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研究生:尤怡人
研究生(外文):YU, I-REN
論文名稱:藉由週期性雙氧水刺激提高重組酵母菌生產鄰位氫氧化金雀異黃酮產量之研究
論文名稱(外文):Improving 3'-hydroxygenistein production in recombinant Pichia pastoris by a periodic hydrogen peroxide-shocking strategy.
指導教授:張德生張德生引用關係
指導教授(外文):CHANG, TE-SHENG
口試委員:江建民丁秀玉吳聲祺
口試委員(外文):CHIANG, CHIEN-MINDING, HSIOU-YUWu, SHENG-CHI
口試日期:2016-03-01
學位類別:碩士
校院名稱:國立臺南大學
系所名稱:生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:51
中文關鍵詞:3'鄰位氫氧化金雀異黃酮
外文關鍵詞:3'-Hydroxygenistein
相關次數:
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  • 下載下載:24
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金雀異黃酮(Ge)是異黃酮的一種,主要發現於大豆、紅花苜蓿等植物,具有抗氧化、預防癌症、糖尿病、動脈粥狀硬化等疾病的功效,也可以抑制酪胺酸脢的活性來抑制黑色素生成。此外,鄰位氫氧化的金雀異黃酮(3'-OHGe),生物活性更為提升。
在本研究中,我們使用之重組酵母菌P. pastoris X-33可將金雀異黃酮生物轉換成3'鄰位氫氧化金雀異黃酮,而3'鄰位氫氧化金雀異黃酮之抗氧化能力極好,藉此特性,以P. pastoris X-33進行週期性雙氧水刺激(Periodic hydrogen peroxide shocking),篩選出一株高產菌P2-D14-5,產生23 mg/l的3'-OHGe,是原始菌株產量12.29mg/l的1.87倍。而5公升發酵槽實驗中,高產菌P2-D14-5產生20.3mg/l的3'-OHGe。qRT-PCR的結果顯示,週期性雙氧水處理所造成的基因突變並沒有直接表現在融合的基因上。

3'-Hydroxygenistein can be obtained from the biotransformation of genistein by the engineered Pichia pastoris X-33 strain, which harbors a fusion gene composed of CYP57B3 from Aspergillus oryzae and a cytochrome P450 oxidoreductase gene (sCPR) from Saccharomyces cerevisiae. We applied periodic hydrogen peroxide shocking to select for higher 3'-hydroxygenistein production by P. pastoris X-33 mutants. One mutant (P2-D14-5) produced 23.0 mg/l of 3'-hydroxygenistein, which is an amount 1.87-fold higher than that produced by the recombinant X-33. In a scale-up experiment, P2-D14-5 produced 20.3 mg/l of 3'-hydroxygenistein in a 5-L fermenter. The P2-D14-5 mutant therefore has high potential for industrial-scale 3'-hydroxygenistein production.
摘要 ----------------------------------------------------------------------i
Abstract -----------------------------------------------------------------ii
致謝 --------------------------------------------------------------------iii
目錄 ---------------------------------------------------------------------iv
圖目錄 -------------------------------------------------------------------vii
縮寫對照表 ---------------------------------------------------------------vii
第 1 章 緒論 --------------------------------------------------------------1
1.1 研究背景 ------------------------------------------------------------1
1.2 研究目的 ------------------------------------------------------------2
1.3 研究架構 ------------------------------------------------------------3
第 2 章 文獻回顧 ----------------------------------------------------------4
2.1 異黃酮 --------------------------------------------------------------4
2.1.1 異黃酮結構之簡介 -------------------------------------------------4
2.1.2 異黃酮之生理活性與功能 -------------------------------------------5
2.2 細胞色素P450 --------------------------------------------------------8
2.3 細胞色素P450應用 ---------------------------------------------------12
2.4 週期性雙氧水刺激 ----------------------------------------------------14
第 3 章 材料與方法 -------------------------------------------------------15
3.1 材料 --------------------------------------------------------------15
3.1.1 藥品 ---------------------------------------------------------15
3.1.2 Primers 序列及訂購公司 ----------------------------------------15
3.1.3 溶液配製 ------------------------------------------------------15
3.1.4 實驗儀器 ------------------------------------------------------16
3.1.5 菌株來源 ------------------------------------------------------16
3.2 方法 --------------------------------------------------------------17
3.2.1 DPPH ---------------------------------------------------------17
3.2.2 菌株培養 ------------------------------------------------------17
3.2.3 菌株活化 ------------------------------------------------------17
3.2.4 週期性雙氧水刺激 -----------------------------------------------18
3.2.5 高產菌株篩選 --------------------------------------------------19
3.2.6 超高效液相層析法(Ultra Performance Liquid Chromatography, UPLC) -19
3.2.7 雙氧水耐受性測試 -----------------------------------------------20
3.2.8 去氧核醣核酸(RNA)萃取後倒轉錄合成cDNA ---------------------------20
3.2.9 聚合酵素連鎖反應(Polymerase chain reaction,PCR) ----------------22
3.2.10 即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction, Q-PCR) ---------------------------------------------------------23
3.2.11 5L發酵槽操作 --------------------------------------------------24



第 4 章 結果 -------------------------------------------------------------26 4.1 3'-Hydroxygenistein 抗氧化能力測試 -----------------------------------26
4.2 重組P. pastoris X-33進行週期性雙氧水處理 -------------------------------28
4.3 篩選P. pastoris X-33高產菌株 ---------------------------------------31
4.4 P. pastoris X-33和P2-D14-5進行雙氧水耐受性測試 ----------------------32
4.5 P. pastoris X-33和P2-D14-5之mRNA含量分析 ---------------------------33
4.6 P2-D14-5在5公升發酵槽大量生產之產率 ------------------------------------35
第 5 章 結論與未來展望 ----------------------------------------------------36
5.1 結論 --------------------------------------------------------------36
5.2 未來展望 -----------------------------------------------------------36
第 6 章 參考文獻 ---------------------------------------------------------37
附錄----------------------------------------------------------------------40

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