臺灣博碩士論文加值系統

帶有RPLKPW降血壓胜肽基因的大腸桿菌轉殖菌株，以液體培養可產生RPLKPW降血壓胜肽。本研究目的在於探討利用發酵技術製造RPLKPW胜肽的可行性。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肽的能力並不穩定，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。為尋求能提高胜肽產量的培養基，經培養基試驗，發現添加碳源無助於胜肽的產生，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，結果降血壓胜肽產量為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^2
And the equation was derived from the data of response surface methodology.

中文摘要------------------------------------------------------------ I
英文摘要----------------------------------------------------------- II

壹、前言------------------------------------------------------------ 1

貳、文獻回顧--------------------------------------------------------- 2
一、血管收縮素I轉換酵素(Angiotensin I-converting enzyme, ACE)、血管收縮素I轉換酵素抑制劑(Angiotensin I-converting enzyme inhibitor, ACEI)與血壓三者的關係 ----------------------------------------------------------- 2
1.ACE與血壓的調控---------------------------------------------------- 2
2.ACE與ACEI的關係--------------------------------------------------- 4
3.常見ACEI藥物------------------------------------------------------ 6

二、降血壓胜肽的來源------------------------------------------------- 7
1.從食物當中利用酵素水解取得------------------------------------------ 9
2.應用基因重組技術合成胜肽------------------------------------------- 11
3.降血壓胜肽RPLKPW的發展過程---------------------------------------- 13

三、降血壓胜肽生產菌株培養方法之探討---------------------------------- 19
1.生產RPLKPW重組菌株之性質------------------------------------------ 19
2.培養基之最適化---------------------------------------------------- 22
3.發酵槽培養技術---------------------------------------------------- 25

參、材料與方法------------------------------------------------------ 27
一、材料----------------------------------------------------------- 27
1.菌種------------------------------------------------------------- 27
2.營養培養基------------------------------------------------------- 27
3.化學無機鹽類------------------------------------------------------ 28
4.抗生素----------------------------------------------------------- 28
5.分子篩管柱層析試劑------------------------------------------------ 28
6.親和性管柱層析試劑------------------------------------------------ 28
7.總蛋白質含量試劑-------------------------------------------------- 29
8.硫酸十二酯鈉-聚丙烯醯胺膠體電泳分析 (Sodium dodecyl sulfate poly-acrylamide gel electrophoresis analysis, SDS-PAGE) --------------- 29

二、儀器----------------------------------------------------------- 30

三、試驗方法-------------------------------------------------------- 33
1.種菌的培養------------------------------------------------------- 33
2.分子篩層析管柱方法------------------------------------------------ 34
3.親和性管柱層析方法------------------------------------------------ 34
4.SDS-PAGE電泳步驟------------------------------------------------- 36
5.總蛋白質含量測量方法(Micro Assay) -------------------------------- 38
6.比較重組菌株E. coli p274在不同時間下的表現------------------------- 38
7.以發酵槽大量培養轉型菌株------------------------------------------- 39
7.1.培養條件------------------------------------------------------- 39
7.2.步驟----------------------------------------------------------- 40
8.質體對培養基添加不同醣類的表現情形---------------------------------- 40

四、試驗設計-------------------------------------------------------- 41
1.使用的回應曲面法決定最佳之培養基------------------------------------ 41
1.1.Plackett-Burman(PB)試驗設計篩選組成份--------------------------- 41
1.2.部分因子試驗設計判斷關鍵組成份----------------------------------- 41
1.3.陡升試驗設計尋求組成份濃度之最適範圍------------------------------ 41
1.4.中心混成設計決定培養基最適組成份---------------------------------- 42
2.使用軟體--------------------------------------------------------- 42
3.實驗步驟--------------------------------------------------------- 42
4.液態培養發酵方法-------------------------------------------------- 43

肆、結果與討論------------------------------------------------------ 49
一、發酵液中融合蛋白(ZZ-RPLKPW)之回收-------------------------------- 49
二、以發酵槽大量培養重組大腸桿菌p274---------------------------------- 54
三、轉型菌株融合蛋白表達之穩定性-------------------------------------- 59
四、培養基成分對融合蛋白產量之影響------------------------------------ 64
(一)不同碳源之影響-------------------------------------------------- 64
(二)最適培養基組成分之探討------------------------------------------- 67
1.有效組成份之篩選-------------------------------------------------- 67
2.關鍵組成份之確定-------------------------------------------------- 72
3.尋找主要組成份之最佳濃度範圍--------------------------------------- 76
4.尋找最適培養基組成分---------------------------------------------- 78

伍、結論----------------------------------------------------------- 93

陸、參考文獻-------------------------------------------------------- 95

柒、附錄---------------------------------------------------------- 103

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