Purification And Characterization Of Acetylcholinsterase From Periplaneta Americana

Periplaneta americana head was chosen as the experimental enzyme source material inthis dessertation. Firstly, the AChE enzyme activity assay conditions of Periplanetaamericana head was optimized with the form of ortogonal experiments. Secondly,procainamide hydrochloride pro and chromatography were adopted to isolate and purify theactivity of acetyl cholinesterase (AChE) in the head of Periplaneta americana. Finally, thepurified enzyme to the susceptibility of16kinds of pesticides was measured. Meanwhile,electric eel AChE and crude enzyme were compared to provide experimental basis forscientific assessment of whether it can be used as sensitive enzyme source. The main findingsare as follows:1. Optimization of the periplaneta americana AChE activity measurement conditionsThe conditions of periplaneta americana basis of AChE activity and the determinationof optimal conditions for enzyme reaction system were: substrate concentration1.0M,lyophilized enzyme concentration0.05g/mL, temperature35℃, time10min, pH7.5. Thedescending order from primary to secondary of the effects of these three factors were: pH>reaction temperature> reaction time.2. The stydy of purification and characterization of AChE activity in periplanetaamericana headProcainamide hydrochloride chromatography were adopted to isolate and purify theactivity of acetyl cholinesterase (AChE) in Periplaneta americana head. Purification multipleswas172.04times, the yield was16.18%. The relative molecular weight was about78kDa,which was approximate to other literature reports. The Km value of periplaneta AChE was0.0402M, the Km value of house fly was0.0784M, of whose affinity to substrate ATChIapproached.3. The study of the AChE activity in periplaneta americana head to the susceptibility ofcommon pesticides(1) There was no significant difference about the two kinds of AChE’s IC50totrichlorfon, dichlorvos, carbaryl, carbo-furan and metol-carb. The susceptibility to the fourpesticides were basically the same, except the carbo-furan. The susceptibility of AChE fromthe head of Periplaneta Americana to carbo-furan was higher than that from electric eel.TheIC50values are9.1×10-8mol/L and2.28×10-7mol/L, respectively. For the two pure enzyme,Periplaneta Americana head AChE and electric eel AChE, there were700times,180timesdifference between the most sensitive mevinphosphosphorus and the susceptibility of theweakest omethoate IC50, which has huge span range. (2) There was no significant difference about the susceptibility of these two kinds ofAChE to these seven kinds of carbamate pesticides: metol-carb, carbaryl, methomyl, aldicarb,carbo-furan, benfuracarb and carbosulfan. They are maintained in the same order ofmagnitude, but existed a discrepancy of magnitude. The IC50value of periplaneta AChE,electric eel AChE to isoprocarb were6.4×10-8、8.6×10-7M respectively. For these two pureenzyme, periplaneta AChE and electric eel AChE, the discrepancy between the IC50of thehighest susceptibility and the weakest susceptibility were41times and33times respectively.(3) The degree of sensitivity between these two kinds of AChE and those16kinds oforganic phosphorus pesticides, and the degree of sensitivity between these two kinds of AChEand Carbamate pesticides are more or less consistent. The degree of sensitivity to carbamatepesticides is higher than that to organic phosphorus pesticides. The span range of the degreeof sensitivity to organic phosphorus pesticides is bigger than that to carbamate pesticides.(4) The degree of sensitivity is improved after the purification of PeriplanetaAmericana AChE. The degree of sensitivity to dichlorvos, trichlorfon, carbofuran areimproved by three times, six times, three times respectively, while the degree of sensitivity tocarbaryl, metolcarb stay the same.(5) To some pesticides, the degree of sensitivity of Periplaneta Americana AChE ishigher than electric eel AChE, which can be used as enzyme inhibition method instead ofenzyme source to be further studied.