【摘要】 采用CEA亲和层析法对家蝇AChE进行纯化,测定了不同温度、pH值、ATChI浓度、DTNB浓度对AChE活性的影响,并测定了不同杀虫剂对纯酶和粗酶的抑制作用。结果表明,CEA纯化倍数为672.36,产率为34.68%;当温度为35℃时,粗酶和纯酶的AChE活性最高;当pH=7.2时,纯化后AChE的活性最高,随后随着pH的升高AChE的活性反呈下降趋势;当pH=7.8时,粗酶AChE活性最高;当底物浓度的2500μmol·L-1时,纯化AChE活性达到最大值,当底物浓度为1500μmol·L-1时,粗酶活性达到最大值;粗酶和纯化的AChE活性的两个波峰出现在DTNB浓度为0.2mol·L-1和0.4mol·L-1时;仲丁威、乙酰甲胺磷、速灭威、甲胺磷、毒死蜱、克百威、抗蚜威、灭多威和辛硫磷等9种杀虫剂对粗酶和纯酶AChE的IC50比值都大于6,甲萘威对纯酶和粗酶AChE的IC50比值为4.826,二嗪磷的为1.184,三唑磷的为0.099。更多还原

【Abstract】 Acethlcholinesterase (AChE, EC3.1.1.7)is an important enzyme sources in enzyme-based methods for pesticide instant determi- nation. In this study, 3-carboxyphenyl ethydimethyl ammonium(CEA)method was adopted to purify AChE from housefly(Musca domestica). The effects of reaction temperature,pH,ATChI concentration,and DTNB concentration on the purified and crude AChE activities were in- vestigated. Also, the inhibitory effects of different insecticides on the purified and crude AChE activities were measured. The results showed that AChE purification fold can be reached to 672.36 when the yield was 34.68%. Significantly highest purified AChE activity was observed when the experiment conditions were 35 ℃,pH7.2,2 500 μmol·L-1 ATChI and 0.2 mol·L-1 DTNB. In addition, significantly highest crude AChE enzyme activities were observed at 35 ℃,pH7.8,1 500 μmol·L-1 ATChI,0.4 mol·L-1 DTNB. The IC50 ratio of crude and purified AChE, caused by fenobucarb, acephate,metolcarb,methamidophos,chlorpyrifos,carbofuran,pirimicarb,methomyl,phoxim were more than 6. The IC50 ratio were 4.826,1.184 and 0.099 caused by carbaryl, diazinon and triazophos, respectively. The results showed that purified AChE is more sensitive to tested insecticides than crude AChE, but the sensitivity may depend on chemical structure of insecticides and re- action conditions, which indicated the purified AChE-based methods for instant pesticides determination are more effective and useful at optimized conditions.更多还原