The Study Of Cometabolism And Enzymes Involved In Bacterial Degradation Of PAHs

Polycyclic aromatic hydrocarbons (PAHs), one class of organic pollutants consisting of two or more fused benzene rings, are widely distributed in the coastal environment. As a new way to study the PAHs biodegradation, differentially expressed protein technology can help to investigate PAHs cometabolism process and its degrading mechanism.In this research, growth curve, electron transport system activity(ETSA), degradation rates, catechol dioxygenase activity and differentially expressed protein of degrading-bacteria US6-1 had been determined. In former study, we found that there were some relationship between the MCD and the PAHs degradation rate of bacteria US6-1. The gradient electrophoresis was used to find the differentially expressed proteins, which were induced by different PAHs in the cometabolism.Detection of differentially expressed protein in PAHs cometabolism and discovery of the degrading-enzymes were very helpful to analyze the mechanisms of PAHs biodegradation and cometabolism.The main results are listed as follows:1. Several methods were used to extract the whole proteins of US6-1, and the results of acrylamide gel electrophoresis showed the optimal protein extraction buffer was 0.1 mmol/L PMSF, 1 mmol/L DTT, 0.05 %NP-40 and 20%glycerol.2. Compared with linearity separate gel electrophoresis, application of the gradient gel electrophoresis could get better whole protein expression profile. In addition, it was helpful and referential to study of degradation process and induction of functional enzymes. The time distribution character of the differentially expressed protein of US6-1 during the PAHs biodegradation was studied, and the results showed that it would lead to a higher level of expression in 80KDa after MCD was added. 3. Several important parameters of US6-1, including growth curve, metabolism activity, degradation rate, activity of dioxygenase and differentially expressed proteins, were measured to reveal the effect of MCD on the growth and phenanthrene degradation. The results indicated that the cell growth and metabolism activity of US6-1 were greatly improved by MCD. In different culture conditions—2216E-Phe and 2216E-MCD-Phe culture medium, the highest degradation rate of the degrader US6-1 was attained to 13.32 ppm and 21.02 ppm per day.4. The activity of ring cleave enzymes (catechol-1,2-dioxygenase and catechol-2,3- dioxygenase ) was measured, and the phenanthrene degradation pathway of US6-1 was predicted. The results showed that catechol-2,3-dioxygenase, but not catechol-1,2-dioxygenase was detected in US6-1 in the experimental process. Therefore, when degraded by US6-1, phenanthrene was divided into pyruvic acid and aldehyde in by meta cleave pathway, then turned into tricarboxylic acid cycle. The molecular weight of catechol-2,3- dioxygenase was estimated to be about 25KDa to 38KDa, by recovering and measuring activity of catechol-2,3- dioxygenase from gel.