• 作者： Chang, Jo-Shu; Chao, Yun-Peng; Fong, Yin-Ming; Hwang, Yuh-Ping; Lin, Ping-Jei
• 中文摘要： 本研究將質體pHP45Ω-Hg上所含源自跳躍基因Tn501之抗攻基因（汞操縱子）剪接鑲入含tac啟動子之質體pHP118EH上，並將該基因重組質體轉形至大腸桿菌 VJS632QA，以建構具抗汞性之基因重組菌體大腸桿菌PWS1。該基因重組菌體可抵抗高達80mg/L的汞離子，而其比汞還原活性亦較野型之綠膿桿菌PU21為高。本研究探討誘導劑（IPTG與汞離子）對PWS1菌體之汞還原活性的影響，並決定該菌體全細胞、穿透細胞、細胞粗萃液及純化酵素之汞還原動力學模式。結果顯示，汞操縱子基因之表達受到IPTG與汞離子之誘導，而0.4mM IPTG對汞還原活性之誘導強度優於5mg/L之汞離子。PWS1菌體之全細胞酵素在起始汞離子濃度為16mg/L時有最大之汞還原活性，而其它三種酵素型態之最佳活性則出現在起始汞離子濃度為10-12mg/L時。由於穿透細胞之活性大於全細胞，顯示汞離子穿越細胞膜進入胞內之質傳阻力明顯地影響整個汞還原作用之速率。純化酵素之最大活性為0.41μg Hg/mg total protein/sec，約為粗萃酵素最大活性之120倍。本研究亦發現汞還原活性與起始汞離子濃度之關係可利用基質抑制與酵素失活模式來描述。
• 英文摘要： Transposon Tn501-originated mercury resistance determinant (mer operon) located in plasmid pHP45.OMEGA.-Hg was subcloned into plasmid pHP118EH, containing the tac promoter, and was subsequently transformed into Escherichia coli VJS632QA. The resulting recombinant strain E. coli PWS1 was able to tolerate Hg/sup 2+/ concentration up to 80mg/L and also exhibited higher specific mercury reduction activity than that of wild-type mercury-resistant strain Pseudomonas aerugionosa PU21 (Rip64). The effects of inducers (IPTG and Hg/sup 2+/) on the mercury reduction activity of the PWS1 strain were identified. The mercury reduction kinetics were determined for the intact cells, permeabilized cells, crude cell extract, as well as purified mercuric reductase. The results show that the expression of mer operon in E. coli PWS1 was induced by IPTG and mercuric ions, while the addition of 0.4mM IPTG resulted in a better enhancement on the enzyme activity than the addition of 5mg/L Hg/ sup 2+/. Maximal mercury reduction activities occurred at initial Hg/sup 2+/ concentration of 16mg/L for intact cells and around 10-12mg/L for the others. The activity of permeabilized cells was better than that of intact cells, suggesting that the transport of Hg/sup 2+/ across the cell membrane appreciably retards the overall mercury reduction rate. The maximal specific activity of the purified enzyme was 0.41mg Hg/mg total protein/sec, approximately 120-fold higher than that of the crude cell extract. The dependence of mercury reduction activity on substrate (Hg/sup 2+/) concentrations can be described by substrate inhibition and enzyme deactivation models.
• 中文關鍵字： 酵素還素; 汞還原; 重組基因技術; 酵素動力學; 大腸桿菌; 汞
• 英文關鍵字： Enzymatic Reduction; Mercuric Reductase; Recombinant Dna Technology; Enzyme Kinetics; Escherichia Coli; Mercury (Hg)