技术领域technical field
本发明涉及一种采用聚乙烯吡咯烷酮修饰小麦酯酶的制备方法，属于小麦酯酶修饰利用领域，具体涉小麦酯酶的修饰方法并提高了酶比活力。The invention relates to a preparation method for modifying wheat esterase with polyvinylpyrrolidone, which belongs to the field of modification and utilization of wheat esterase, in particular to a method for modifying wheat esterase and improving the specific activity of the enzyme.
背景技术Background technique
有机磷类及氨基甲酸酯类农药的使用广泛，是我国常用农药种类。因此，使用不当时，常导致农产品残留，而影响食品安全质量。Organophosphorus and carbamate pesticides are widely used and are commonly used in my country. Therefore, when used improperly, it often leads to residues of agricultural products, which affects food safety and quality.
目前，有机磷类及氨基甲酸酯类农药的检测方法包括色谱法、酶联免疫分析法、酶抑制法等。色谱法需要特殊、昂贵的仪器且检测费用高。酶联免疫分析法，具有灵敏度高、特异性强、检出限低、操作简便快捷的优点，但有一定的局限性，因为酶联免疫法特异性强，一般一种试剂盒只能检测一种有机磷，不能检测到有机磷的总量，检测过程中具有一定的盲目性，且有机磷抗体制作过程难度较大，并且检测成本相对较高。酶抑制法是目前研究比较多且比较成熟的一种对有机磷和氨基甲酸酯类农药残留快速检测的一种技术。所用的酶包括动物来源的胆碱酯酶及植物来源的酯酶。动物胆碱酯酶抑制法具有快速、简便、灵敏等优点，但其来源于动物体，取材不易，且保存条件苛刻。植物酯酶具有酶源丰富、取材方便、成本低和保存方便的优势，而且农药的灵敏度和检出限与动物胆碱酯酶相当，精密度甚至更好。对小麦酯酶修饰并对其稳定性的提高，利于酶抑制法检测有机磷类及氨基甲酸酯类农药。At present, the detection methods of organophosphorus and carbamate pesticides include chromatography, enzyme-linked immunoassay, and enzyme inhibition. Chromatography requires special, expensive equipment and detection costs are high. Enzyme-linked immunoassay has the advantages of high sensitivity, strong specificity, low detection limit, simple and quick operation, but has certain limitations. Because of strong specificity of enzyme-linked immunosorbent assay, generally one kit can only detect one The total amount of organic phosphorus cannot be detected, and the detection process has a certain degree of blindness, and the production process of organic phosphorus antibody is relatively difficult, and the detection cost is relatively high. Enzyme inhibition method is a kind of rapid detection technology for organophosphorus and carbamate pesticide residues that has been studied more and more maturely. Enzymes used include cholinesterases of animal origin and esterases of plant origin. The animal cholinesterase inhibition method has the advantages of rapidity, simplicity, and sensitivity, but it is derived from animals, and it is not easy to obtain materials and the storage conditions are harsh. Plant esterase has the advantages of rich enzyme source, convenient material acquisition, low cost and convenient storage, and the sensitivity and detection limit of pesticides are comparable to animal cholinesterase, and the precision is even better. The modification of wheat esterase and the improvement of its stability are beneficial to the detection of organophosphorus and carbamate pesticides by enzyme inhibition method.
对小麦酯酶的研究包括不同品种小麦比较、酶的提取制备方法以及用于有机磷农药检测方法的开发等。相关专利有快速测定有机磷农药的植物酯酶片(CN99114268.3)、残留有机磷农药快速测试盒及制造方法(CN00135716.6)、快速检测痕量有机磷和氨基甲酸酯农药的方法(CN01144343.X)、一种小麦酯酶生物传感器的制备方法(CN200910072241.6)、一种快速检测食品中农药残留量的试纸条(CN201120345140.4)、一种检测微痕量克百威的方法(CN201210346004.6)、一种检测微痕量甲基磷酸二甲酯的方法(CN200710092751.0)等。The research on wheat esterase includes the comparison of different varieties of wheat, the extraction and preparation method of enzyme, and the development of detection method for organophosphorus pesticides, etc. Related patents include plant esterase tablets for rapid determination of organophosphorus pesticides (CN99114268.3), rapid test kit for residual organophosphorus pesticides and its manufacturing method (CN00135716.6), method for rapid detection of trace organophosphorus and carbamate pesticides ( CN01144343.X), a preparation method of wheat esterase biosensor (CN200910072241.6), a test strip for rapid detection of pesticide residues in food (CN201120345140.4), a method for detecting trace amounts of carbofuran method (CN201210346004.6), a method for detecting trace amounts of dimethyl methyl phosphate (CN200710092751.0), etc.
小麦酯酶及其他植物酯酶是实现酶抑制法检测农药残留的关键材料之一。通过一定方法提高小麦酯酶比活力及稳定性是开发农药残留检测方法、降低检测成本等关键之一。通过对小麦酯酶进行修饰是提高小麦酯酶比活力及稳定性的有效手段之一。Wheat esterase and other plant esterases are one of the key materials to realize the detection of pesticide residues by enzyme inhibition method. Improving the specific activity and stability of wheat esterase by a certain method is one of the keys to developing pesticide residue detection methods and reducing detection costs. Modification of wheat esterase is one of the effective means to improve the specific activity and stability of wheat esterase.
发明内容Contents of the invention
本发明的目的是为了获得适于有机磷类及氨基甲酸酯类农药的检测的小麦酯酶修饰酶，提高现有制备的小麦酯酶的比活力及热稳定性，降低小麦酯酶法检测有机磷类及氨基甲酸酯类农药的检测成本。同时提供一种新的小麦酯酶修饰方法。The purpose of the present invention is in order to obtain the wheat esterase modification enzyme that is suitable for the detection of organophosphorus and carbamate pesticides, improve the specific activity and thermostability of the wheat esterase of existing preparation, reduce the wheat esterase method to detect organic matter. Testing costs for phosphorus and carbamate pesticides. At the same time, a new wheat esterase modification method is provided.
本发明的技术方案为：聚乙烯吡咯烷酮修饰小麦酯酶的制备方法。其具体步骤为：对提取制备的小麦酯酶用氧化的聚乙烯吡咯烷酮进行修饰，从而提高小麦酯酶的比活力及热稳定性。降低酶抑制法检测有机磷类及氨基甲酸酯类农药的成本。The technical scheme of the invention is: a preparation method of polyvinylpyrrolidone modified wheat esterase. The specific steps are: modifying the extracted wheat esterase with oxidized polyvinylpyrrolidone, thereby improving the specific activity and thermal stability of the wheat esterase. Reduce the cost of detecting organophosphorus and carbamate pesticides by enzyme inhibition method.
其中所述的提取制备的小麦酯酶为从小麦面粉中以0.05～0.15mol/L的NaCl的溶液，料液体积比1∶2～6(m∶v)，在28～40℃的振荡提取40～120min，再经双层纱布过滤，取得的滤液在3000～6000r/min，4℃离心5～10min，收集上清液；进而以20％-70％硫酸铵盐析，4℃条件10000r/min离心15～20min收集沉淀，将沉淀采用截留分子量为8000～14000Da透析袋，将透析袋放入蒸馏水透析脱除硫酸铵。即为制备的小麦酯酶。The wheat esterase prepared by extracting described therein is a solution of NaCl of 0.05～0.15mol/L from wheat flour, the volume ratio of solid to liquid is 1:2～6 (m:v), and the vibration extraction at 28～40°C 40-120min, then filtered through double-layer gauze, the obtained filtrate was centrifuged at 3000-6000r/min at 4°C for 5-10min, and the supernatant was collected; then salted out with 20%-70% ammonium sulfate, 10000r/min at 4°C Centrifuge for 15 to 20 minutes to collect the precipitate, use a dialysis bag with a molecular weight cut-off of 8,000 to 14,000 Da, and put the dialysis bag into distilled water for dialysis to remove ammonium sulfate. It is the prepared wheat esterase.
所述的氧化的聚乙烯吡咯烷酮为将高碘酸钠与聚乙烯吡咯烷酮(2～4∶1，m∶m)溶于40～100mL蒸馏水中，25℃避光搅拌反应20～36h后，将所得产物用截留分子量为8000～14000Da的透析袋在蒸馏水中透析10～20h，得到聚乙烯吡咯烷酮的氧化溶液。The oxidized polyvinylpyrrolidone is obtained by dissolving sodium periodate and polyvinylpyrrolidone (2-4:1, m:m) in 40-100mL distilled water, stirring and reacting at 25°C for 20-36 hours in the dark, and then dissolving the obtained The product is dialyzed in distilled water for 10-20 hours with a dialysis bag with a molecular weight cut-off of 8000-14000 Da to obtain an oxidized solution of polyvinylpyrrolidone.
所述的小麦酯酶用氧化的聚乙烯吡咯烷酮进行修饰为将制备得到的小麦酯酶与氧化的聚乙烯吡咯烷酮溶液按体积比为1∶1～20(v∶v)混合后，在避光25℃下，振荡10～20h后，加入0.1～0.3mol/LNaBH4溶液1～2mL继续振荡4～6h终止反应，调至pH7.0～8.0，得到聚乙烯吡咯烷酮修饰小麦酯酶.The wheat esterase is modified with oxidized polyvinylpyrrolidone so that after mixing the prepared wheat esterase with oxidized polyvinylpyrrolidone solution in a volume ratio of 1:1 to 20 (v:v), it is protected from light for 25 After shaking for 10-20 hours at ℃, add 1-2 mL of 0.1-0.3 mol/LNaBH4 solution and continue shaking for 4-6 hours to terminate the reaction, adjust to pH 7.0-8.0, and obtain polyvinylpyrrolidone-modified wheat esterase.
所述的修饰的小麦酯酶米氏方程中Vmax值增大，Km值减小，小麦酯酶对底物乙酸-1-萘酯的亲和力增强，酶比活力提高至原酶活的130～150％，25℃～45℃酶活在100min内保持稳定。In the modified wheat esterase-Menten equation, the Vmax value increases, the Km value decreases, the affinity of the wheat esterase to the substrate -1-naphthyl acetate is enhanced, and the specific activity of the enzyme is increased to 130-150 of the original enzyme activity. %, the enzyme activity remains stable within 100 min at 25°C to 45°C.
原酶与修饰酶的酶学性质：Enzymatic properties of original enzyme and modified enzyme:
修饰酶的Km为原酶1/3，(以乙酸-1-萘酯为底物)，为1.1mmol/L。最大反应速率较原酶增加33～40％。The Km of the modified enzyme is 1/3 of the original enzyme (with 1-naphthyl acetate as the substrate), which is 1.1 mmol/L. The maximum reaction rate is 33-40% higher than that of the original enzyme.
原酶与修饰酶的热稳定性分别为15～65℃。The thermal stability of the original enzyme and the modified enzyme is 15-65°C, respectively.
原酶与修饰酶的反应最适温度为35～45℃；在65℃下，经过120min处理后，其相对酶活只有初始状态下酶活的59.6％。The optimal temperature for the reaction between the original enzyme and the modified enzyme is 35-45°C; at 65°C, after 120 minutes of treatment, the relative enzyme activity is only 59.6% of the initial enzyme activity.
原酶与修饰酶的反应最适pH为6.4。对酸碱的稳定性较原酶有所提高。The optimum pH for the reaction between the original enzyme and the modified enzyme was 6.4. The stability to acid and alkali is improved compared with the original enzyme.
修饰酶的活力是原酶的130～150％The activity of the modified enzyme is 130-150% of the original enzyme
本发明的优点：Advantages of the present invention:
1)原料为小麦面粉，来源容易、价廉；1) The raw material is wheat flour, which is easy to source and cheap;
2)修饰过程较为简单；2) The modification process is relatively simple;
3)修饰酶的活力增高，降低酶抑制法检测有机磷类及氨基甲酸酯类农药的成本。3) The activity of the modified enzyme is increased, and the cost of detecting organophosphorus and carbamate pesticides by the enzyme inhibition method is reduced.
具体实施方式Detailed ways
实施例1：Example 1:
1)小麦面粉中加入0.07mol/L的NaCl的溶液，料液体积比1∶2(m∶v)，在29℃的振荡提取120min，再经双层纱布过滤，取得的滤液在6000r/min，4℃离心10min，收集上清液；进而以20％-70％硫酸铵盐析，4℃条件10000r/min离心15min收集沉淀，将沉淀采用截留分子量为8000～14000Da透析袋，将透析袋放入蒸馏水透析脱除硫酸铵。即为制备的小麦酯酶。1) Add 0.07mol/L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:2 (m:v), shake and extract at 29°C for 120min, then filter through double-layer gauze, and the filtrate obtained is at 6000r/min , centrifuge at 4°C for 10min, collect the supernatant; then salt out with 20%-70% ammonium sulfate, centrifuge at 10,000r/min at 4°C for 15min to collect the precipitate, use a dialysis bag with a molecular weight cut-off of 8,000-14,000Da, put the dialysis bag dialyzed into distilled water to remove ammonium sulfate. It is the prepared wheat esterase.
2)将高碘酸钠与聚乙烯吡咯烷酮(2∶1，m∶m)溶于40mL蒸馏水中，25℃避光搅拌反应20h后，将所得产物用截留分子量为8000～14000Da的透析袋在蒸馏水中透析20h，得到聚乙烯吡咯烷酮的氧化溶液。2) Sodium periodate and polyvinylpyrrolidone (2:1, m:m) were dissolved in 40 mL of distilled water, and after stirring for 20 hours at 25°C in the dark, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000 to 14,000 Da. Dialyzed for 20 hours to obtain an oxidized solution of polyvinylpyrrolidone.
3)将制备得到的小麦酯酶与氧化的聚乙烯吡咯烷酮溶液按体积比为1∶1.5(v∶v)混合后，在避光25℃下，振荡10h后，加入0.1mol/LNaBH4溶液1mL继续振荡4h终止反应，调至pH7.0，得到聚乙烯吡咯烷酮修饰小麦酯酶.3) Mix the prepared wheat esterase with the oxidized polyvinylpyrrolidone solution at a volume ratio of 1:1.5 (v:v), shake for 10 h at 25°C in the dark, and then add 1 mL of 0.1 mol/L NaBH4 solution Continue shaking for 4 h to terminate the reaction, adjust to pH 7.0, and obtain polyvinylpyrrolidone-modified wheat esterase.
4)修饰的小麦酯酶活力为原酶的135％。其他酶学特性不变。4) The activity of the modified wheat esterase is 135% of that of the original enzyme. Other enzymatic properties remain unchanged.
实施例2Example 2
1)小麦面粉中加入0.15mol/L的NaCl的溶液，料液体积比1∶6(m∶v)，在40℃的振荡提取40min，再经双层纱布过滤，取得的滤液在3000～6000r/min，4℃离心5min，收集上清液；进而以20％-70％硫酸铵盐析，4℃条件10000r/min离心19min收集沉淀，将沉淀采用截留分子量为8000～14000Da透析袋，将透析袋放入蒸馏水透析脱除硫酸铵。即为制备的小麦酯酶。1) Add 0.15 mol/L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:6 (m:v), shake and extract at 40°C for 40 minutes, and then filter through double-layer gauze. /min, centrifuge at 4°C for 5min, collect the supernatant; then salt out with 20%-70% ammonium sulfate, centrifuge at 10,000r/min at 4°C for 19min to collect the precipitate, and use a dialysis bag with a molecular weight cut-off of 8,000-14,000Da to dialyze the precipitate The bag was put into distilled water and dialyzed to remove ammonium sulfate. It is the prepared wheat esterase.
2)将高碘酸钠与聚乙烯吡咯烷酮(4∶1，m∶m)溶于45mL蒸馏水中，25℃避光搅拌反应35h后，将所得产物用截留分子量为8000～14000Da的透析袋在蒸馏水中透析19h，得到聚乙烯吡咯烷酮的氧化溶液。2) Sodium periodate and polyvinylpyrrolidone (4:1, m:m) were dissolved in 45 mL of distilled water, and after stirring for 35 hours in the dark at 25°C, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. Dialyzed in medium for 19 hours to obtain an oxidized solution of polyvinylpyrrolidone.
3)将制备得到的小麦酯酶与氧化的聚乙烯吡咯烷酮溶液按体积比为1∶20(v∶v)混合后，在避光25℃下，振荡20h后，加入0.25mol/LNaBH4溶液2mL继续振荡6h终止反应，调至pH8.0，得到聚乙烯吡咯烷酮修饰小麦酯酶.3) Mix the prepared wheat esterase with the oxidized polyvinylpyrrolidone solution at a volume ratio of 1:20 (v:v), shake for 20 hours at 25°C in the dark, and then add 2 mL of 0.25mol/L NaBH4 solution Continue shaking for 6 h to terminate the reaction, adjust to pH 8.0, and obtain polyvinylpyrrolidone-modified wheat esterase.
4)修饰的小麦酯酶活力为原酶的149％。其他酶学特性不变。4) The activity of the modified wheat esterase is 149% of that of the original enzyme. Other enzymatic properties remain unchanged.
实施例3Example 3
1)小麦面粉中加入0.07mol/L的NaCl的溶液，料液体积比1∶3(m∶v)，在30℃的振荡提取60min，再经双层纱布过滤，取得的滤液在30000r/min，4℃离心7min，收集上清液；进而以20％-70％硫酸铵盐析，4℃条件10000r/min离心16min收集沉淀，将沉淀采用截留分子量为8000～14000Da透析袋，将透析袋放入蒸馏水透析脱除硫酸铵。即为制备的小麦酯酶。1) Add 0.07mol/L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:3 (m:v), shake and extract at 30°C for 60min, then filter through double-layer gauze, and the filtrate obtained is at 30000r/min , centrifuged at 4°C for 7 minutes, and collected the supernatant; then salted out with 20%-70% ammonium sulfate, centrifuged at 10,000 r/min at 4°C for 16 minutes to collect the precipitate, and used a dialysis bag with a molecular weight cut-off of 8,000 to 14,000 Da, and put the dialysis bag into dialyzed into distilled water to remove ammonium sulfate. It is the prepared wheat esterase.
2)将高碘酸钠与聚乙烯吡咯烷酮(2.5∶1，m∶m)溶于90mL蒸馏水中，25℃避光搅拌反应30h后，将所得产物用截留分子量为8000～14000Da的透析袋在蒸馏水中透析19h，得到聚乙烯吡咯烷酮的氧化溶液。2) Sodium periodate and polyvinylpyrrolidone (2.5:1, m:m) were dissolved in 90 mL of distilled water, and after stirring for 30 hours in the dark at 25°C, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. Dialyzed in medium for 19 hours to obtain an oxidized solution of polyvinylpyrrolidone.
3)将制备得到的小麦酯酶与氧化的聚乙烯吡咯烷酮溶液按体积比为1∶4(v∶v)混合后，在避光25℃下，振荡10h后，加入0.25mol/LNaBH4溶液1.2mL继续振荡4h终止反应，调至pH8.0，得到聚乙烯吡咯烷酮修饰小麦酯酶.3) After mixing the prepared wheat esterase and oxidized polyvinylpyrrolidone solution in a volume ratio of 1:4 (v:v), shake for 10 hours at 25°C in the dark, and then add 0.25mol/L NaBH4 solution 1.2 mL continued to shake for 4h to terminate the reaction, adjusted to pH 8.0, and obtained polyvinylpyrrolidone-modified wheat esterase.
4)修饰的小麦酯酶活力为原酶的142％。其他酶学特性不变。4) The modified wheat esterase activity is 142% of the original enzyme. Other enzymatic properties remain unchanged.
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| CN201310300705.0ACN103333877B (en) | 2013-07-18 | 2013-07-18 | Preparation method of polyvinylpyrrolidone modified wheat esterase |
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| CN201310300705.0ACN103333877B (en) | 2013-07-18 | 2013-07-18 | Preparation method of polyvinylpyrrolidone modified wheat esterase |
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