Purification And Characterization Of Nonylphenol Degrading Enzyme

Nonylphenol(NP),to be one of environmental endocrine disruptors,its threat to animals and plants has caused the extensive concern.In recent years,eliminating NP from environment, especially biodegradation of NP,has been a hot issue in national and international study.Many researchers screened out strain which can degrade NP,but,there are few reports on separation and purification of NP degrading enzyme and its characteristic analysis.This research aims to purify a Nonylphenol degrading enzyme from Pseudomonas putida X-8,study its characterization and investigate possible degradation way of NP.The given research has been accomplished based on selecting a highly efficient NP degradation bacteria Pseudomonas putida X-8 from activated sludge.The present research cultured Pseudomonas putida X-8 in NP-urea medium at 30℃for 26 hours,and then the cells were collected by centrifugation at the speed of 8000 r/min,at which the cell wall can be cracked by ultrasonic disintegrated at 0℃,and thus the crude enzyme can be extracted with buffer.Then the crude enzyme can be purified to SDS-PAGE homogenous by ammonium sulfate precipitation, dialysis,sephadex G-200 gel filtration and DEAE-sepharose fast flow chromatography.The result of SDS-PAGE shows that the molecular weight of the enzyme was 37 kDa.Characterization of the NP degrading enzyme was analysed by taking 4-NP as substrate.The optimum reaction factors of the enzyme proves at pH 7.0 and 30℃,and the enzyme remains stable during 10⁵0℃and pH 7.0¹0.0.After measuring,Km of the enzyme is 24.9 mmol/L.It is tested that enzyme activity is influenced by some of metallic ion and composition,for example, enzyme activity will be improved by Mg²⁺ and Ca²⁺;and will be restrained completely by Cu²⁺ with low concentration;also,it will be restrained to a certain degree by some other metallic ions. Metal chelating agent EDTA can be used as complexing agent in solution,which has some certain restraint on enzyme activity.In lower concentration,the enzyme activity will be restrained slightly, but when content of EDTA is up to 20 mmol/L,enzyme activity only accounts for 12%of control group.Besides,activity of the NP degrading enzyme will be restrained to a certain degree by anionic surface-active agent SDS and reducing agent dithiothreitol(DTT).Acrylamide won't effect natural structure of protein,it has small effect on activity of the NP degrading enzyme.Activity of the NP degrading enzyme will be effected slightly by potassium iodide with low concentration, and restrained completely with concentration of no less than 800 mmol/L.Possible degradation way of NP is studied in this thesis.Firstly,degradation of 4-NP is analyzed by UV spectrophotometry.Removing rate of 4-NP will exceed 73%when it has been degrading for 3 days.At same time,based on UV scanning spectrogram,it is concluded that degradation way of 4-NP is not meta position open loop,but adjacent position open loop. Secondly,intermediate product and final product made in degradation of 4-NP are tested by GC-MS.Therefore,it is concluded that possible way which 4-NP is degraded by microorganism. When 4-NP is catalyzed by crude enzyme,the nonyl chain is fragmentized,produced two kinds of intermediate products:4-octylphenol and 4-propylphenol.Then,small molecule substances,for example,CO₂ and H₂,etc,will be made through further adjacent position open loop.The purified NP degrading enzyme purified from this research has strong activity,which has high rate for catalytic reaction and high stability in normal temperature,it can be used for biodegradation of nonylphenol in sewage.When it is used for degrading Nonylphenol,the enzyme is separated only in the first two steps,and in so doing the process has been simplified with the high yield.