Functional Analysis Of A Hydroxyquinol 1,2-Dioxgenase Gene Involved In Catabolism Of P-Nitrophenol By Pseudomonas Putida Strain DLL-E4

p-Nitrophenol(PNP) have been widely utilized in the agricultural, and chemical industries. Due to their stability and high water solubility, these pollutants spread rapidlyand then persist in the environment. However, the large quantities of nitrophenols into the environment has enabled some microorganisms to develop the ability to degrade these compounds. So far, several strains which can degrade nitrophenols have been isolated, such as Pseudomonas Putida DLL-E4.The strain Pseudomonas putida DLL-E4 degrading methyl parathion (MP) was isolated from soil polluted by MP for long term, and it can use p-Nitrophenol(PNP) as sole carbon and nitrogen sources.SEFA PCR was used to clone Flanking DNA Sequences of pnpC which has been cloned by Deng Haihua. Analyses of potential open reading frames (ORFs) and comparison of amino acid sequences (or nucleotide sequences) were performed with the ORF finder and BLAST programs on the NCBI website. The pnpC nucleotide sequence indicated that the DNA fragment was 873 bp with an open reading fragment, encoding 290 amino acids and showed a significant level of homology (67%) to hydroxyquinol 1,2-dioxygenase of Burkholderia ambifaria AMMD 17616 (GeneBank accession No.EAO48077).The pnpC gene was expressed using a vector pEÎ¥-29b in Escherichia coli BL21.A expected 32kD protein was detected from a positive clone(pET-pnpC) by SDS-PAGE.As described above, pnpC was presumed to encode a hydroxyquinoll,2-dioxygenase(PnpC). Spectroscopic characteristics were found during conversion of hydroxyquinol with a distinctive peak at 244 nm,which indicated the formation of maleylacetate. The specific activity of PnpC was 0.777±0.014μmol/minute/mg protein.The rate of catechol transformation by PnpC is 7.13% of the rate of hydroxyquinol cleavage. During cleavage of catechol, the absorbance maximum at 275nm decreases and absorbance at 260 nm increases. PnpC is inactive against hydroquinone and resorcinol. The optimal reaction temperature of PnpC is 30℃. The optimal reaction pH of PnpC is 7.5, the activity of PnpC in alkaline pH range is stronger than that in acidic pH range. Activity of PnpC drop for original 35.84% by addition of EDTA, but Li⁺,Zn²⁺ Mn²⁺,Fe³⁺,Cu²⁺,Fe²⁺have certain activation of activity of PnpC.W e conclude PnpC is a metal enzyme. Oppositely, Cd²⁺,Ni⁺,Co²⁺ inhibit the activity of PnpC.A hydroxyquinol 1,2-dioxygenase gene (pnpC) insertional inactivated mutant (DLL-ApnpC1) and a pnpC knock-out mutant (DLL-ApnpC) were constructed by homologous recombination from Pseudomonas putida DLL-E4. Insertional inactivated mutant (DLL-ApnpC1) lost the ability of degrading p-nitrophenol(PNP) and hydroquinone (HQ). But pnpC knock-out mutant (DLL-ΔpnpC) recovered the ability of utilizing PNP and HQ at a lower degradation rate compared with DLL-E4. These results suggested that pnpC was related genes in catabolic PNP in DLL-E4. For preparation of the crude cell extracts of DLL-ΔpnpC and DLL-E4, two strains were induced by PNP and catechol respectively. The difference between DLL-ΔpnpC and DLL-E4 on crude enzyme's activity against catechol was studied with ammonium sulfate precipitation method. The result indicated that there is another dioxygenase in DLL-ΔpnpC which substitute the function of pnpC in catabolism of PNP.The reported catechol 1,2-dioxygenase genes from Pseudomonas were aligned using Clustalx software and degenerated primers were designed according to the conserved sequence and cordon bias of Pseudomonas. Two diffenent catechol 1,2-dioxygenase was amplified from DLL-E4 genomic DNA.