The Protemic Research Of Aromatic Hydrocarbon Biodegradation Pathway

Aromatic hydrocarbon is a capital polluted organic and widely distributes in environment, which causes diseases of human and is harmful to ecological environment. Due to unique physical chemistry characteristic, Aromatic hydrocarbon can be clean up by biotechnology. In the postgenome era, the center of bioresearch changes from global genetic information to biological function. Recently, Protemics becomes a hot spot, which aims at function and interaction of protein. Because Aromatic hydrocarbon is a kind of xenobiotics in environment, degradation microorganism will form inducible enzyme to have new metabolism. Meanwhile, degradation microorganism will be faced with multicontamination factors in practical. Through the analysis of protein expression stressed with pressure, stress response protein and enzyme participated in metabolism of environmental pollutant can be identified, which will helpful to our deep understanding of biodegradable. It also provides good evidence to evolutionary trend and potential mechanism of evolution. In this thesis, one strain, named ZX16, which could use phenanthrene as sole carbon source was isolated from petroleum and heavy metal contaminated soil. The differential protein expression of degradation strain stressed by pollutants was analyzed by 2D-gel. This project also observed the change of surface structure of cell membrane in nano level. Inverse PCR approachment was employed to sequence the gene encoding the inducible putative GDO. The detail result of this paper was listed: (l)One strain which could use phenanthrene as sole carbon source was isolated from petroleum and heavy metal containminated soil, which was obtained from old industry depot in northeast China. Based on morphological and physio-biochemical characteristics and homology identification of 16S rDNA sequence, the strain ZX16 was dientified as Sphingomonas aromaticivorans. In the mineral salts medium under initial phenanthrene concentration of 1500mg/L the removal rates of phenanthrene was 98. 2% in 72h. Tolerance of ZX16 to phenanthrene exceeded 2500mg/L and ZX16 was relatively to the cell growth at neutral pH value. The effect of different heavy metal on degrading rate was Cu²⁺>Cd²⁺>Zn²⁺>Pb²⁺ in order. Microstructure of cellsurface screening by Atomic Force Microscopy showed that the volume and morphous of strain had significant changes and the surface also changes from rough to smooth and glossy. The proteome profiles of P25X and mutant strain SNZ28, grown in the presence and absence of the aromatic induced gentisate, were compared after 2D-PAGE. Fifteen distinctive protein spots which were observed only in induced cells of P25X and mutant SNZ28 but absent in non-induced cells of both were further analyzed by MALDI-TOF and Q-TOF. Of the 15 proteins, 12 showed significant sequence similarity to proteins with assigned function in other microorganisms. The identification of protein P4 that showed positive identification to a gentisate dioxygenase from Ralstonia species indicated the putative role of this protein to encode gentisate 1, 2-dioxygenase in P. alcaligenes.The differential proteins expression of ZX16, grown in the presence and absence of phenanthrene and heavy metal(Cd) was separated by 2D-gel method. Among these spots,21 new expressed and 20 increased protein spots were obtained and analyzed by MALDI-TOF/TOF. The protemic profiles included transcriptional regulator, heat shock protein and solute-binding protein.(3) A pair of degenerated primers was designed according to the Q-Tof and N-terminal sequencing of this protein spot. Partial GDOII PCR product was amplified and cloned into p-GEMT. Inverse PCR approach was employed to sequence the gene encoding the putative inducible GDOII. One sequence of 1,047bp was obtained which show high homology with Ralstonia sp. U2. The putative GDOII enzyme was also compared with other GDOs. This study provided a basis for further studying of the evolutionary relationship and ancestral relatedness between GDOII and GDOI or other GDOs in aromatic hydrocarbon degradation pathway.