| Summary: | 碩士 === 國立交通大學 === 生物科技系所 === 93 === Air pollutants, especially H2S, have caused serious environmental problems. To solve this problem, our lab has developed a system which has combined the advantages of both the chemical oxidation and the biological regeneration trying to remove H2S. The bacteria used in the biological regeneration is Acidithiobacillus ferrooxidans ATCC 19859. But the slow growth rate of this bacteria has limited the efficiency of this system. Therefore, we hope to improve this system through investigating the ferrous oxidation mechanism of this bacteria.
The outer membrane protein Cyc2 and the periplasmic protein rusticyanin (Rus) were proposed to play key roles in the ferrous oxidation pathway in A. ferrooxidans ATCC 33020. Hence, we tried to identify whether they play similar roles in A. ferrooxidans ATCC19859.
Cyc2 and Rus expression plasmid were constructed, expressed and purified in E. coli. for activity assay. It was found that the recombinant Rus proteins were expressed in periplasm in soluble form and the purified Rus proteins were able to be reconstituted with CuSO4. Alternatively, the recombinant Cyc2 proteins were almost in the form of inclusion bodies. Moreover, no cytocrome c activity which is characteristic of Cyc2 can be detected from the extracted E. coli outer membrane by spectra and heme staining. In order to solve this problem, we have tried to reconstitute Cyc2 activity by heme addition in vitro and in vivo but still in vein. It is possible that the different growth condition between A. ferrooxidans and E. coli causes the Cyc2 proteins expressed in non-functional form in E. coli.
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