臺灣博碩士論文加值系統

摘要

乳酸菌代謝的主要產物為乳酸(Lactate)，但是其副產物丁二酮(Diacetyl)是相當昂貴的乳製品調味料，附加價值較高，但是其產量相當小。在過去有釵h乳酸菌的相關研究，可惜大部分的研究均屬於單一部分的實驗，或者是利用試誤法來提升丁二酮的產量。如果能配合代謝工程的技術，便能找出最佳基因突變決策。
故本研究應用通量平衡分析(Metabolic flux analysis, MFA)的方法，配合反置通量分析(Inverse flux analysis)方法來分析整體內部調控的情況。文中也利用基本通量模式(Elementary flux modes)的二項式最適化分析，來建構乳酸菌的發酵代謝的生理通量路徑分布(Physiological flux distributions)，試圖找出有效生成丁二酮的主要途徑，並且對重要基本通量路徑進行酵素交集分析，分析每個基本通量模式各自的角色及弁鄐峔銗眸歲q過的必需路徑(Essential route)。

Abstract

Lactate is one of the major fermented products from lactic acid bacteria. Furthermore, diacetyl is a small amount of valuable byproduct for dairy food and pharmaceutical application. In the past decade, the reserchers only focus on doing parts of the experiments in order to understand the regulatory relationship of lactic acid bacteria metabolism and raise the productivity of diacetyl by trial and error processes. These take a lot of money and time but in vein. If employing the technology of metabolic engineering, we can make a better mutation strategy for producing diacetyl.
In this study, metabolic flux analysis (MFA) and inverse flux analysis are introduced to analyze the impact of the objective flux when the other flux is slightly perturbed. In addition, we use the quadratic programming optimization to construct the physiological flux distributions of lactic acid bacteria metabolism. Further, we compared the enzyme sets of elementary flux modes with each other to characterize the functional routes and the essential route in lactic acid bacteria metabolism.

目錄

中文摘要 ……………………………………………………………………....Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖目錄 Ⅴ
表目錄 Ⅶ
第一章 緒論…………………………………………………………………..1
1.1 前言………………………………………………………………….1
1.2 文獻回顧…………………………………………………………….6
1.3 研究動機………………………………………………………...…10
1.4 組織章節……………………………………………………………11
第二章 代謝通量分析與反置通量分析.…………………….………….…..12
2.1 代謝通量分析的簡介及其運用…………………………………...12
2.2 反置通量分析及靈敏度分析...………………………………...….15
2.3 路徑分析方法及原理………………………………………….…..17
第三章 乳酸菌代謝模式分析………………………….………...…………22
3.1 系統描述…………………………………………………...………22
3.2 代謝通量分析方法之結果………………………………...………42
3.3 反置通量分析方法之結果…………………………………...……46
第四章 基本通量模式分析…………………………..……………...….…53
4.1 基本通量模式………….………………………………...………53
4.2 二項式最適化方法………………………………………………55
4.3 基本通量模式之弁鄔吨尷R……………………………………58
第五章 結論與建議………………………………………………..………74
參考文獻………………………………………………...……...……………76













圖 目 錄

圖1.1 代謝工程的概念流程圖..…………….………………………………5
圖1.2 代謝路徑分析流程圖………………………………………………..11
圖2.1 簡單生物系統的基本通量模式及其極路徑分析…………………..19
圖2.2 Convex basis vector之示意圖……………………………………….21
圖3.1 乳酸菌代謝路徑圖…………………………………………………..28
圖3.2 分別對所有通量做5%的擾動對丁二酮的影響關係圖……………49
圖3.3 分別對所有通量做5%的擾動對乙醯乳酸的影響關係圖…………50
圖3.4 對丁二酮通量的靈敏度分析圖……………………………………..50
圖4.1 基本通量模式之酵素交集統計圖…………………………………..63
圖4.2 EM8的路徑圖………………………………………………………..64
圖4.3 EM11的路徑圖………………………………………………………65
圖4.4 EM15的路徑圖………………………………………………………66
圖4.5 EM21的路徑圖………………………………………………………67
圖4.6 EM22的路徑圖………………………………………………………68
圖4.7 EM33的路徑圖………………………………………………………69
圖4.8 EM37的路徑圖………………………………………………………70
圖4.9 EM46的路徑圖………………………………………………………71
圖4.10 EM109的路徑圖……………………………………………………72
圖4.11 EM322的路徑圖……………………………………………………73
















表 目 錄

表3.1 乳酸菌代謝反應方程式……………………………………………..29
表3.2 M-M反應動力方程式……………………………………………….30
表3.3 M-M反應動力方程式之符號對照表……………………………….35
表3.4 參與反應之中間代謝物及產物符號說明………………………….38
表3.5 參與反應之酵素符號說明…………………………………………..39
表3.6 通量守恆方程式…………………………………………………….40
表3.7 乳酸菌代謝系統之計量矩陣 ….…………………………………..41
表3.8 的矩陣架構(Wild-type)………………………………………...…43
表3.9 的矩陣架構(Wild-type)…………………………………………...44
表3.10 由MFA方法推得的通量結果(Wild-type)…………………………45
表3.11 反置通量分析中 的矩陣架構(對 做擾動)…………………….47
表3.12 反置通量分析中 的矩陣架構(對 做擾動)……………………48
表3.13 反置通量分析之通量結果(減少 通量5%)…………………….49
表4.1 重要的基本通量模式及每個通量的化學反應式………………….60
表4.2 重要基本通量模式的總反應方程式……………………………….61
表4.3 野生菌株與減少50% 酵素活性的加權比較表………………..61
表4.4 野生菌株與增加50% 酵素活性的加權比較表………………..62
表4.5 野生菌株與減少50% 酵素活性的加權比較表………………..62
表4.6 野生菌株與減少50% 酵素活性的加權比較表………………..63

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