从恶臭假单胞菌(Pseudomonas putida)X-8中分离纯化出一种壬基酚降解酶,研究其酶学特性。将该菌株X-8在NP-尿素培养基中振荡培养26h(30℃),其细胞经超声波破壁,得到粗提酶液。粗提酶液经硫酸铵分级盐析、透析脱盐、Sephadex G-200分子筛柱层析、DEAE—Sepharose Fast Flow离子交换层析,得到凝胶电泳均一的NP降解酶。SDS—PAGE电泳结果表明,该酶相对分子质量为37000±1,适宜作用条件为30℃、pH=7.0。该酶在pH=7.0—10.0、10—50℃时非常稳定。本研究得到的壬基酚降解酶活力强,常温下反应速度快、稳定性高,可用于污水中壬基酚的生物降解。将壬基酚降解酶纯化至电泳纯时工艺复杂、收率较低,但实际应用中只需对该酶进行粗分离,其工艺简单、收率高,因此该酶可用于含NP废水的处理。
The present paper aims to introduce an approach to the purification and analysis of the characteristic features of a nonylphenol degrading enzyme from Pseudomonas putida. The nonylphenol degrading enzyme mentioned here can be purified into a SDS- PAGE homogenous state. The actual process of purification can be stated as follows: at the beginning, it was proceeding very complicated with low yield, but when used to degrade nonylphenol, the enzyme can just be sorted out in the first two steps. If so, the purification process could be made simplified with high yield. From what is said above, it carl be seen that the isolation or purification method is feasible and economically practical suppose the enzyme obtained can be applied for sewage treatment with NP. Mention is also needed that this research has been conducted based on selecting high efficient NP degradation bacteria, known as Pseudomonas put/da X - 8 from the activated sludge. The Pseudomonas put/da X - 8, cultured in urea medium at 30℃ for 26 hours, and then the with the cells collected by centrifugation at a speed of 8 000 r/min, the cell wall tends to be cracked by the ultrasonic disintegration at 4 ℃, at which the crude enzyme can be extracted with buffer. Then the crude enzyme can be purified into homogenous SDS - PAGE by ammonium sulfate precipitation, dialysis, Sephadex G - 200 gel filtration and DEAE - Sepharose Fast Flow. The result of SDS - PAGE shows that the molecular weight of the enzyme is 37 000 ± 1, and the optimal reaction factors of the enzyme proves at pH = 7.0 and 30 ℃. In such a situation, the enzyme purified remains stable at the temperature between 10- 50 ℃ with pH=7.0- 10.0.