Cloning And Expression Of Dioxygenase Gene From Aeromonas Sp.XJ-6 And Promoting Degradation Of Tyr

Dioxygenase is a sort of enzymes from bacteria,which was involved biodegradati on of the aromatic hydrocarbons.The enzyme can active oxygen in the air,and double oxidize of aromatic hydrocarbons on the ortho-position carbon. For example,In aer obic conditions,Bacteria attacks the benzene ring usually by starting dioxygenase,adds two oxygen atoms to substrate formed in the two-plus oxygen ethane,further oxidation became cis-dihydrotestosterone ethanol later,Hydrochlorothiazide ethanol can continue to be oxidized intermediate metabolites catechin,protocatechuic and gentisic acid,etc.. And then the benzene ring is disconnected,produce succinic acid,fumaric acid,acetic acid,pyruvic acid and acetaldehyde.The products of degradation are used to synthesize their own biological energy by microorganisms,while producing water and CO2.This is a kind of enzyme with biological function and application prospect due to its important role in the process of degradation of aromatic hydrocarbons.Based on the above,a strain of pyrene with strong degradation was obtained in th e surface soil of the oil well of Karamay Lu Liang oil field of Xinjiang Uygur Autono mous Region,namely Aeromonas.sp XJ-6(GenBank accession number:KT952518),this study have been cloned a segment of dioxygenase gene dio6 from genomic DNA by strain-specific primers(GenBank accession number:KT952519).A recombinant E.coli expression system which can efficiently express the soluble double enzymes was constructed,and the dio6 with high purity and high viability was obtained.The cloning and expression of dio6 in E.coli expression system was achieved.the recombinant pro tein was expressed and purified,and its degradation function and basic properties of amino acid tyrosine(Tyr) were determined.the basic structure of the recombinant prot ein was predicted. Finally,the main results are as follows:(1)In this study,the surface soil of oil well is the source of bacteria,after a step by step acclimation,screening,isolation,10 strains of pyrene degrading bacteria were obtained.Morphological properties and 16 S rDNA were used to identify the isolated strains.the strains that have formed a clear transparent circle were selected,and the degradation ability of pyrene was detected by HPLC,and the highest degradation ability of the str ain was Aeromonas sp.XJ-6.(2)Based on the gene sequence of Aeromonas sp. XJ-6(GenBank accession number:KT952518),we cloned partial DNA sequences encoding dioxygenase protein of XJ-6from genemic DNA through PCR amplification,and connecting the dioxygenase gene dio6 to pMD-19 T.Sequence analysis have showed the amplified fragment of the dioxy genase dio6 gene.Through the pET-28 a and pMD-19T-dio6 respectively double enzy me digestion and T4 ligase to construct recombinant plasmid pET-28a-dio6,by polym erase chain reaction(PCR) and a small amount of enzyme digestion successfully ide ntified and sent to sequencing jointly identified recombinant plasmid construction is completed.(3)The prokaryotic expression vectors of dio6 were transformed into expression host strain BL21(DE3),the cells were cultured to exponential phase,adding IPTG,the final concentration is controlled at 100 uM,high expression of protein dio6 was obtained at16 °C under the induction culture of 16 h.SDS-PAGE showed that there was a dio6 ba nd,and the presence of soluble recombinant proteins in the form of easy to be purified;Metal chelate affinity chromatography(MCAC) expression product size was about 44.9 kDa after purification of dio6.HPLC identification showed that dio6 had a strong effect on the degradation of Tyr.TLC and HPLC tests showed that the Tyr was degrad ed rapidly under the conditions of 60 L(0.6 mg/mL) and 30 C reaction temperature.Mg2+and Ca2+ can slightly inhibit the enzymatic reaction, Mn2+、Zn2+、Cu2+、Fe2+ and Ca2+ can promote the degradation of substrate.Among them, Mn2+ have the biggest effect for dio6.LC-MS analysis under the action of dioxygenase dio6,Tyr degradation products are mainly fumaric acid.(4)The Aeromonas sp.XJ-6 dio6 was analyzed using the bioinformatics software, including the partial sequence of the vector,a complete ORF containing 1194 bp nucleotid es,encoding 397 amino acids.ATG plays a start codon,TAA plays termination codon.Predicted by the Prot Param online, dio6 derived from the theory of molecular mass is44995.8 Da,isoelectric point(PI) is 5.86,the extinction coefficient in aqueous solution is 70275 M-1cm-1 in 280 nm,the total average hydrophilicity is-0.351.Hydrophobicity analysis software Protscaletool/Kyte&Doolittle predicts that the dio6 gene N terminal,C terminal are hydrophilic;Based on the method of TMHMM can predict transmembr ane domain of dio6,the protein is a type of membrane exocrine protein.The secondary structures of dio6 were forecasted mainly α-helix(15.34%),extended strand(27.14%),beta turn(12.98%) and random coil(44.54%) by Predict Protein method.It is indicates that the protein of dio6 protein belongs to mixed type secondary structure.Containing the signal peptide in the 1-26 sequence of dio6.At last,Application online tools of Rap torX predicts the tertiary structure of dio6.