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研究生:唐維泰
研究生(外文):Taung,Wei-Tai
論文名稱:以Stizolobiumhassjoo細胞培養有效生產二次代謝產物L-DOPA
論文名稱(外文):Effective Production of Secondary Metabolite L-DOPA by Stizolobium hassjoo Cell Culture
指導教授:黃世佑黃世佑引用關係
指導教授(外文):Huang,Shih-Yow
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:1993
畢業學年度:81
語文別:中文
論文頁數:87
中文關鍵詞:多巴植物細胞培養黎豆
外文關鍵詞:L-DOPAPlant cell cultureStizolobium hassjoo
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本研究以無菌栽培之Stizolobium hassjoo植株為驗材料,由其葉片誘導
癒合組織,再由此癒合組織建立懸浮細胞之培養,生產二次代謝產物L-
DOPA。於懸浮培養時,其懸浮細胞中 L-DOPA 含量最高可達細胞乾重
之6.25﹪。並且藉由量測懸浮培養期間諸多變數如:沉降體積﹑L-DOPA
﹑ L-tyrosine 含量 ﹑Phenoloxidase ( P.O.) 酵素活性及殘留於培養
液中之蔗糖濃度等,相互間變化之關係來了解懸浮細胞生長之行為,進而
嘗試以半批式饋料(Fed-Batch)方式培養懸浮細胞。在17日之半批式饋料
懸浮細胞培養過程中,於第9日開始饋進新的培養基,發現除第10日其L-
DOPA含量降低外,自第11日以後測得之 L-DOPA量均高於第 9日且有逐漸
增加趨勢。由此結果可知半批式饋料培養方式為可行之策略。固定化之植
物細胞是將已培養七日之 S.hassjoo懸浮細胞以 40% (v/v) 的細胞量包
埋於海藻酸鈣 (Ca-alginate) 擔體中,進行將 L-tyrosine生物轉換成L-
DOPA,由實驗發現其合成之L-DOPA大部份可釋放至反應液中,最高約占全
量的百分之九十。同時經由改變基質濃度(L-tyro sine) 探討其對 L-
DOPA 生成速率之影響,並且比較反應液之酸鹼值、成膠時氯化鈣
(CaCl2) 濃度及抗壞血酸鈉(Sodium ascorbate )滅菌方式等對L-DOPA 生
成之影響。
The plant Stizolobium hassjoo which grows in Taiwan is adopt-
ed as an investigation system. The callus was induced from the
leaves with MS medium. The aim of this work is to produce the
L-DOPA (L-dihydroxyphenylalanine) by suspension culture of S.
hassjoo cells. Suspension cultures were conducted mainly in 250
ml Erlenmeyer flask with initial working volume of 75 ml
medium. During suspension culture, cell volume (Settling volume
S.V.) concentrations of L-DOPA and L-tyrosine, the activity of
Phenoloxidase (P.O.) and the residual sucrose concentration
were determined. Generally, the trend of increase of S.V. and L-
DOPA was the same. This indicates that the more the growth of
the cell the higher the production of L-DOPA. A fed-batch
culture of suspension cells was conducted with an aim of
elasting the cell growth by feeding the medium to meet their
demand. It was found that the concentration of L-DOPA increased
1.63 folds compared to that not conducting fed-batch culture.
Biotransformation of L-DOPA from L-tyrosine was carried out by
entrapping the 7 day cells of S.hassjoo in Ca-alginate beads
whose size was approximately 3.5 mm in diameter. The percentage
transformation of tyrosine was 27% in 22 hr reaction. The cor-
responding percentage of L-DOPA was 14% at the initial dosage
of 2 mM L-tyrosine.
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