氧化亞鐵硫桿菌之rus與cyc2基因選殖及表現

Abstract

臭味及毒性氣體是近幾年來常見的環保問題，尤其以硫化物 (H2S) 為惡臭空氣之主要物質。在硫化氫處理法中，化學反應具有高反應性及高污染性，而生物反應則是反應性及污染性均較低，本實驗室先前的研究將兩者結合，進行化學反應之高氧化性及生物反應之再生循環，以達到除臭/再生/減廢之積極目的。 然而自營性氧化亞鐵硫桿菌(Acidithiobacillus ferrooxidans ATCC 19859)之生長速率非常緩慢，使得生物反應中再生作用趕不及化學反應之硫化氫處理，而使系統效率受到限制。希望能藉由了解其亞鐵氧化分子機制，進而利用相關酵素之重組蛋白質來進行研究並取代細菌之反應， 本實驗以A. ferrooxidans ATCC 33020亞鐵氧化途徑作為參考模式，針對細胞外膜蛋白質Cyc2及細胞膜間蛋白質Rus進行研究。利用分子選殖的生物技術，已構築了Rus及Cyc2之表現質體，並於大腸桿菌中大量表現，純化並進行活性測試。實驗結果得到Rus重組蛋白質成功表現於大腸桿菌之細胞膜間並可利用CuSO4進行活性恢復。Cyc2重組蛋白質在大腸桿菌的表現狀況則多為inclusion bodies，自大腸桿菌萃取之細胞外膜蛋白質以分光光度計及heme stain呈色法亦不具cytochrome c 之活性，利用in vitro及in vivo 外加heme 的方式仍是無法得到具有活性之Cyc2，推測是因E. coli 與A ferrooxidans 生長環境差異太大 使得Cyc2的表現為無活性之構型。

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.