【作者】 李建科；

【导师】 胡秋辉；

【作者基本信息】 南京农业大学 ， 食品科学， 2006， 博士

【摘要】 本文以酶检测法为出发点，寻找良好的植物酶源替代动物乙酰胆碱酯酶，系统研究了大豆植物酯酶的提取、分离和纯化，并进行了较为系统的酶学特性表征，研究和建立了多种形式的大豆酯酶农药残留快速检测方法和体系。主要研究结果如下：1．大豆酯酶水提取的最佳条件为：大豆种子粉碎，料液比1：5，振荡或搅拌处理30min左右，4℃冰箱静置过夜，过滤离心即得大豆酯酶粗酶液；大豆酯酶的磷酸盐缓冲液抽提和分离纯化条件为：大豆种子经拣选、粉碎，以1：5料液比加0.3moL／LpH 7.0PBS抽提，震荡或搅拌处理30min，4℃冰箱静置浸提过夜，纱布过滤，差速离心（6000rpm-1000rpm），60％硫酸铵盐析沉淀，透析，超滤浓缩得粗酶液，经DEAE-32离子交换层析，收集酶活性峰即得大豆酯酶纯酶制品，再经浓缩得浓缩酶液，或冷冻干燥得大豆酯酶冻干粉，-20℃低温冷冻保存。所得酶活力为62.48 U／mL，比活力为6.455 U／mg．pro．经各步纯化及浓缩后，对酶的提纯倍数可达90.92倍，酶的回收率为6.32％。2．研究了大豆酯酶的酶学特性：大豆酯酶的Km值为2.97μmoL／L；酶促反应的适宜温度为25℃-40℃，最适温度为30℃，酶促反应的适宜pH值范围为7.0-8.0，最适pH值为7.5；在50℃-60℃下1h后，活力降低50％左右，12h后，活力降低1／4；酸性环境下（pH≤6）酶易失活；缓冲液离子强度在0.2moL／L-0.3moL／L的体系中酯酶活力较大，最适缓冲液离子强度为2.5moL／L。大豆酯酶适宜在-20℃以下保存，-20℃保存2个月、4个月、6个月时，酶相对活力分别为87.4％、64.8％和42.6％。大豆酯酶由两种分子量不同的亚基组成，其分子量分别为44.67KD和69.18KD；对大豆酯酶酶促反应体系的显色稳定性研究表明，以a-乙酸萘酯为底物，固兰B盐为显色剂，10min内显色液较为稳定，酶的活性测定应在10min内完成。3．建立了基于大豆酯酶的酶抑制-光度法农药残留快速检测方法。结果表明，大豆酯酶对除倍硫磷外的其他24种有机磷农药和氨基甲酸酯类农药的敏感性均较好，最低检测限（LOD）都远远低于其MRL值，对18种有机磷农药的LOD在0.03125mg．kg^-1-0.0625 mg·kg^-1范围之间，其中，对毒死蜱、乐果和杀扑磷的LOD甚至达到了其MRL的1／32；对6种氨基甲酸酯类农药的LOD值为0.03125 mg·kg^-1-0.25 mg·kg^-1。完全可以满足除倍硫磷以外的其他24种有机磷和氨基甲酸酯类农药残留检测要求。对于苹果样品中5种有机磷类农药加标回收率在78.5％-102.1％，变异系数在3.1％-5.1％范围内，对3种氨基甲酸酯类农药加标回收率为87.4％-94.0％，变异系数在2.3％-3.8％范围内，与GC检测结果的符合率达100％。4．建立了基于大豆酯酶的酶抑制-电位法农药残留快速检测方法。用所建立的方法检测了水样中20种有机磷及8种氨基甲酸酯类农药的敏感性和最低检测限，结果表明，对有机磷农药的LOD可达0.000625 mg·kg^-1-0.0625 mg·kg^-1，对氨基甲酸酯类农药的LOD为0.015 mg·kg^-1-0.3125 mg·kg^-1。经与国家规定的最大残留限量（MRL）相比较，除倍硫磷之外的27种农药的最低检测限均可达到MRL限值要求。27种农药在苹果样品中的回收率在82.5％-92.3％之间，变异系数在2.03％-5.22％之间。对果蔬样品的检测与GC检测结果的符合率达83％以上。5．获得了硝酸纤维（NC）膜固定化大豆酯酶的制备技术路线和工艺参数，进行了固定化酶特性和固定化效率研究，并测试了酶片检测性能。经60个批次的固定化酶片试验，各批次之间标准差为0.031，变异系数为4.36％，说明该固定化方法的重现性和精密度良好；所制备的NC膜固定化大豆酯酶片，酶活回收率为15.37％；经7次重复使用后仍有60％以上的活性，表明固定化酶反复使用的工作稳定性较好；固定化酶最适pH为8.0，比游离酶提高了0.5个pH单位，最适温度为35℃，比游离酶提高了5℃；固定化酶的保存稳定性比游离酶明显提高。对所制备的NC-大豆酯酶膜进行了超微结构和红外光谱表征及固定化机理探讨。扫描电镜观察表明，使用戊二醛交联剂可使大豆酯酶有效地固定化于NC膜，与单纯的NC膜上承载大豆酯酶比较，酶在膜上的分布较多且较均匀；对NC膜固定化大豆酯酶前后的红外谱图解析表明，NC膜上的氨基经戊二醛连接臂与酶分子中的氨基酸残基上的游离基团产生了共价交联而实现了酶在膜上的固定化；固定化前对NC膜的PBS浸泡处理可使膜表面孔径明显增大，膜表面游离基团充分暴露，有利于酶的固定化。建立了NC-大豆酯酶膜农药残留快速检测方法，并进行了对农药残留检测效果试验。用本研究所制备的大豆酯酶NC膜片，采用目测法对水样中29种有机磷和氨基甲酸酯类农药残留的最低检测限在0.4 mg·kg^-1-5 mg·kg^-1之间；比色法对水样中8种农药检测限为0.00625 mg·kg^-1-0.0625 mg·kg^-1，远远低于其MRL值，相关系数为0.9584-0.9932；对苹果样品中8种农药的回收率在85.3％-91.8％之间，变异系数均小于5.21％，表明本方法具有较好的准确度和精密度。6．探索性研究了魔芋葡苷聚糖（KGM）膜固定化大豆酯酶方法，对所制备的KGM-大豆酯酶膜进行了超微结构和红外光谱表征及固定化机理探讨，并测试了KGM酶膜性能。KGM大豆酯酶膜制备的主要参数为，1％的KGM溶胶用Na₂CO₃调整pH为8进行改性，添加1％的甘油作增塑剂，再加入大豆酯酶，制膜。所制备的KGM大豆酯酶膜有良好的感官性能，如超薄、透明、柔韧、可塑性强等；对所制备的KGM-大豆酯酶膜性能试验表明，酶活回收率可达50％以上，与同一批游离酶活性测定表明，固定化酶比游离酶活性几乎没有损失，表明大豆酯酶在KGM膜中可高活性表达。环境扫描电镜观察表明，大豆酯酶被完整包埋于KGM膜中，并且在膜中有较均匀的分布；对KGM膜固定化大豆酯酶前后的红外谱图解析表明，酶分子中的氨基酸残基与KGM膜中的羰基以氢键方式或共轭效应方式结合，这种固定化方式较为温和，有利于维持酶的活力。由于KGM酶膜的上述良好特性，以及酶在膜中的高活性表达，有望成为生物传感器法检测农药残留的新型膜材料，同时，也可作为其他酶传感器膜材料制备的方法学参考。更多还原

【Abstract】 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.更多还原