Screening Of PAH-degrading Bacteria And Degrading Mechanism Study Based On The Structure Of Target Enzyme

In this study, we isolated two high-effective PAHs degrader from crude oil, and the biodegradation ability, degrading products, biological activity and utilization level for substrates were studied. The 3D crystal structure of target enzyme was obtained by homology modeling, and thus the binding status and interaction mode between PAHs and target enzyme were analyzed. The detailed research contents were discussed as follows.1. In this study, two high-effective pyrene- and fluoranthene-degrading strains were isolated from Dagang Oilfield. The identification results of molecular biology showed that the pyrene- and fluoranthene-degrading strains were belonged to the Pseudomonas and Microbacterium, and named as Pseudomonas. sp. JPN2 and Microbacterium paraoxydans JPM1, respectively.2. The strain JPN2 could degrade 82.88% of pyrene after 25 d culture. Five degrading products including 4,5-dihydroxy-4,5-dihydropyrene,4-phenanthrol, 1-hydroxy-2-naphthoic acid,1-hydroxynaphthalene and phthalic acid were detected in the liqiud degradation system. And thus we deduced that the degradation of pyrene in JPN2 started from the dihydroxylation at C4-C5, and a phthalic acid pathway was involved in the degradation process of pyrene. The strain JPN2 possessed an extensive range for substrates. It could utilize the other 15 aromatic and non-aromatic compounds as growth substrates. Among them, JPN2 was able to utilize salicylic acid as sole growth substrate, and it indicated that the degradation pathway of salicylic acid simultaneously existed in the strain JPN2. In addition, the result of microcalorimetry displayed that the strain JPN2 had high tolerance for pyrene, and the growth activity of JPN2 was almost unaffected at the 100-400 mg/L concentrations of pyrene.3. A nahAc gene encoding a subunit of naphthalene dioxygenase in the strain JPN2 was amplified by PCR. The amino acid sequence of target enzyme was obtained by translation based on ORF analysis of gene sequence. A crystal structure model of a subunit of naphthalene dioxygenase (JPN2-NDO) was built by homology modeling using a template 107N. The optimization of molecular dynamics and evaluation of structural rationality by Ramachandran plot were performed for JPN2-NDO.4. The interaction mechanism between the active site of JPN2-NDO and pyrene was studied by molecular docking method. The theoretical analysis displayed that the C4 and C5 atoms on the pyrene ring were the closest positions to the two oxygen atoms, and the interactions were formed between them. In theory, the sites were easy to occur dihydroxylation reaction by the activated oxygen atoms. This theoretical result is line with the identification of degrading products of pyrene. Finally, the biodegradation mechanism of pyrene in the strain JPN2 was illuminated based the results of identification of metabolites and theoretical analysis.5. The biodegradability of fluoranthene in the strain JPM1 was positively correlated with the growth activity of this bacterium. After 25 d cultures, the degradation percent of fluoranthene was 91.78% by the strain JPM1. Four metabolites including 9-fluorenone-l-carboxylic acid,9-fluorenone, phthalic acid and benzoic acid were detected in the degradation process of fluoranthene. The strain JPM1 was able to utilize the other 12 carbonaceous compounds as sole growth substrates. The results of microcalorimetry displayed that the low concentration (25 mg/L) of pyrene could promote the growth activity of JPM1 No significant inhibition for the growth activity of JPM1 was observed at 100-400 mg/L fluoranthene. The inhibition percents increased to 12.80% and 30.34% at 1600 and 3200 mg/L fluoranthene, respectively. At the low concentration of pyrene and fluoranthene, JPN2 and JPM1 showed similar inhibition level, but significant differences were observed at their high concentrations.6. The structure alignment displayed that six sites of amino acid were different between target JPN2-NDO and template 1O7N. The hydrophilic Thr308 in 1O7N was replaced by hydrophobic Val232 in JPN2-NDO. This replacement could increase the hydrophobicity of the active site, and it was beneficial for the combination of pyrene. Meanwhile, Ser225, Met306, Try358 and Glu359 were replaced by Glnl49, Ala230, Tyr267 and Ser268. The replaced residues were smaller in size, but their polarities remained unchanged. Additionally, a hydrophilic Tyr207 was replaced by hydrophobic Leu131 with a smaller size. This replacement was beneficial for reducing the steric hindrance effect of the active cavity and the cavity entrance. It is favorable for the combination of high-molecular-weight PAHs with the active site. The structural differences in the active sites of enzymes may be a major factor leading to the different bioactivity in the diverse of bacteria. This study provides a corresponding theoretical basis for the further gene modification and transformation, as well as the practical application of PAHs degrader in the polluted sites.