为了获得甲醚菊酯农药降解菌株及其降解酶,采用平板划线培养的方法从湖北省襄阳市农药厂下水道活性污泥中分离细菌,随后使用DEAE-52和CM-52阴阳离子纤维素交换层析柱对细菌中甲醚菊酯降解酶进行部分纯化,并研究纯酶的特性.结果表明,获得了对甲醚菊酯农药有降解作用的菌株XU-3,经过 VITEK -32全自动细菌鉴定仪分析,综合其形态、生理生化特性和16S rDNA序列分析结果,将其鉴定为阴沟肠杆菌(Enterobacter cloacae).阴沟肠杆菌XU-3可以生长于甲醚菊酯作为唯一碳、氮源的无机盐培养基中,培养时间为60 h时,能够获取最大量的甲醚菊酯降解酶,降解酶活力达到8.5 U/mL.阴阳离子纤维素交换层析后,该酶纯化了4.4倍,酶活力回收率为66.8%.对收集到的纯酶进行SDS-PAGE不连续凝胶电泳,通过和标准蛋白质Marker比较,其分子质量为74.8 ku.对甲醚菊酯降解酶的特性研究发现,其酶促反应最适温度为35 ℃,在25~40 ℃内热稳定性良好;酶促反应最适pH值为8.5,在pH值7.5~9.0内酸碱稳定性良好.反应动力学测定发现,该酶对底物甲醚菊酯的米氏常数Km为0.825 mmol/L,最大反应速率Vmax为0.748 mmol/(L·min).%In order to obtain methothrin degrading strain and purify the degrading enzyme,the method of plate streaking was used to isolate bacteria from the sewage sludge of Xiangyang pesticide factory in Hubei province,and the DEAE-52 and CM-52 ion exchange cellulose chromatography columns were used to purify the methothrin degrading enzymes.The results showed that the bacterium stain XU-3 was isolated, which had capacity of methothrin degradation. Through VITEK-32 automatic bacterial identification instrument analysis,based on the morphology,physiological and biochemical characteristics and 16S rDNA sequence,the bacterium strain XU-3 was identified as Enterobacter cloacae.The XU-3 could grow in the inorganic salt medium with methothrin as the sole carbon and nitrogen source.When the incubation time was 60 h,the largest amount of methothrin degrading enzyme could be obtained,and the activity of degrading enzyme was 8. 5 U/mL. After the chromatography purification,the degrading enzyme was purified by 4.4 times,and the recovery rate of enzyme activity was 66.8%.The purified enzyme was subjected to SDS-PAGE discontinuous gel electrophoresis. Compared with the standard protein,the purified XU-3 methothrin degrading enzyme had a molecular weight of 74. 8 ku. Studies on the properties of the enzyme found that the optimum temperature was 35 ℃ for enzymatic reaction,with the thermal stability of 25—40 ℃,and the optimum pH value was 8.5,with the pH stability of 7.5—9.0. The reaction kinetics showed that the Km of the enzyme was 0. 825 mmol/L,and the Vmax was 0.748 mmol/(L·min).
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