分离纯化提高鸡肝酯酶对农药抑制响应的研究

Improving Pesticide Inhibition of Chicken Liver Esterase via Enzyme Purification

本文对鸡肝酯酶粗酶液分别进行了硫酸铵分级盐析、硫酸铵分级盐析+透析和硫酸铵分级盐析+排阻色谱三种分离纯化操作,并采用四种结构不同的有机磷、氨基甲酸酯类农药,考察了不同的分离纯化操作对鸡肝酯酶对农药抑制响应的影响。发现经过这三种分离操作后,酯酶对农药抑制的响应程度显著提高,其被四种农药抑制的程度分别提高了6.49~11.30%、14.30~26.26%和10.95~39.87%,而敏感酯酶回收率分别为86.07~89.96%、66.60~73.57%和33.01~41.62%。由于分级盐析+排阻色谱的回收率过低,分级盐析+透析操作对于大规模的检测用酶制备更为实用。此外,研究还发现对这四种农药较敏感的酯酶均集中地分布在相同的分离部分,因此推测鸡肝酯酶粗酶液中对农药抑制敏感的酯酶可能是一种酶或几种溶解性质及分子量都比较接近的酶的混合。

Chicken liver esterase was purified using three different methods: including fractional ammonium sulfate precipitation, fractional ammonium sulfate precipitation + dialysis, and fractional ammonium sulfate precipitation + exclusion chromatography. Four types of organophosphate with different structures and carbamate pesticides were used to detect the effects of these purification methods on the sensitivity of purified chicken liver esterase to pesticide inhibition. The results showed that the sensitivity of the esterase to pesticide increased significantly after purification. Compared with crude esterase, pesticide inhibition rates of purified esterase increased by 6.49%~11.30% with fractional ammonium sulfate precipitation, 14.30%~26.26% with fractional ammonium precipitation + dialysis, and 10.95%~39.87% with fractional ammonium precipitation + exclusion chromatography. The recovery rates of sensitive esterase were 86.07%~89.96%, 66.60%~73.57%, and 33.01%~41.62%, respectively. Due to the low recovery of sensitive esterase obtained by fractional ammonium sulfate precipitation + exclusion chromatography, fractional ammonium sulfate precipitation + dialysis is preferable for the large-scale preparation of enzymes. In addition, the esterase sensitive to the four different types of pesticides was always in the same fraction, regardless of the purification method. Thus, the sensitive esterase might be one or several kinds of esterases with similar solubility and molecular weight.