Comparative Study On The Detction Methods Of B-esterase

Pesticide is refers to the hazard prevention and control of agricultural and sideline products of insect pests,weeds and to regulate plant growth agents,and plays an important role in agricultural production.However the use of pesticides excessive and inappropriate will pollute the environment and agricultural products,resulting in human and animal poisoning and death.Carbamate and organophosphate pesticides are pesticides commonly used in agricultural production,which residues are also worth attention.In this thesis were selected three kinds of organophosphorus pesticides(dichlorvos,trichlorfon,monocrotophos)and 2 carbamate pesticide(methomyl,aldicarb),select B-esterase three races enzyme electric 'eel acetylcholinesterase(acetyl cholinesterase,AChE),horse serum butyrylcholinesterase(butyryl cholinesterases,BChE),kidney beans esterase as the enzyme source.Cholinesterases is capable of catalyzing the hydrolysis of cholinesterases and have different professional hydrolase,into AChE and BChE,the main source of animal,expensive,difficult to save.Kidney bean enzyme is a plant esteraes,an enzyme source phytoesterase rich and low cost,easy to store,by experiment activity kidney beans purified relatively good.This paper uses two methods and colorimetric assay,comparing the two methods common five kinds of pesticides on B-esterase sensitivity and detection limits,and the two methods used in vegetable samples and found in the study cyclodextrin derivative of the fluorescence sensitizing effect.Comparison of three enzymes by these two methods commonly used for 5 pesticides sensitivity and detection limits of different vegetables to use in the study sample,can provide a better method for the rapid detection of pesticides and new varieties in the practical application of the sample ideas,provide the basis for enzyme inhibition assay enzyme source.The main findings are as follows:1.Determination of enzyme inhibition assay flower kidney beans esterase optimum temperature AChE,BChE activity was 40?,35?,35?,the optimal pH for the 6.5,7.5,8.0,the best times are 15 min.2.The electric eel AChE,horse serum BChE,kidney beans esterase fluorescence spectroscopy,the optimal pH of the reaction,the temperature and time respectively 6.5,35?,15 min.3.Colorimetry and fluorescence spectrophotometry pesticides respectively flower kidney beans esterase,AChE,colorimetry and fluorescence detection IC50 values of BChE five pesticides on electric eel AChE,kidney beans esterase,horse serum BChE of inhibition,with five kinds of pesticides negative logarithm of the concentration increases,the final concentration of pesticides decreasing inhibition strength diminishing.Fluorescence detection of three enzymes 5 pesticides sensitivity assay is more sensitive than the detection limit lower.4.Fluorescence and colorimetric determination of electric eel AChE,horse serum BChE,kidney beans esterase five pesticides detection limit,measured by fluorescence detection limits lower than the assay more sensitive.5.5 kinds of vegetables homogenized after grinding acetone treated with the enzyme inhibition assay enzyme influence on little red rapeseed affect more than 30% the maximum effect.Enzyme inhibition method can be applied in vegetables of organophosphorus pesticide residues and carboxylic acid esters.6.The reaction solvent different concentrations vegetables have fluorescence quenching effect,different vegetable sample was diluted concentration of fluorescence quenching effect is different.7.Vegetables liquid solution of 4-methylumbelliferone and indole acetate have quenching effect,indicating that vegetables were not specific to the substrate,the phosphor has a quenching effect.8.HP-?-CD solution concentration of 400mg/mL,the best effect of fluorescence enhancement,fluorescence increased 5-fold.Trimethyl-Beta-cyclodextrin concentration at 40 mg /mL fluorescence enhancement best,fluorescence increase 3.5 times.9.The reason caused the fluorescence quenching phenomenon are many,the mechanism is very complex,as a result of the molecule and a quencher molecule fluorescent substances collision loss of energy.Fluorescence enhancers can relieve part of fluorescence quenching,fluorescence continued to improve but not strong.In the experiment also used a network thickener polyvinyl pyrrolidone,xanthan gum,ethylene diamine tetra-acetic acid,cetyl trimethyl ammonium bromide and other vegetables to reduce solution fluorescence interference,although the fluorescence enhancing effect but the effect is not strong,when applied in real samples ineffective.