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• Main Authors: Li-An Chen, 陳麗安
• Other Authors: Teh-Liang Chen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/k7p6de

碩士 === 國立成功大學 === 化學工程學系碩博士班 === 90 === Numerous foreign proteins are secreted to periplasmic space of recombinant Escherichia coli. This work was to extract a periplasmic enzyme, creatinase, without releasing cytoplasmic proteins. We adapted osmotic shock that releases protein solely by changing environment osmotic pressure. Addition of EDTA helps to reducing diffusion resistant by chelating divalent cation out off the outer membrane. To improve the enzyme recovery, we tried to increase the divalent cation concentration of outer-membrane surface. One approach was to pretreat cells with Ca+2, Mg+2 solution. The enzyme recovery was increased by 63% and 43%, respectively; purity could be increased by 30% and 27%. Another approach was to incubate the cells in medium abundant in Ca+2 or Mg+2. Although it also increased the enzyme recovery and purity, the high concentration of cation used decreased product yield. Comparing the effects of Ca+2 and Mg+2, the former had a better recovery. Using Ca+2 and Mg+2 simultaneously, the enzyme recovery could not be improved; this is due to the absorptions of Ca+2 and Mg+2 to outer membrane are competitive. The existence of Mg+2 decreases the surface concentration of Ca+2, there fore; it’s better to use Ca+2 as the only cation to pretreat the cell.