| 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 8th～12th.The largest activity was in the 11th 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. |
PDF Full Text Request