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標題: Development of Novel Whole Cell Biocataylast
作者: 萬曉明
關鍵字: biocatalyst;生物觸媒;Cell surface display;Cyclodextrin glucanotransferase;細胞表面表現;環狀糊精葡萄糖基轉遺
出版社: 化學工程學系
本研究是利用細胞表面表現(cell surface display)的技術,製備一種全細胞生物觸媒(whole cell biocatalyst)。利用基因轉殖技術經發酵大量生產該基因重組細胞,取得有酵素表面表現的菌體,即成為所需的生物觸媒。此一技術可降低傳統製造生物觸媒程序的繁瑣與成本高的缺點。質體pSD192上具有Lpp-OmpA表面表現系統,我們利用具有Lpp-OmpA表面表現系統的質體pSD192當成基因選殖的載體,將標的的酵素基因cgt,連接在表面表現系統Lpp-OmpA的基因後,成為Lpp-OmpA-CGTase三段融合基因,此構築而成的質體稱為pCS005。以大腸桿菌JM109當成宿主,進行轉形作用,將質體pCS005送入細胞中。接下來我們培養含有pCS005的大腸桿菌至對數生長期並且取菌體進行CGTase酵素活性分析,但發現全細胞並無酵素活性,於是利用西方點墨法偵測蛋白,發現Lpp-OmpA-CGTase三段溶合蛋白的確有表現,進一步再以蔗糖密度梯度分離大腸桿菌細胞的內胞膜與外胞膜,並進行西方點墨法的蛋白偵測,結果顯示Lpp-OmpA-CGTase三段融合蛋白的確位於細胞的外胞膜區域。為了偵測酵素CGTase是否表現在胞外,又進行蛋白部分切除(protease partial digestion)實驗,實驗結果顯示酵素並沒有表現在胞外。因此推斷,Lpp-OmpA-CGTase三段融合蛋白雖然有固定在外胞膜上,但是酵素卻是表現在細胞間質區,而非胞外。接下來我們嘗試以隨機突變的方式,作為改良酵素的方式,冀望能獲得有CGTase表面表現的菌株。

Cell surface display systems were used to develop a novel whole cell biocatalyst. This approach eliminates the many difficulties, including protein purification and enzyme immobilization, encounterd in conventional ones for the preparation of biocatalysts.
In this study, surface display system with Lpp-OmpA hybrid protein was adopted for the preparation of whole cell CGTase biocatalyst. A recombinant plasmid, pCS005, encoding lpp-ompA-cgt tripartite fusion gene was constructed and used to transform E. coli JM109. The recombinant E. coli containing pCS005 was grown to late log phase and subsequently harvested for activity analysis. However, no CGTase activity was detected on the recombinant whole cells. Western blotting assay, isopycnic surcose gradient centrifugation and partial protease digestion were performed to elucidate the location of the expressed CGTase. The results from western blotting assay and isopycnic surcose gradient centrifugation indicated that E. coli JM109(pCS005) cells did express the Lpp-OmpA-CGTase trifusion protein and successfully targeted to outer membrane. However, the failure of partial protease digestion in releasing any CGTase fragments into the reaction buffer indicated that CGTase was not localized on the cell surface. It was, therefore, concluded that CGTase domain of the trifusion is not exposed on the cell surface but located within the periplasmic space of the cell. To tackle this problem, a random mutagenesis experiment was conducted in hope of obtaining mutants with surface-displayed CGTase.
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