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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30267

Title: 由藍細菌Synechocystis sp. Strain PCC6803 菌株所產藍藻蛋白合成酶之基因在乳酸菌Nisin 控制的基因表現系統中之表現與藍藻蛋白產量探討
Studies on the Expression of Synechocystis sp. PCC6803 Cyanophycin Synthetase in Lactococcus lactis Nisin-Controlled Gene Expression System (NICE) and Cyanophycin Production
Authors: Kai-Chun Chang
張凱淳
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 藍藻蛋白;藍藻蛋白合成酶;乳酸鏈球菌素;乳酸乳球菌
cyanophycin;cyanophycin synthetase;nisin;lactococcus lactis
Date: 2011
Issue Date: 2011-11-25T07:38:03Z
Abstract: 藍藻蛋白 (Cyanophycin) 是一種天然來源的類蛋白聚合物,主要 是由天門冬胺酸 (L-aspartic acid)、精胺酸 (L-arginine) 所組成的共聚 物,透過化學方法將分支精胺酸還原去除後,便可得到由聚天門冬胺 酸 (polyaspartic acid) 所組成的骨幹,目前被廣泛應用在工業與生醫材 料等多種領域。因此如何低價、大量生產藍藻蛋白就成為一個引人注 目的研究方向。本實驗首次利用乳酸菌生產藍藻蛋白,藉由RF-cloning 法將藍細菌Synechocystis sp. strain PCC6803 所產的藍藻蛋白合成酶 (cyanophycin synthetase) 基因cphA 重組至載體pNZ8149 上,再轉形至 乳酸菌Lactococcus lactis subsp. cremoris NZ3900 並利用nisin-controlled gene expression system (NICE) 表現系統進行藍藻蛋白的生產。於培養 過程中加入不同濃度的nisin 進行誘導,並改變不同誘導時間,再添加 不同濃度的天門冬胺酸與精胺酸於培養基中做為基質,並對基因cphA 尾端進行修飾,找出藍藻蛋白最佳的生產條件。實驗結果顯示,將cphA 基因尾端插入1 個glycine 的轉形株培養於20 mM 精胺酸與10 mM 天 門冬胺酸的500 ml M17L 培養基內,培養至生長對數期時以250 ng/ml 的nisin 誘導5 小時後進行藍藻蛋白的純化,可得到水溶性藍藻蛋白 0.0127±0.0010 mg/ml 與非水溶性藍藻蛋白0.1759±0.0011 mg/ml,非水 溶性藍藻蛋白的產量比起原生型組別提高了9.16 倍,非水溶性藍藻蛋 白佔細胞乾重之比例可達19.01±0.16%,比起原生型組別提高了10.34 倍。
Cyanophycin is a natural source protein-like polymer which consisting of aspartic acid and arginine. By use of chemical method reduce branche arginine, we can obtain backbone polyaspartic acid which can be widely used in industry and biomedical area. How low-cost, mass production cyanophycin become an attractive research direction. This study first uses lactic acid bacteria producting cyanophycin, we use RF-cloning by recombinant Synechocystis sp. strain PCC6803 cyanophycin synthetase gene cphA to vector pNZ8149 and transform to Lactococcus lactis subsp. cremoris NZ3900, and product cyanophycin by nisin-controlled gene expression system (NICE) system. Modified gene cphA terminal and different concentration of nisin and aspartic acid and arginine as well as different induction time were used to find the optimum condition for cyanophycin production. It shows that the group which insert 1 glycine to the end of gene cphA growth in 500 ml M17L medium (plus 20 mM arginine and 10 mm aspartic acid) to log phase, and induce with 250 ng/ml of nisin 5 hours, and purifies cyanophycin, we can obtain 0.0127±0.0010 mg/ml soluble cyanophycin and 0.1759±0.0011 mg/ml insoluble cyanophycin. The yield of inoluble cyanobacteria than the wild type group increased 9.16 times, and insoluble cyanophycin of dry cell weight is 19.01±0.16%, compared to wild type group increased by 10.34 times.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98320055
http://ntour.ntou.edu.tw/handle/987654321/30267
Appears in Collections:[食品科學系] 博碩士論文

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