Expression，Purification And Characterization Of The Gentisate 1，2-dioxygenase Gene From A Halophilic Martelella Strain

PAHs (Polycyclic Aromatic Hydrocarbons, PAHs) are the typically toxic compounds, widespread in the soil polluted with oil, oil pollution aways resulting in high sality, high pH and high concentration of PAHs. Normal bacteria were introduced to remove the PAHs in saline-alkali soil, but they can not work well. Considering the low efficiency of bioremediation of PAHs in saline-alkali soil, there is a growing concern to the use of moderately halophilic bacteria to degrade the contaminated soils. So, it is the prerequirzation to illuminate the mechanism of PAHs degradation and key enzyme catalytic mechanism, for biodegredation.In this study, Martelella sp. AD-3 was selected, it can degradate PAHs rapidly at high salt conditions. GDO gene was cloned and expressed in E.coli21(DE3), based on the genomic sequence, and GDO was purified by affinity chromatography, then its enzyme activity was detected. To learn the mechanism of degradation and salt-tolerant for sp. AD-3, different proteomics researches were preformed. AD-3 were cultured at different conditions:one condition was used PAHs as the sole carbon sources under high salinities, the other one used glycerol as the sole carbon source under low salinity. Then, analysis the different expression of proteins in cultured Martelella sp. AD-3 by Label-Free. At last, the gene-knockout system for Martelella sp. AD-3 was constructed, based on the CRISPR-Cas9 system. Concluded as follows:(1) A 1,077 bp long GDO gene was cloned from sp. AD-3, and expressed in E.coli21(DE3), the molecular weight of GDO was detected by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and native-polyacryl-amide gel electrophoresis, its unnative molecular weight was 38 kDa and its native molecular weight 120 kDa, it was proposed that GDO was a homologous trimer. The optimal reaction conditions for GDO was pH 7.0, temperature 30℃ and salinity 12%, its Km and kcat values to gentisate were 26.64 μM and 161.29 s-1. According to the isotope labeling experiment, it was proposed that the enzyme-catalyzed O2 attacked on the aromatic ring of gentisate were from H2O and O2, respectively. Additionally, four highly conserved histidine residue were substituted to neutral lysine residue, enzyme analysis indicated that any substitution of these four histidine residues had resulted in the complete loss of catalytic activity, so all the four highly conserved histidine residue were improtant for GDO.(2) Analysis of the differential expression of proteins in strain AD-3, which was cultured at different conditions.187 up-regulated proteins were indentified, include 16 proteins for the degradation of PAHs, and 8 proteins for salt-tolerant; Combined the result of differential proteomic with the genome sequence of strain AD-3, an important initial enzyme related to the degradation of PAHs was found, it contains two subunits α and β, their foldchanged times were 149.7 and 41.0, respectively; Proteins for salt-tolerant were also found, including an improtant gene related to synthesis of glycine betaine, its expression increased 19.1 times, and a gene related to glycine/betaine tranpoter ABC, its expression increased 6.6 times.(3) The plasmid pTC-gdo-3580 was constructed for knockouting GDO gene in sp. AD-3, based on the CRISPR-Cas9 system. Electransproted the plasmid pTC-gdo-3580 to strain AD-3 competent cells, the monoclone was filtered by antibiotics ampicillin. After induced by IPTG, the mutant strain AD-3-pCT-gdo-3590 was generated. The wild type AD-3 can use gentisate as solar carbon source with great color change of medium. Compared with wild AD-3, the color of medium used for cultivating AD-3-pCT-gdo-3590 had not changed obviously. It means that GDO gene was knocked out. This work can provide a useful tool to identify the function of more unknown genes in strain AD-3.