Study On The Extraction Of Acetylcholinesterase From Chicken Brain Organ And Its Characteristics

In this paper, different sources were compared with specific activity for acetylcholinesterase（AChE） extraction. Chicken brain organ was found as the perfect AChE source. The enzymatic characteristics of AChE extracted from chicken brain organ were studied. The main results were as follows:The influence factors of extraction condition and reaction system were studied. The optimum extraction conditions were as follows: the phosphate buffer containing 0.5% TritonX-100 was the extracting solution at pH=8.0, the ratio of material to liquid was 1:4,. The optimum reaction system was: pH8.0, temperature 37℃, 15 min and substrate concentration 5mg/mL.Aqueous two-phase system（ATPS） was established based on PEG-1000 and ammonium sulfate to separate and purify AChE. The optimum conditions of aqueous two-phase extraction（ATPE） were: PEG-1000 concentration 15.5%, ammonium sulfate concentration 14.9%, pH6.3, enzyme concentration 14.9%. The optimum conditions for separating PEG-1000 were: the volume ratio of sodium citrate buffer to upper solution 8:1, p H 5.0. Compared with the purification results of ammonium sulfate precipitation, DEAE-52 ion exchange chromatography and Sephadex-200 gel filtration chromatography, ATPE has the advantages of high purification fold, high yield, short time and low cost.The enzymatic characteristics of AChE extracted from chicken brain organ were studied. The AChE showed pI 5.15, Km 5.67×10^-5mol/L, Vm 0.20×10²mol/（min·L）. AChE showed good thermal stability at the temperature below 40℃. AChE exhibited pH stability from pH5 to 9. The sensitivity experiments of chicken brain AChE, crude AChE and housefly AChE were carried out to detect six cabarmate toxins（carbofuran methomyl, carbaryl, aldicarb, metolcarb, ethyl carbamate）. The results indicated that ethyl carbamate had no inhibition effect on chicken brain AChE, crude AChE and housefly AChE, while chicken brain AChE had more sensitivity than crude AChE. Besides, the inhibition dynamics, half inhibitory concentration（IC50） and bimolecular rate constant（K_i） showed chicken brain AChE had more sensitivity than housefly AChE. The results showed that chicken brain AChE has the potential application for detecting the residual cabarmate toxinsat low concentration.