帶有RPLKPW降血壓胜?基因的大腸桿菌轉殖菌株，以液體培養可產生RPLKPW降血壓胜?。本研究目的在於探討利用發酵技術製造RPLKPW胜?的可行性。菌種穩定性是發酵菌種的基本要求，經試驗結果得知此轉殖菌株表達胜?的能力並不穩定，在-80°C甘油儲存下經過一年以上，有些菌可能喪失了轉殖菌株的特性，於LB斜面培養基保存於4°C下一個月，轉殖菌株特性些微減少，兩個月後，特性迅速失去，故以-80°C甘油長期保存轉殖菌株為宜。為尋求能提高胜?產量的培養基，經培養基試驗，發現添加碳源無助於胜?的產生，雖會增加轉殖菌株菌數和分泌之總蛋白質含量，但對融合蛋白分泌較不顯著，甚至抑制融合蛋白生成。應用Plackett-Burman與部分因子試驗設計所得的數據，確認tryptone與Yeast Extract(YE)是關鍵性培養基成分，進一步使用回應曲面法尋找這兩種成份的最佳組合，結果可得回歸方程式Protein = -1.614 + 3.030*tryptone + 0.099*YE – 1.316*tryptone^2 + 0.150*tryptone*YE – 0.175*YE^2經SAS分析轉換得到的正則方程式為Y =0.237－0.079 Z1 ^2－0.112 Z2 ^2(Y=融合蛋白產率，Z1=tryptone，Z2=YE)。依據回歸方程式，推算最佳組合為tryptone=1.20%、YE=0.80%，利用此最適組成份進行三角瓶發酵，結果降血壓胜?產量為0.0144 μg/ mL。 The recombinant strain of E. coli p274 possesses the capability to produce RPLKPW, an anti-hypertensive peptide, in a submerged culture. The objection of this study is to explore the feasibility of producing RPLKPW by fermentation technique, with this recombinant strain. Usually, the genetic stability is the fist criterion for a fermentation strain, but unfortunately this strain lost part of capability to generate RPLKPW after storage in 20% glycerol at -80°C for 1 year and in LB agar slant for months. The medium studies revealed that extra carbon sources such as sugars and glycerol did not improve the productivity of RPLKPW, some of sugars even repressed the peptide gene; tryptone and Yeast Extract have been decided as indispensable in gradients of the medium, and the optimal amount used in the medium were 1.20% and 0.80%, respectively. The optimal composition was figured out according to the regressive equation:Protein = –1.614+ 3.030*tryptone + 0.099*YE– 1.316* tryptone^2 + 0.150*tryptone*YE– 0.175*YE^2And the equation was derived from the data of response surface methodology.
