Identification of Methothrin Degrading Bacterium Strain and Partial Purification of Methothrin Degrading Enzyme

YAN Hua; WANG Hui; YU Bo; HUANG Shengmou; LI Yunjie; WU Jinju; LI Yuqi

doi:10.15933/j.cnki.1004-3268.2018.02.017

Abstract：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).