Biodegradation Of Aromatic Compounds And Construction Of Genetically Engineered Bacterium For Aromatic Compounds Biodegradation

Three aromatic compounds degrading bacterial strains ZD 2, ZD 4-1 and ZD 4-3 were isolated from the sludge of pesticide manufacturing factory and the soil around the coal gas factory. All these isolates are Gram-negative, aerobic, non-spore-forming rods. Based on phenotypic characteristics and 16S rDNA sequence alignment, strains ZD 2, ZD 4-1 and ZD 4-3 were identified as Sphingobacterium multivorum, Comamonas testosteroni and Pseudomonas aeruginosa, respectively. The result of phylogenetic analysis showed that strain ZD 4-1 and ZD4-3 were positioned in two different subclusters. Cometabolism of pentachlorophenol (PCP) was studied by using Sphingobacterium multivorum ZD 2 to investigate the metabolism mechanism of PCP. The effect of PCP degradation with the phenol or glucose as growth substrates was compared. The results revealed that not only the PCP but also the phenol inhibited the bacterial cells and glucose was better than the phenol as a growth substrate for PCP cometabolism because there was no competitive inhibition between the glucose and PCP. Protein electrophoresis results showed that the unspecific enzymes for PCP degradation were induced by the non-growth substrate PCP itself when glucose was served as growth substrate. Gas chromatograph-Mass (GC-MS) analysis indicated that phenol was one of intermediate products during PCP cometabolism, suggesting that the dechlorination reaction increasingly happened even on the condition of aerobic biodegradation process.To investigate the characteristic and biochemical mechanism about the biodegradation of aromatic compounds by bacteria, bacterial strains ZD 4-1 and ZD 4-3 were used to degrade phenol. The identification of the intermediates and the detection of the corresponding catabolic enzymes in crude extracts indicated that the strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and wefar-pathways, respectively. The results of induction experiment showed that the catechol dioxygenases, both catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O), were all inducible. Finally, the results of biodegradation and enzyme assays showed that the biodegradation efficiency of phenol by meta-Tpathways was higher than that by ortho-pathways but the former is in an inferiorityposition in terms of adaptation to pH fluctuation and of growth ability on other aromatic compounds compared with the latter.C23O is one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta-position of dihydroxylated aromatic substrates. C23O is able to catalyze the conversion of catechol to 2-hydroxymuconic semialdehyde (HMS). Based on curing experiment, PCR identification and Southern Hybridization, the C23O encoding gene pheB was localized on a 3.5 kb EcoRI/BamHI fragment and was then cloned from P. aeruginosa ZD 4-3 that was able to degrade both single and bicyclic compounds via a meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it has one open reading frame (ORF) encoding 307 amino acids, which showed a strong overall amino acid similarity to the known Gram-negative bacterial mesophilic C23Os. The heterogonous expression of the pheB gene in E. coll BL21 indicated that the activity of C23O was 3.5617U/mg protein and was higher that that of the C23O in wild bacterium.In order to probe the relationship of the structure and function of C23O encoded by pheB gene, the alignment was performed by comparing the homology between the pheB deduced amino acids sequence and the other extradiol dioxygenases by the Bioinformatics software. The alignment analysis showed that 12 homology areas aroused during the whole sequences and these homology areas mainly distributed near the C-end and N-end. According to three-dimension crystal structure of the known extradiol dioxygenases, the amino acids Gly-31, His-157, Leu-184 were speculated to be the active sites of the C23O in this study. Moreover, there are evident differences between the extradiol dioxygenases which degrade single ring aromatic compounds, and those bicyclic...