Purification And Characterization Of Flavin Reductase Involvedin Desulfurization By Mycobacterium Goodii X7C

Sulfur dioxide released into the atmosphere during combustion of fossil fuels is a serious environmental problem because it contributes to air pollution and is a major cause of acid rain. Currently, hydrodesulfurization (HDS) is the leading industrial measure used to remove sulfur compounds from fossil fuels. What the process need are high temperature, pressure and metal catalyzers, and it is effective to remove not only inorganic sulfur but also simple organic sulfur compounds. Nevertheless, when it comes to the condensed heteroatom sulfur, HDS is not so efficacious. So this means tends to be of fewer and fewer effect, as the legislative limit on emission of sulfur becomes more and more severe. Recently, the biodesulfurization (BDS) develops as an alternative method for desulfurization, because of its specificity for organic sulfur and availability of all temperature. What's more is that BDS could remove sulfur compounds remained after hydrodesulfurization. A bacterial strain of Mycobacterium goodii, which could grow and desulfurize DBT at 45℃, was obtained by our group. This strain is a rod-shaped bacterium with the dimension of 0.8-0.1x3-4 μm; It is not readily stained by Gram's method, usually weakly Gram positive; Acid fast, nonmotile, nonsporing; without conidia or capsules; Aerobic and catalase negative. This strain had glutamic acid, alanine and the meso form of diaminopimelic acid (DAP) in the cell wall. The major cell wull sugars were arabinose and galactose. It can grow on MacConkey agar without crystal violet and in the presence of 5 % NaCl. After ultrasonication, the activity of flavin reductase from M. goodii X7C was higher than that of Rhodococcus erythropolis IGTS8.Because of being endocellular enzyme, it must be released byultrasonication first. According to former experiences, we gain the optimization of ammonium sulfate precipitation. The enzyme was purified to electrophoretic homogeneity by protamine sulfate precipitation, Butyl-Sepharose Fast Flow chromatography (two times), Sephadex G-100 chromatography , Superdex 75 chromatography and Q-Sepharose Fast Flow chromatography with the recovery of 1.9% activity. The 9 amino acid sequence of N-terminal was VNQQQADKM, which has no homologous sequence in GenBank. Therefore, the gene of Flavin Reductase should be gotten in a genomic library of M. goodii X7C.DszD from M. goodii X7C is confirmed to be facultative thermophilic enzyme whose optimal pH is 8.0 and optimal temperature is 45°C. Different from DszD of R. erythropolis IGTS8, the substrates of it are all of NADH, FMN, FAD and NAPH. It owns the uniform activity when NADPH or NADH exists. While its activity by FAD is 30% of FMN. And the Km of NADH was 490.1 uM. The native molecular weight is found to be around 35 KDa by gel filtration and the Flavin Reductase is presumed to be a single subunit.It's a conventional means to screen gene sequence with genomic library after the primer was designed according to the N-terminal of this protein, The pUC19 was used as the vector and the size of inserted fragment was between 4 kb and 6 kb. The genomic DNA of stain X7C digested by Sau3A I beforehand was ligated to pUC19 which was digested by BamU I and treated by Alkanine Phosphatase previously. Successful conversion of long fragments made the screening work to be achieved. Furthermore, the verification of hybridization signal is approaching.