【作者】 王涛；

【导师】 王振宇；

【作者基本信息】 东北林业大学 ， 食品科学， 2009， 硕士

【摘要】 本文对小麦酯酶的提取,分离和纯化工艺进行了优化,对使用溶胶凝胶法对小麦酯酶进行固定化进行了探索,对使用固定化小麦酯酶膜组装成的生物传感电极探头进行了研究,并对两种常用的有机磷农药乐果和敌百虫进行了检测。主要试验结果如下:1.小麦酯酶的提取工艺研究。以小麦为原料,采用盐溶法对小麦酯酶进行提取,在单因素试验的基础上,根据Box-Behnken中心组合试验设计原理,选取中性盐浓度、提取时间和提取温度3个因素的3个水平进行响应面分析。建立小麦酯酶活性的二次回归模型,得出最佳提取工艺条件,即:NaCl浓度0.164mol/L,提取温度37.27℃,提取时间101.21min,此条件下酶活的预测值为3.47×10⁶U/mL,验证值为3.44×10⁶ U/mL。2.小麦酯酶的分离纯化工艺研究。结合使用饱和硫酸铵分级沉淀盐析法和SephadexG-100凝胶色谱法对提取后的小麦酯酶粗酶液进行纯化,得出结论:饱和硫酸铵盐析分离纯化小麦酯酶的最佳浓度范围为45%～55%。当饱和硫酸铵的浓度为55%时,小麦酯酶的活力最高,为5.25×10⁶U/mL,纯化倍数为1.51倍。经SephadexG-100凝胶层析分离纯化小麦酯酶的最佳范围为8～12管。其中11管的酶活最大,为9.30×10⁶U/mL,是纯化前小麦酯酶酶活的2.68倍,纯化效果明显。3.小麦酯酶的固定化工艺研究。使用溶胶凝胶法对纯化后的小麦酯酶进行固定化,经筛选溶胶原料,TEOS最适合对小麦酯酶进行固定化,其酶活保存率达到46.50%。之后使用单因素结合响应面的方法,分析出小麦酯酶溶胶凝胶固定化过程中,最佳的条件为:R值8.25,TritonX-100浓度9.84%,PEG-400浓度5.41%和PBS的pH值6.6,此条件下酶活保存率最大,预测值为56.86%。经验证试验表明,真实值为57.74%,达到预测值的97.76%,结果与模型高度相符。最后对固定化后的生物敏感膜的特性进行测试,得出生物敏感膜在蒸馏水中的溶胀率为5.89%,在丙酮中的溶胀率为2.64%。4.用于检测有机磷农药的生物传感电极探头的研究。使用固定化后的小麦酯酶酶膜,将之固定在精密酸度计电极的玻璃探头部位从而组装成生物传感电极探头,对检测中涉及到的各项参数进行了优化,得出结论:每张酶溶胶凝胶传感膜上最佳小麦酯酶的负载量为2g,溶胶凝胶与小麦酯酶体积比为0.4,进行有机磷农药检测时体系中缓冲溶液的pH值为7.0,底物浓度为0.8mmol/L,温育时间选择为10min。之后对在农业上使用率最高的两种有机磷农药乐果和敌百虫进行了检测,结果为:乐果的浓度在1×10^-8～1×10^-3mol/L内与小麦酯酶的抑制率呈线性关系,回归方程为y=13.02x+118.7,相关系数为0.995,最低检出限（LDC）为1.7×10^-9mol/L;敌百虫的浓度在1×10^-8～1×10^-3mol/L内与小麦酯酶的抑制率呈线性关系,回归方程为y=13.08x+112.7,相关系数为0.997,最低检出限（LDC）为1.1×10^-8mol/L。进行实际样品检测,对照国家标准方法得出的结论,采用检测方程得出的乐果的检测结果回收率为真实值的88.9%～92.3%,标准偏差为3.05%;敌百虫的检测结果回收率为真实值的87.9%～91.7%,标准偏差为3.24%。最后对生物敏感膜的重复使用次数进行了试验,得出结论使用20次以内其酶活保存率为初始酶活的90%左右。更多还原

【Abstract】 This paper the extraction,separation and purification process of wheat esterase were optimized,explored the usage of sol-gel method on wheat esterase immobilizeation,stud-ied on using immobilized wheat esterase assembled into a biosensing electrode probe and detected the two kinds of usual organophosphorus pesticides—Dimethoate and Metrifonate.The mainly results in this paper were as follows:1.Studies on the Extraction process of wheat esterase.We extracted wheat esterase with tihe method of neutral salt-dissolution,on the basis of single-factors experiments,the Box-Behnken center-united experimental design principles were used to design the experiment and the responsive surfaces analysis of 3 factors（neutral salt concentration,extraction temperature and extraction time） and 3 levels was adopted.The quadric regression model for enzymatic activity was established,and the optimum extraction conditions was obtained as follow:sodium chloride concentration 0.164mol/L,extraction temperature 37.27℃and extraction time 101.21 minutes,the enzymatic activity was maximum with estimated value was 3.47×10⁶U,and verified value was 3.44×10⁶U/mL.2.Studies on the separation and purification processes of wheat esterase.A combination way of saturated ammonium sulfate fractional salting-out precipitation and Sephadex G-100 gel chromatography purified the crude enzyme solution after extraction of the wheat esterase and concluded:the best concentration range of saturated ammonium sulfate salting-out for separation and purification of the wheat esterase is 45%to 55%.When the concentration of ammonium sulfate was 55%,the highest activity of wheat esterase is 5.25×106U/mL with a multiple of 1.51.Using Sephadex G-100 gel chromatography for separating and purificating the wheat esterase,the best range of tube is 8^th～12^th.The largest activity was in the 11^th With the activity of 9.30×10⁶U/mL and it was 2.68 times to unpurified wheat esterase.So the effect of this method is very well.3.Studies on the immobilization process of wheat esterase.We used sol-gel method to immobilize purified wheat esterase,through filtering the sol materials,we found that TEOS was most suitable for wheat esterase immobilized and the preserved activity is 46.50%.Then we designed the experiment with the combined method of single-factors and responsive surfaces analysis and found the best conditions of immobilization were that R value was 8.25,TritonX-100 concentration was 9.84%,PEG-400 concentration was 5.41%and PBS’s pH was 6.6.At this condition,the predictive preserved activity of wheat-esterase was 56.86%,actual value was 57.74%,the results were highly correspond to model prediction.Finally,we texted the characteristics of bio-sensitive film and derived that the swelling rate of bio-sensitive membrane in distilled water is 5.89%and in acetone it was 2.64%.4.Studies on the biosensing electrode probe of detecting organophosphorus pesticides.The immobilized wheat esterase membrane was fixed at the glass electrode precise acidimeter probe to form a biosensor,and we optimized the parameters involved in detection and concluded that the best load of each wheat esterase sol-gel sensing membrane was 2g,the ratio of sol-gel to the volume wheat esterase is 0.4,the pH value of PBS in organophosphorus pesticides detection is 7.0,the concentration of substrate is 0.8mmol/L and the responsed time is 10min.Then detected two widely organophosphorus pesticides of Dimethoate and Metrifonate,the result was in the range of Dimethoate concentration 1×10^-8～1×10^-3mol/L it was linear relation with wheat esterase’s inhibition rate.Regression equation was y=13.02x+118.7,correlation coefficent was 0.995,limit detectability（LDC） was 1.7×10^-9 mol/L;In the range of Metrifonate concentration 1×10^-8～1×10^-3mol/L it was linear relation with wheat esterase’s inhibition rate.Regression equation was y=13.08x+112.7,correlation coefficent was 0.997,limit detectability（LDC） was 1.1×10^-8mol/L;In the actual sample detection,compared to the national standard method of control,we concluded that the the recovery rate of in the result from using Dimethoate detection equation was 88.9%～92.3%of the true value,standard deviation was 3.05%;the recovery rate of in the result from using Metrifonate detection equation was 87.9%～91.7%of the true value,standard deviation was 3.24%.Finally,we texted the repeated used number of bio-sensitive membrane and resulted that in 20 times using the preserved activity was 90%of initial’s.更多还原