately studied in this paper. It indicated that SEI2 is better heat-resisting than SEI1 and SEI3. They all have high tolerance to wide pH, especially stabe in the condition of alkalinity. They can be better preserved at 4℃, and in BSA, saccharose and glycerol.
4. The effects of organic solvents, metal ions, chelators and surfactant on three SEIs were studied in this papar. It indicated that almost all of them can active or inactive the enzyme actitivity.
5. The detection system of sepectrophotometric method for pesticides has been improved in this paper.1.450mL phosphate buffer,0.5mL enzyme liquid and 0.5mL pesticides are mixed and incubate for 10 min at 30℃. Subsequently, put in 50μL a-Naphthyl acetate as a substrate, and incubate at 30℃for 15min. Next, it is mixed with 0.5mL fast blue B salt. React at 30℃for 10min. Finally, the absorbance was measureed at 595nm by spectrophotometer, and phosphate buffer subtitutes the enzyme and pesicides as control test.
6. The sensitivities to Organophosphate and Carbamate Pesticides of three isozmes were tested. The result indicated that the sensitivitives to pesticides of SEI2 was better than SEI3, and SEI1 did not sense to the pesticides. Thus, SEI2 can be selected as the enzyme to detect pesticide residues.
酶抑制法具有需时短,成本低,技术要求不高等优点,易于在农产品生产基地和批发市场推广,适于现场快速检测。目前市场上的速测卡、检测箱、pH测量、传感器法及酶催化动力学光度法等都是基于酶抑制所建立起来的方法。植物酯酶酶原丰富,取材方便,对农药的敏感性与动物乙酰胆碱酯酶相当,因而近年来备受关注。本研究选用本实验室筛选的大豆酯酶作为农药快速检测用生物识别元件,对大豆中酯酶同工酶进行了分离纯化,制得纯的大豆酯酶同工酶制品,并分别研究了各种大豆酯酶同工酶的酶学特性及对有机磷和氨基甲酸酯类农药的敏感性。研究内容及结果如下：
(1)确定了大豆酯酶同工酶的分离纯化工艺路线。即：大豆种子粉碎后,按1：5料液比加0.3mol／L,pH 7.0的磷酸盐缓冲液,搅拌30min,4℃冰箱中浸提过夜,2-3层纱布过滤后,上清液依次进行1000r／min、2000r／min、4000r／min、6000r／min的差速冷冻离心,合并有酶活的部分,用60%硫酸铵盐析1h,沉淀溶解并透析,真空冷冻干燥制成酶粉,然后依次进行纤维素DEAE-32离子交换层析和葡聚糖Sephadex G-100凝胶过滤层析,分别收集酶活力峰,最终得到三种纯的大豆酯酶同工酶(Soybean Esterase isozyme, SEI)制品,分别记为SEI1,SEI2和SEI3。
(2)对三种大豆酯酶同工酶的理化性质和动力学特性分别进行了研究。研究表明：SEI1的亚基相对分子量为40.7KDa；SEI2含有两种亚基,相对分子量分别为37.2KDa和21.4KDa；SEI3的亚基相对分子量为74.1KDa。三种酯酶催化底物α-乙酸奈酯的米氏常数Km分别为22.7μmol／L,58.6μmol／L和13.4μmol／L,最适温度分别为30℃,30℃和25℃,最适pH分别为6.5,6.0和6.5。三种大豆酯酶同工酶对α-乙酸萘酯和β-乙酸萘酯有催化能力,对乙酰胆碱和有机磷酸酯类化合物甲基对硫磷无催化能力。
(3)考察了三种大豆酯酶同工酶的热稳定性、酸碱稳定性和保存稳定性,并确定了液态大豆酯酶同工酶的有效稳定保护剂。结果表明：SEI2的热稳定性好,SEI1和SEI3较差；三种酶的pH适应范围均较广,且碱性条件下相对稳定；三种酶在4℃的保存效果比—20℃好,且BSA、蔗糖和甘油可以有效地保护酶的稳定性,保存效果BSA>蔗糖>甘油。
(4)研究了常见有机溶剂、金属离子、螯合剂及表面活性剂对三种大豆酯酶同工酶的影响。结果表明：乙腈、甲醇、丙酮、二氯甲烷、乙酸乙酯、苯、正己烷等对酶的活性影响很大,且极性溶剂的影响大于非极性溶剂；金属离子除CaCl2外对三种酯酶均有很大的影响；螯合剂EDTA和表面活性剂SDS对三种酯酶均有抑制作用。
(5)改进了分光光度法检测有机磷和氨基甲酸酯类农药的检测体系。即：1.45 mL的磷酸缓冲液中依次加入0.5 mL酶液和0.5 mL的农药,混匀后在30℃水浴中反应10 min,取出试管,加入50μLα-乙酸萘酯丙酮溶液,混匀,在30℃水浴中反应15 min,再加入0.5mL固兰B盐溶液,混匀放入30℃水浴中反应10min,然后在595 nm波长处测定吸光度,用缓冲液代替酶和农药作空白,调零。
(6)进行了三种酯酶同工酶对有机磷和氨基甲酸酯类农药的敏感性试验,结果表明：SEI2对有机磷和氨基甲酸酯类农药的敏感性最好,SEI3其次,而SEI1无敏感性。故而选择SEI2作为快速检测有机磷和氨基甲酸酯类农药的生物识别元件。
Since Organochlorine pesticide was forbidden, Organophosphate and Carbamate Pesticides as succedaneums have been used widely. However, being backward in making technique and being back of the specification in using pesticides led them abused, which not only destroyed agricultrural ecology environment and restricted agricultrural economic development, but also brought serious problems of food safety. At present, events about food poisoning for eating food polluted by pesticides had happened repeatedly. So, it is badly in need to study and develop a rapid detection technology for pesicides which is simple, convenient, fast, reliable, sensitive and practical. This technology is suitable for our country's producing and selling characteristics and can stop the agricultural products containing high pesiticides residues outside of the market.
Due to its less detection time, low cost and low technology demanding, Enzyme-inhibition methods are suitable for on-site rapid detection for pesiticids and can be spreaded in producing base and wholesale market of agricultural products.At present, the detection methods in market almost are on the base of Enzyme-inhibition principle. Due to its similar sensitivity to cholinesterase, easier gaining and abundant source, phytoesterase was paid close attention in the past few years. In this study, esterase from Soybean seeds was selected for a biomarker for rapid detection of pesticide residues. Then the separation and purification, the characteristics, the sensitivities to Organophosphate and Carbamate Pesticides of different Soybean Esterase isozymes were studied explicitly in this paper.
The results are as follows:
1. The technical process on purification of phytoesterase from soybeans is as follows:soybean flour was surged for 30 min with 5 times phosphate buffer(0.3mol/L, pH 7.0), then, put it at 4℃for more than 12 hours and filtrate by 2～3 layers gauze. Next, centrifugalize the filtrate at 1000r/min to 6000r/min by differential centrifugation. The supernatant fluid was collected, selt-out with 60% ammonium sulfate saturation 1h and dialysed. The active enzyme was further purified by DEAE-32 ion exchange column chromatography and Sephadex G-100 gel filtration column chromatography, with collecting the enzyme active parts. At last, Three Soybean Esterase isozymes (SEIs)of electrophoresis purity were obtained, marked with SEI1, SEI2 and SEI3.
2. The physico-chemical and kinetics characteristics were separately studied of three SEIs. It demonstrated that the molecular weight of SEI1 is 40.7 KDa, SEI2 has two kinds of subunits with the molecular weight 37.2 KDa and 21.4 KDa, and the molecular weight of SEI3 is 74.1 KDa.The Km of SEI1 is 22.7μmol/L, with the optimal temperature 30℃and pH 6.5.The Km of SEI2 is 58.6μmol/L, with the optimal temperature 30℃and pH 6.0.The Km of SEI3 is 13.4μmol/L, with the optimal temperature 25℃and pH 6.5. They all have catalyzine ability to a-Naphthyl acetate and P-Naphthyl acetate, and have no catalyzine ability to AChE and methyl paraoxon.
3. The storage stability, temperature stability, acid alkali stability and storage condition of Soybean Esterase for pesticide residues rapid detection were separately studied in this paper. It indicated that SEI2 is better heat-resisting than SEI1 and SEI3. They all have high tolerance to wide pH, especially stabe in the condition of alkalinity. They can be better preserved at 4℃, and in BSA, saccharose and glycerol.
4. The effects of organic solvents, metal ions, chelators and surfactant on three SEIs were studied in this papar. It indicated that almost all of them can active or inactive the enzyme actitivity.
5. The detection system of sepectrophotometric method for pesticides has been improved in this paper.1.450mL phosphate buffer,0.5mL enzyme liquid and 0.5mL pesticides are mixed and incubate for 10 min at 30℃. Subsequently, put in 50μL a-Naphthyl acetate as a substrate, and incubate at 30℃for 15min. Next, it is mixed with 0.5mL fast blue B salt. React at 30℃for 10min. Finally, the absorbance was measureed at 595nm by spectrophotometer, and phosphate buffer subtitutes the enzyme and pesicides as control test.
6. The sensitivities to Organophosphate and Carbamate Pesticides of three isozmes were tested. The result indicated that the sensitivitives to pesticides of SEI2 was better than SEI3, and SEI1 did not sense to the pesticides. Thus, SEI2 can be selected as the enzyme to detect pesticide residues.
引文
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