R-工程菌表达产物酯酶B1的纯化及其特性

A RESEARCH ON PURIFICATION AND PROPERTIES OF ESTERASE Bl FROM R-ENGINEERED BACTERIA

为将酯酶B1应用于果蔬农药残留的降解,对其进行分离纯化,并对其酶学特性进行研究。R-工程菌在LB液体培养基中37℃震荡培养14 h,细胞经石英砂冰浴研磨破壁,粗提酶液经硫酸铵分级盐析、Sephadex G-50分子筛柱层析脱盐、DEAE-Sepharose FastFlow离子交换层析、Sephadex G-75分子筛柱层析得到凝胶电泳均一的酯酶B1。SDS-PAGE结果表明,该酶分子量为67 kDa。酯酶B1的纯化倍数为43.4,收率为2.94％。该酶的最佳作用条件是37℃、pH=7.O,常温(25℃)下,酯酶B1的半衰期为62 h,42℃时该酶仍然十分稳定。酶促反应动力学曲线采用双倒数法,37℃时最大酶促反应速度Vm=0.73 mg/(mL·min),米氏常数Km=3.89 mg/mL。酯酶B1活力强、在常温下反应速度快、稳定高,说明其用于果蔬农残降解在技术上是可行的。本文将酯酶B1纯化至电泳纯,因而工艺复杂、收率较低,但在实际应用中,只需进行粗分离,工艺简单,收率高达70％,因此,该酶的开发应用在经济上也是可行的。

The present paper aims to present the author's research results on the purification and analysis of esterase Bl properties from R-engineered bacteria. In this research, esterase Bl is purified to SDS-PAGE homogenous. At the beginning, the process of purification was very complicated and therefore the yield was also low. But when used to degrade the pesticides, esterase Bl was separated only in the first two steps, and in so doing the process has been simplified with the yield reaching over 70%. From what is said above, it can be seen that the author's method is feasible and economically applicable when esterase Bl is used for degrading the residual pesticides to kill pets in vegetables and fruits. The given research has been accomplished based on the work of Doctor YAN Yan-Chun, who successfully cloned Esterase Bl gene into E. coli HB101 and constructed R-engineered bacteria. As is known, R-engineered bacteria can degrade organophosphorous pesticides, organic chloride pesticides and some other pesticides. The present research cultured R-engineered bacteria in LB-medium at 37 ℃ for 14 hours, and then the cells were collected by cen-trifugation at the speed of 3 500 r/min, at which the cell-wall can be cracked by grinding along with quartz sand at 0 'C , and thus the crude enzyme can be extracted with buffer A. Then the crude enzyme can be purified to SDS-PAGE homogenous by ammonium sulfate precipitation, sephadex G-50 gel filtration, DEAE-sepharose fast flow chromatography and Sephadex G-75 gel filtration. The result of SDS-PAGE shows that the molecular weight of esterase Bl was 67 kDa. The purification period of esterase Bl is 43. 4 and the yield is 2. 94%. On the contrary, the optimum reaction factors of esterase Bl proves at pH=7. 0 and 37 C , with the half-life of esterase Bl was 62 hours at normal temperature, and the enzyme remains stable at 45 C. The kinetic curve of enzyme-catalyzed reaction has also been reported. Esterase Bl can be used to clear out the residual pesticides in vegetable to degrade the pesticides in the polluted water.