The Isolation And Purification And Characterization Of Sulfite Acceptor Oxidoreductase

The biofilm immobilized on the magnetic porous beads obtained from the aerobic magnetically stabilized bed (aMSFB) was enriched cultured under the aerobic condition by using culture medium for thiobacillus thioparus, and isolated and purified repeatedly to get the isolated bacteria. The isolated bacteria was identified as thiobacillus thioparus of thiobacillus according to《Bergey's Manual of Systemaic Bacteriology》through the observe of the bacteria and the morphology of the bacteria clone and a series of physiological and biochemical index.The isolated and purified thiobacillus thioparus bacteria were broken by using ultrasonic,and then desalted condensed. sulfite acceptor oxidoreductase was obtained through isolation and purification by using DEAE-52 ion-exchange chromatography and Sephadex G-150 molecular-exclusion chromatography and was characterized for its properties. The spectra analysis indicated that the enzyme had no other characteristic adsorption peak except in 280nm. SDS-PAGE showed that the enzyme was aαβheterodimer constituted by two subunits with molecular weights 33.9kDa and 42.2 kDa. Activity analysis indicated that 92% of enzyme existed in the cell membrane and the enzyme was a membrane protein. The optimum pH of the enzyme was 7.5, the optimum temperature of the enzyme was 40℃and the enzyme activity had good stability and the enzyme activity could maintain up to 70% within the temperature range of 3℃-50℃. The Vmax and Km of the enzyme to sodium sulfite as substrate were 22.15μM/min and 86.09μM respectively. The enzyme had high substrate specificity and could not be influenced by metal ions. EDTA and NaOH had obvious inhibition to the enzyme and the oxidative product was sulphate.According to the latest study of sulphur oxidative bacteria, the cytoplasm extracts and the membrane extracts of the isolated bacteria were fetched and the activity of the key enzymes in the physiological and biochemical metabolism was determined to judge the location of the enzymes. The possible sulphur metabolic path in thiobacillus thioparus was conferred through the analysis of the reactive paths catalyzed by different enzymes and the reference to the relative literature.The sulphur oxidative system model of thiobacillus thioparus was put forward based on the above facts.