Purification And Characterization Of The Polychlorinated Biphenyls Degrading Enzyme

Polychlorinated biphenyls (PCBs), to be one kind of persistent pollutants, its threat to environment or living creature in environment has been not ignored for a long time. The microbiological restoration of PCBs pollution in the environment is the hotspot in the domestic and global research all the time. Most of researchers filtered some bacterial strains degrading PCBs from the environments that had been polluted by PCBs, but there are fewer reports about purification and characterization of the PCBs degrading enzyme. This research aims to purify a PCBs degrading enzyme from Staphylococcus epidermidis, study its characterization and investigate possible degradation way of PCBs.The given research has been accomplished based on selecting a highly efficient PCBs degradation bacteria strain Staphylococcus epidermidis from activated sludge. The present research cultured Staphylococcus epidermidis in medium at 30℃for 20 hours, and then the cells were collected by centrifugation at the speed of 8000rpm, 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 by ammonium sulfate precipitation, dialysis and Sephadex G-150 gel filtration.Characterization of the PCBs degrading enzyme was analyzed by taking PCBs as substrate. The optimum reaction factors of the enzyme proves at 40℃and pH 7.0. And the enzyme is stable during 30-60℃and pH 6.0-9.0. After measuring, michaelis constant Km of the enzyme is 4.96mmol/L, maximum reaction velocity Vm of the enzyme is 2.03mmol/(L·min). It is tested that enzyme activity is influenced by some of metallic ion and composition. Cu+ and Fe2+ have the most lager influence of enzyme activity, and the enzyme activity is restrained by them; K+,Mg2+, Ca+ and Zn2+ less influent enzyme activity; enzyme activity will be improved by Na+ and Al3+. Possible degradation way of PCBs is studied in this thesis. Ultraviolet-visible spectrophotometry and GC-MS are used to analyze serial enzymes and intermediate products during the degradation way. And the degradation way can be surmised according to the information about these enzymes and intermediate products. Basic on the results of ultraviolet-visible spectrophotometry and GC-MS, the possible way of the bacteria strain Staphylococcus epidermidis degrading biphenyl is the typical aerobic PCBs/biphenyl degradation way. It is that:it catalyzes the oxidation of biphenyl by biphenyl-dioxygenase, biphenyl is converted into 2,3-dihydro-2,3-dihydroxybiphen--yl. With the catalytic effect of 2,3-dihydro-2, 3-dihydroxybiphenyldihydrogenase 2,3-dihydro-2,3-dihydroxybiphenyl loses two atoms of hydrogen, and converted into 2,3-dihydroxylbiphenyl. One benzene ring on 2, 3-dihydroxylbiphenyl is opened by 2,3-dihydroxybiheny-dioxygenase, and it turns into the yellow substance that is 2-hydroxy-6-oxo-6-phenylhexo-2,4-dienoate (HOPDA). HOPDA is hydrolyzed by 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic hydrolase to benzoic acid.The purified PCBs 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 the microbiological restoration of PCBs pollution in the environment. When it is used for degrading PCBs, the enzyme is separated only in the first two steps, and in so doing the process has been simplified with the passable yield.