Naphthalene Degradation Pathway Study And Cloning,Expression Of Partial Degradation Genes From Rhodococcus Ruber OA1

Polycyclic aromatic hydrocarbons(PAHs)are a kind of pollutants widely exiting in the environment.PAHs have toxic effects on humans,and some classes of PAHs can even cause cancer.The soil has become the hardest hit of PAHs cause the low water solubility of PAHs.Up to now,microbial remediation has become one of the most efficiency treatment method to the contaminated soils,and it were also attracted the attention of scholars gradually.Rhodococcus ruber OA1 was a PAHs degrader isolated by our lab.Our previous study revealed OA1 could utilize naphthalene as sole carbon and energy source for growth.The possible naphthalene degradation pathway of OA1 was deduced by genomic sequence analysing of OA1.The genome sequencing results revealed that the genome of strain OA1 contained 5317putative coding sequences,6 r RNA genes and 46 t RNA genes.A total of 46 genes encoding dioxygenases and 19 hydroxylase genes as well as 14 aldolase genes were predicted that may be related to the degradation of xenobiotic compounds,including PAHs.In order to study the expression feature of key enzyme genes in the process of naphthalene degradation in OA1,a quantitative real-time RT-PCR(q PCR)analysis method were used to analysis the expression level of genes of those key enzymes like naphthalene dioxygenase(NID)and protocatechuic 3,4-dioxygenase(3,4-PCD)et al.The specific primers were designed based the genomic sequence of OA1.The c DNA used in this study were obtained from the thallus which was induced by naphthalene.The results showed that the expression of NID and 3,4-PCD genes was induced by naphthalene but the other genes involved in this study were defined as uninduced for constitutive expression.In this study the catechol 1,2-dioxygenase(C12O)from R.ruber OA1 was successfully expressed in E.coli BL21(DE3)for the first time and its catalytic characteristics were explored in detail.The results showed that C12O had the ability to degrade catechol to cis,cis-muconic acid with a specific enzyme of 231.4 U/mg protein at 25?,p H 7.4.The optimal reaction conditions of C12O were 25? and p H 7.0.Besides,Mn²⁺could increase the activity of OA1-C12O,while Mg²⁺and NH₄⁺inhibited its activity.The acetaldehyde dehydrogenase(ALDH)from R.ruber OA1 was successfully expressed in E.coli BL21(DE3)for the first time and its catalytic characteristics were explored in detail.The results showed that ALDH of R.ruber OA1,not only had ALDH activity,but also had the activity to catalyze the hydroxylation of formaldehyde,acetaldehyde and benzaldehyde.This is the first report of ALDH from Gram-positive bacterium that can catalytic salicylaldehyde and have a better catalytic efficiency to aromatic aldehydes than to aliphatic aldehydes.Based on the comparative analysis of the ALDH amino acid sequence and the structure of ALDH,it was found that there were two residues Ser122 and Ala124 were close to the putative active site.This might affect the characteristic of the enzyme and its catalytic ability.This study was aimed to elucidate the mechanism and the key enzymes revealed in the process of naphthalene degradation of strain OA1.This study has significance to alteration of gene engineering strain which will contribute to the integrated control of environmental pollution like PAHs.