Expression, Purification And Characterization Of Cysteine Desulfurase From Acidithiobacillus Ferrooxidans (AF3), Sulfobacillus Acidophilus

Acidothiobacillus ferrooxidans (abbreviation:A. ferrooxidans) has an important position in the bioleaching. It can oxidize ferrous, sulfure and patially oxidize sulfur compounds. And A. ferrooxidans is the model species of bioleaching bacteria. In recent years, there have been many researches on ferrous and sulfure oxidation of A. ferrooxidans. In the previous work of our laboratory, cysteine desulfurase IscS was upregulated, when A. ferrooxidans utilizes elemental sulfur as its sole energy source. IscS, a cysteine desulfurase, is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the conversion of L-cysteine to both L-alanine and sulfan sulfur by forming a protein-bound cysteine persulfide intermediate on a conserved cysteine residue.In this study the IscS gene of A. ferrooxidans was cloned and expressed in Escherichia coli. Whether on the LB agar plate or in the LB medium, the pET-His-IscS/BL21 grows faster than the pET-His/BL21 (the vector control). IscS can promote the growth of E.coli in the LB medium. In the LB medium adding 2.5% sodium thiosulfate the pET-His-IscS/BL21 can grow but pET-His/BL21 can not. The results suggested that the IscS can promote the growth of the bacterial and help the bacterial to metabolize sulfur. The reason may be that sulfur activation must occur before oxidation because elemental sulfur consists of a stable octasulfane ring system (S8) that forms orthorhombic crystals with extremely poor water solubility. The activation reaction could be an opening of the S8 ring by nucleophilic reagents that causes the formation of linear inorganic. The cysteine residue of IscS can attack the S8 to the linear inorganic. Further explored by immunoprecipitation, there is a protein Short-chain dehydrogenases/reductases which interact with the IscS in vivo.However, with the demand for mineral resources increasing, the growth temperature of bacteria have become more and more important. So the moderate thermophilic acidophic bacteria has an important position in the bioleaching slowly.In the study, same research methods were used in the cysteine desulfurase SufS of Sulfobacilhs acidophilus(TPY). The results showed that SufS also can promote the growth of E. coli in the LB medium. The expression of the protein increased with the growth of bacteria by the western verify from the molecular level. And E.coli can grow faster, when purified protein was added in LB medium. In the LB medium adding 3% sodium thiosulfate the pET-His-SufS/BL21 can grow but pET-His/BL21 can not. The results suggested that the SufS can also promote the growth of the bacterial and help the bacteria to metabolize sulfur. And the mechanism of sulfur metabolism was similar to IscS.In vitro, the activity of SufS was determined by Methylene blue method. This suggested that the purified protein have activity in vitro. Further explored by immunoprecipitation, there is a protein which interact with the SufS in vivo. But the results of mass spectrum are not present.