The effect of hydrogenase2 expression on physiology in Escherichia coli

• Main Authors: Wu, Hui Fan, 吳惠芬
• Other Authors: Lin, Chan Shing;Liu, Jong Kang
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/51835077710436367962

碩士 === 國立中山大學 === 海洋資源學系 === 84 === Under aerobic metabolism, Escherichia coli produces reductants via the electron transport chain in which oxygen is used as a terminal acceptor to form H2O and generates energy by oxidative phosphorylation. However, in anaerobic condition, E. coli will shift its metabolic pathway to utilize hydrogen as a energy source. Hydrogenase-1 and hydrogenase-2 catalyzed the oxidation of hydrogen and generated energy via the electron transport pathway and substrate level phosphorylation. The hyb D, which belongs to the fourth gene of hyb operon gene products, was suspected to be involved in processing the activation of the large subunit of hydrogenase-2 based upon its homology to hya D. However, no experimental data were provided. In this study, I cloned hyb D gene and studied the hyb D effects on hydrogenase-2 activity. It was found that the culture of pHYD203 and pHYBD under IPTG induction will reduce the hydrogenase activity de novo. In the expression of hyb D alone, one can obviously observe a 17.5 KD protein in the SDS- PAGE. The HYBD protein was purified by anion exchange chromotography and gel filtration chromotography. Casein-FITC and hydrogenase-2 crude extract were used as subtrate to determine HYBD protease activity. In vitro HYBD can process hydrogenase-2 to be mature and high activity.