臺灣博碩士論文加值系統

Isoamyl acetate為發酵性酒精飲品之重要香味化合物，其由Saccharomyces cerevisiae之alcohol acetyltransferase (AATase)催化isoamyl alcohol與acetyl-CoA所生成。本研究乃將S. cerevisiae之ATF1 架構於一持續表現載體(pGAPZA)，轉殖進入P. pastoris GS115，以大量表現ATF1來生產天然芳香化合物isoamyl acetate，並探討表現ATF 1，生產isoamyl acetate之最適培養條件，冀能提高芳香化合物isoamyl acetate 之產量
吾人以不同的基質濃度isoamyl alchol探討其最適條件，發現當培養液額外添加20mM isoamyl alchol可獲得最高isoamyl acetate之含量，約為0.87 mM，而在碳源的試驗上則是以2 % glucose為最佳。Yeast extract之濃度試驗則發現，當yeast extract添加濃度為1~3 %時，其isoamyl acetate之含量則無顯著性差異。而在peptone之濃度試驗則發現，培養液之isoamyl acetate含量並不受到peptone濃度之影響。因此，根據本研究結果得到，當轉型酵母菌P. pastoris培養於含2% glucose、1 % yeast extrace與20 mM isoamyl alcohol時，可獲得最佳isoamyl acetate之產量，約為0.87 mM。

Isoamyl acetate is an important flavor compound in wine and beverage.
It is formed from isoamyl alcohol and acetyl-CoA catalyzed by alcohol
acetyltransferase (AATase) in Saccharomyces cerevisiae. In this study, the
gene ATF1 coded for AATase from S. cerevisiae BCRC 21731 was cloned
and constructed into a constitutive expression vector pGAPZA. A
recombinant Pichia pastoris strain was obtained express high level natural
flavor to produce isoamyl acetate. The optimal culture condition to enhance
the production of isoamyl acetate by recombinant P. pastoris was also
investigated.
The results showed that recombinant strain GS115/GAPZA-ATF1
produced twice as much isoamyl acetate as GS115/GAPZA (control strain).
When glucose, other than glycerol or methanol, was used as carbon source,
recombinant strain produced the highest amount of isoamyl acetate. The
optimal medium composition for isoamyl acetate production by recombinant
strain was 2% glucose, 1% yeast extract and 20 mM isoamyl alcohol. The
highest production of isoamyl acetate, is 0.87 mM, was obtained when the
recombinant strain GS115/GAPZA-ATF1 was cultivated in the optimal
medium at 30℃ for 4 days.

中文摘要
壹、前言
貳、文獻回顧
一、芳香酯類之成份
(一)、花
(二)、水果
(三)、發酵食品
二、酯類芳香化合物之生合成方式
(一)、化學性合成
(二)、生物性合成
三、Alcohol acetyltransferase (AATase)之探討
(一)、AATase之催化機制
(二)、S. cerevisiae之AATase之性質
(三)、AATase之isozyme
四、Alcohol acetyltransferase gene (ATF)之性質
(一)、ATF1、ATF2與Lg-ATF1之性質
(二)、ATF之調控
五、ATF之應用
(一)、提高清酒釀造之風味
(二)、利用轉殖技術使非酵母菌生產芳香化合物
六、利用轉殖微生物生產天然香料
(一)、Vanillin之產生
(二)、mesifurane之產生
(三)、melon之esters之產生
(四)、Cheese芳香化合物carboxylic acid之產生
(五)、豐富Yeast在發酵過程中的芳香化合物
七、嗜甲醇酵母菌 (P. pastoris)之表現系統
(一)、P. pastoris之簡史
(二)、P. pastoriss對甲醇的代謝路徑
(三)、Alcohol oxidase promoter
(四)、glyceraldehydes-3-phosphate dehydrogenase promoter
(五)、表現質體嵌入(integration)染色體之表現方式
(六)、P. pastoris之轉型
(七)、P. pastoris之高密度發酵培養
参、材料與方法
ㄧ、實驗材料
(ㄧ)、菌種
(二)、培養基
(三)、試驗藥劑
(四)、商業試驗套組
(五)、儀器設備
二、實驗方法
(一)、菌種之冷凍保存
(二)、Saccharomyces cerevisiae BCRC 21731之培養
(三)、RNA之抽取
(四)、水平式膠體電泳
(五)、Total cDNA之合成
(六)、ATF1 DNA之擴增
(七)、重組質體pGEM-T- ATF1之構築
(八)、重組質體pGEM-T- ATF1轉型至 E. coli DH5��
(九)、表現載體pGAPZA-ATP1之構築
(十)、P. pastoris GS115之勝任細胞製備
(十一)、P. pastoris GS115之轉型
(十二)、含高套數質體pGAPZA-ATF1之重組菌株的篩選
(十三)、利用primer shift PCR方式檢測重組酵母菌P.
pastoris GS115 genome上之ATF1
(十四)、培養重組酵母菌GS115/GAPZA或GS115/GAPZA-
ATF以產生isoamyl acetate
(十五)、GS115/GAPZA-ATF1產生isoamyl acetate之最適
條件
三、分析
(一)、菌株之生長檢測
(二)、培養液pH值之測定
(三)、培養液glucose含量之測定
(四)、isoamyl acetate之收集與產量分析
四、統計分析
肆、結果與討論
ㄧ、重組表現質體 ( pGAPZA-ATF1 )之構築
（一）、Total RNA之萃取
（二）、以反轉錄聚合酶連鎖反應(Reverse transcription
PCR, RT-PCR )擴增S. cerevisiae BCRC 21731之
ATF1
(三)、重組質體pGEM-T-ATF1之構築
(四)、重組表現質體pGAPZA-ATF1之構築
二、P. pastoris GS115之轉型與篩選
(一)、重組表現載體之轉型與重組酵母菌之篩選
(二)、高套數重組酵母菌P. pastoris GS115之篩選
三、利用primer shift PCR檢測重組酵母GS115/GAPZA-ATF1
之genome上之ATF1 gene
四、重組酵母菌GS115/GAPZA-ATF1表現ATF1以產生
isoamyl acetate
五、GS115/GAPZA與GS115/GAPZA-ATF1產生isoamyl
acetate之情形
六、GS115/GAPZA-ATF1產生isoamyl acetate之最適條件探
討
(一)、震盪培養之影響試驗
(二)、Isoamyl alcohol濃度之影響試驗
(三)、Carbon源之影響試驗
(四)、Glucose濃度之影響試驗
(五)、Yeast extract濃度之影響試驗
(六)、Peptone濃度之影響試驗
(七)、溫度之影響試驗
伍、結論
陸、參考文獻
柒、附錄

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