二價錳離子有誘導耐輻射奇異球菌 (Deinococcus radiodurans) 之生長靜止初期細胞再次分裂的現象，稱之為Mn-CD效應 (Mn-induced cell division effect) 。我們發現二價錳離子可誘導此菌對葡萄糖和果醣利用的效率。果醣雙磷酸醛縮酶 (fructose-1,6-bisphosphate aldolase，FBA) 是此菌進行五碳醣磷酸代謝作用 (pentose phosphate pathway) 中，會受二價錳離子影響的關鍵酵素之一。因此本研究乃針對fba基因進行探討。由DNA序列推算fba基因之蛋白質一級結構，包含甲硫胺酸共由306個胺基酸組成，其分子量為32.4kDa、等電點為5.4。以PCR將此基因增幅後得到的9.3 kbp基因片段，與質體pGEX-4T-2、pQE30、pET28a接合以後，植入適當的大腸桿菌 BL21(DE3)RIL、JM109 宿主中進行表現，並分析及探討合成之FBA蛋白質的特性，包括錳離子對活性的影響等。結果發現以Taq DNA polymerase及pfu turbo DNA polymerase進行PCR增幅fba基因，後者產物DNA序列和NCBI上發表的較為接近。轉形菌株pTDA2/BL21(DE3)RIL經0.1mM IPTG誘導，大量表現的融合蛋白為不溶解狀態的包涵體(inclusion body)。轉形菌株pEDA2/BL21(DE3)RIL在18℃低溫中以0.1mM的IPTG誘導至對數期中期，可產生具溶解狀態融合蛋白His-Thromb-T7-FBA，其活性比30℃下以IPTG誘導的高約50倍；顯示降低IPTG誘導溫度可促進融合蛋白的溶解度與活性。

The addition of Mn(II) to an early stationary-phase Deinococcus radiodurans RI culture could induce a new round of cell division (MnCD effect). The addition of Mn(II) could also stimulate the utilization of glucose and fructose in this bacterium. Class II fructose-1,6-bisphosphate aldolase (FBA) is an Mn-dependent key enzyme in pentose phosphate pathway. Therefore, in this research, we focused on the studies of the fba gene. Base on the gene sequence, FBA protein was composed of 306 amino acids, (M.W., 32.4 kDa； pI, 5.4). The expected PCR product size of the fba gene is 9.3 kbp. We had amplified the fba gene by using both Taq DNA polymerase and pfu turbo DNA polymerase. The sequence of the pfu turbo DNA polymerase products showed a higher homology with the fba gene than those of using Taq DNA polymerase. These amplified fba gene was cloned into three expression vectors, pGEX-4T-2, pQE30, and pET28a, and then further expressed in E. coli BL21(DE3)RIL and JM109. The recombinant GST-FBA protein could be overproduced in pTDA2/BL21(DE3)RIL. However, the expressed insoluble protein accumulated as inclusion bodies in the cells and exhibited no enzyme activity. After partial purification, and processing by thrombin protease cleavage, urea treatment, and the addition of Mn(II), this enzyme still showed no activity. The recombinant pEDA2/BL21(DE3)RIL strain cells grew in 18℃ and induced by 0.1mM IPTG could produced a soluble form His-Thrombin-T7-FBA protein which performed a 50X higher activities than those cells grew in 30℃. This result indicated that decreasing the indicatioin temperature could improve the protein solubility and activity.

致謝……………………………………………………I
中文摘要………………………………………………II
英文摘要………………………………………………III
圖目錄…………………………………………………IV
表目錄…………………………………………………V
前言…………………………………………………… 1-10
材料與方法……………………………………………11-25
結果與討論……………………………………………26-36
結論……………………………………………………36-36
參考文獻………………………………………………37-44
圖………………………………………………………42-59
表………………………………………………………60-62
附錄……………………………………………………63-72

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