Research On Purification, Enzymatic Properties And The Optimum Degradation Conditions Of Atrazine By White Rot Fungi Laccase

Laccase (benzenediol:oxygen oxidoreductases; EC1.10.3.2) is a type of copper-containing polyphenol oxidase, the substrate range of which is fairly broad, it can catalyse the oxidation of various phenolic compounds and kinds of compounds contain hydroxy, acidyl and amino group while reduce molecular oxygen to water. In term of sources, laccase can be classified to animal laccase and botanical laccase, thereinto, among fungi from animal laccase,white rot fungi is the major producer of laccase. In recent years, laccase has always been the key research focus of the field that environmental protection, biological detection, the paper industry, food industry and other.Atrazine is a long residual and low toxicity herbicide,widely used around the world.The herbicide,which has a strong polar and is relatively stable in the environment,is commonly used to weeding in dry land,such as corn、sorghum and sugarcane,it can resist the natural biodegradation in water,and pollute surface water and groundwater through following surface runoff,leaching,subsidence and other kinds of ways. Studies show that atrazine,which is listed as environmental hormone substance by WWF, has a powerful influence on the reproductive function of wild amphibian.It is a potential carcinogen and can effect on endocrine disorder.In this thesis studied isolation and purification,enzymatic properties of Coprinopsis cinerea okayama7#130(C.cinerea) laccase and the preferable condition that of atrazine degradation by laccase, the major result is illustrated below:1Isolation and purification of C.cinerea(1)The method of precipitating crude enzyme liquid with ammonium sulfate:remove the precipitation after C.cinerea crude enzyme liquid precipitated by60%saturation ammonium sulfate, then remove supernatant for collecting the precipitation by secondary precipitation of the rest with90%saturation ammonium sulfate.(2)Precipitate the supernatant,gotten through centrifugation of C.cinerea fermentation liquid and remove precipitates, by60%saturation ammonium sulfate, retain supernatant, and discard the pellet.Collect the precipitation,gotten by secondary precipitation of the supernatant with90%saturation ammonium sulfate,and purify it by dialysis and chromatography.Table2-2was the final result gained through calculation:recovery is24.01%, the purification factor was2.42,and the specific activity was4435.63U/mg2The enzymatic properties of C.cinerea laccasethe optimum pH and temperature of C.cinerea laccase was6.5ang40℃. The laccase activity, which wa rested by keep1h under35℃was stable in pH4.6-5.2. C.cinerea laccase was relatively stable at20～60℃ang in the neutral environment. Its activity can be stimulated by Mg2+,Cu2+,but strongly inhibited by Al3+, Fe3+, Fe2+,Ca2+Na+,K+,Zn2+,Co2+,Mn2+effected on the activity was not obvious. The Km of laccase was0.745mol/L with o-Tolidine as substrate.3The result of the influence of C.cinerea laccase for the single factors in atrazine degradation(1) At the same conditions, descending trend of the degradation amount of atrazine was more and more large with the higher initial concentrations of atrazine, but very slowly with the initial concentration from0.1mol/L to0.5mol/L.With the same laccase activity before reaction and the same total volume of the system, laccase activity showed a slight declined tendency with the higher initial concentrations of atrazine; Comprehensive consideration, it was finally confirmed that the initial concentrations of atrazine in whole degradation testing was10mg/L.(2) The degradation rate of atrazine reached maximum when the reaction time was72h, and then remained unchanged.From the angle of saving energy and time whlie ensuring reaction was complete, it was finally confirmed that the the reaction time in whole degradation testing was3d.(3) With the rising of pH value, the degradation rate of atrazine increased at first and then decreased. When the value of PH was7, the degradation rate of atrazine reached maximum.It was finally confirmed that the the optimum pH value of reaction system was7.(4) At20～35℃, the degradation rate of atrazine increased with rising of temperature, it reached maximum at35℃, then decreased slowly, and decreased sharply above45℃.At30～40℃, the degradation effect of laccase on atrazine was relatively stable. In the system without laccase,the basic concentration of atrazine was hardly changed,that was to say,it wounld not lose because of volatilization. In other words, the reduction of concentrations of atrazine was due to the effct of laccase.(5) Taking all things into consideration, the the optimal reaction volume was5ml.(6) When rotating speed was90-180r/min, the degradation rate of atrazine increased with the increment of rotating speed.As rotating speed was over180r/min,it decreased slowly and remained essentially unchanged.Consider energy conservation, the optimal rotating speed was180r/min in this experiment.(7) When laccase activity was50-500U/mL, the degradation rate of atrazine rised with the increase of laccase activity,that was to say, the effect on atrazine degradation improved with the increase of laccase activity. Taken together,it is confirmed that accase activity was400U/mL in atrazine degradation by C.cinerea.4The optimization results of the optimum condition of atrazine degradation by C.cinerea.Response surface methodology were applied to optimize the major factor that influenced atrazine degradation, the optimum degradation conditions was concluded:PH was7,the temperature was38.3℃,the reaction system volume was5.3ml,the rotating speed was180r/min.Under this conditions,predicted value of response was52.41%, compliance value was52.32. The degradation rate raised10%～15%under the optimum condition, that provided scientific support for reducing damage to environment.