Isolation, Purification And Properties Of APS Reductase And Sulfate Reductase From Sulfate-reducing Bacterium

This paper Presents a research on the production , isolation and characterization of the possible rate-limiting enzyme(sAPS reductase and sulfate reductase)in the sulphate reduction metabolic pathway from the immobilized sulfate-reducing bacterium in an MSFB.The fermentation conditions including the optimum enzyme production time, PH and temperature of sulphate reducing bacteria producing APS reductase and sulfate reductase were reported, in order to provide basis for industrialization of these two enzymes.The two enzymes were purified to electrophoretic homogeneity by ammonium sulfate fractionation, dialysis, Sephadex G-150 and DEAE-52 chromatography; A 116-fold-purification of APS reductase was achieved. The specific activity and recovery were 70.17U/mg and 18.36%. As to sulfate reductase , the fold-purification was 132 , the specific activity and recovery of this enzyme were 70.17U/mg and 19.8%;Kinetic studies on the two isolated enzymes indicated that Km of APS reductase is 2.32 mmol/L for K₃Fe(CN)₆ and 2.43mmol/L for Na₂SO₃ ,respectively. As to sulfate reductase , Km of it is 2.06mmol/L; The optimal temperature and pH for the activity of APS reductase and sulfate reductase were 30℃, 34℃and 7.8 , 7.6,respectively. Both the two purified enzymes were very stable. At 70℃, there was still about 55% activity left. They were also very stable at pH 4 to 10, there were still more than 60% activity left ; APS reductase was partially inhibited by its reducing production SO₃^2-,AMP , Nucleotide coenzyme ATP,ADP,cAMP,GTP,cGMP has little influence about it yet. The sulfate reductase activity was strongly inhibited by PCMB,KCN,Ca2+,Co²⁺,Cu²⁺,Hg²⁺, moderately by Zn²⁺,Mn²⁺; SDS-PAGE indicated that both the two Enzyme wereαβ- heterodimers, withαmolecular mass 66.7KDa andβmolecular mass 29.4KDa of the APS reductase and withαmolecular mass 43.6KDa andβmolecular mass32.3KDa of the sulfate reductase , respectively; Spectral analysis revealed that the APS reductase had absorbition maxima at 388nm and 440 nm in accordance with the presence of FAD and two interacting [4Fe-4S] clusters. The sulfate reductase heterodimers contains the sirohemes and [4Fe-4S] clusters.