Isolation, Purification And Properties Of Sulfide Dehydrogenase

Isolation of the bacteria was performed from aerobic magnetic stabilized fluidized bed reactor of flue gas biodesulfurization system. The immobilized biofilm samples were cultured on Thiobacillus enrichment media under aerobic conditions. The isolated cultures were obtained through repeated separation and purification. The isolated bacteria was identified to find out that: the morphology of the bacteria was rod-shaped; the average size of the bacteria was 0.3～0.5×1.1～1.5μm; the optimum growth temperature was 28oC; the optimum PH was 7.0. The results of gram staining for the bacteria indicated that the bacteria were negative with flagella on its side. The colonies on Thiosulphate agar media were round-shaped with bump surface, integrated fringe and the diameter of them is about 1mm-2mm. The colony was transparent first, then it became white for the sulphur sediment and at last the centre of the colony turned brown. After the long-term culture, the media PH dropped and the elemental sulphur was yielded. The film with sag fibers was formed when the samples were cultured stably in the liquid. The bacteria were strictly autotrophic and they derived energy for growth from the oxidation of thiosulphate, sulphide, Na₂S₄O₆ with nitrate and ammonium as nitrogen source to accumulate elemental sulphur particles. Then elemental sulphur particles may be oxidated continually. According to Bergey's Manual of Determinative Bacteriology, the culture was elemently identified as Thiobacillus thioparus of Thiobacillus genus.A novel membrane-bound sulfide dehydrogenase enzyme was purified from the neutrophilic, obligately chemolithoautotrophic Thiobacillus thioparus by means of a four-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 42.6 and 51.3 kDa. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide dehydrogenase heterodimer contains one molecular of haem c and one molecular of flavin. It appeared that the sulfide dehydrogenase is a member of a small class of redox proteins. The pH optimum of the enzyme is 8.5. The Vmax was 4.9±0.1μmol cytochrome c(mg protein×min)^-1, and the Km values for cytochromes and sulfide were 2.1±0.3μM and 6.1±0.8μM, respectively. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product.