Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3533
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dc.contributor.advisor劉永銓zh_TW
dc.contributor.advisorLiu Yung Chuanen_US
dc.contributor.author陳光宇zh_TW
dc.contributor.authorYu, Chang Kaungen_US
dc.date1999zh_TW
dc.date.accessioned2014-06-06T05:32:05Z-
dc.date.available2014-06-06T05:32:05Z-
dc.identifier.urihttp://hdl.handle.net/11455/3533-
dc.description.abstract中文摘要 本研究中,以生物反應網路方法(BRNA)建立大腸桿菌生產盤尼西林醯胺酵素(PGA)之代謝網路方程式。其中大腸桿菌之所有代謝反應依其已解碼之基因編列。而菌體本身生合成之方程式則根據大腸桿菌之組成分計算得之。並根據PGA之基因序列,由20種胺基酸與基本能量ATP列出PGA生合成方程式。 在本研究中,對形成相依反應式之節點給予理論分流率,以代入消去方式,解決傳統BRNA刪除相依方程式導致的誤差,並利用此代謝計算方式,分別探討各重要節點分流率對整體代謝途徑之影響,模擬之結果如下。進入PPP pathway、進入TCA cycle及由PEP流向PYR之代謝通量均會隨著呼吸商數(RQ)之增加而提高;而消耗葡萄糖與產生二氧化碳之比值,對於中心代謝途徑亦有相當大的影響,其中進入PPP pathway之反應式最為明顯,其比值越大,進入P.P.P pathway之流量會減少,而PEP流向PYR之通量隨之增加(除RQ為0.5外),此亦導致流入TCA cycle之通量相對提高;在模擬計算中亦發現PYR產生速率對於進入TCA cycle之通量並無明顯影響,而對流向PPP pathway及PYR兩反應之通量均會隨PYR產生速率之增加而提高流通量;菌體產生之醋酸增加時,流往PYR之流量亦增加,此因PYR可代謝為醋酸之故,而另一方面進入TCA cycle之量卻反之減少,此乃因為越來越多的流量在PYR處代謝成醋酸,導致剩下較少之流量進入TCA cycle。NH3之消耗對於流往PPP pathway反應式有明顯的抑制作用,並於計算中發現,當其消耗增加會緩慢抑制PGA的產量。菌體量對於代謝途徑亦為一影響很大之因素,當其越高時進入TCA cycle的流量就會增加,流向PYR與TCA cycle中之兩反應式流通量隨之增加。 在進行氮源濃度對E.coli醱酵生產PGA之影響時,可發現以3%之無機氮源進行醱酵尚不會對菌體與PGA活性造成影響;但當氮源濃度增加為5%時,即有明顯的抑制現象。正常情況下PYR濃度在醱酵過程中維持一定之濃度,並無較明顯的變化。但是在5%(NH4)2SO4高濃度氮源下,PYR在醱酵後期會大量且快速累積。在高無機氮源條件下(3%以上)培養菌體時,其生長情形有別於一般在低氮(0.5%)培養基的醱酵,在後對數生長期之菌體生長速率均會高於前對數生長期之菌體生長速率。由實驗數據進行代謝計算方式模擬,高氮源培養菌體生成之二氧化碳比例較低氮源者少;且在相同比例之菌量生產時,高氮源培養之菌體其醱酵過程中流入TCA cycle之通量,會略高於低氮源培養之菌體。另外在錐形瓶培養中,發現添加砷化物可提高PGA之活性,此點與模擬計算中若降低進入TCA cycle 之通量時,可得高活性之PGA結果相符。zh_TW
dc.description.abstractAbstract A bio-reaction network algorithm (BRNA) was proposed to analyze flux distributions of E. coli as producing penicillin G acylase (PGA), which was the enzyme wildly applied in the antibiotics industry. The basic metabolic equations were constructed according to the genetic mapping of E. coli, including of the central metabolic pathways, such as EMP, PPP and TCA etc., 20 basic amino acids synthesis pathways along with the biomass synthesis and penicillin G acylase (PGA) formation equations. In this study, the original BRNA method suggested by Vallino et al. was modified. By inserting the split ratios in the singularity nodes in the metabolic pathways, the problems resulting from deleting the singular equations in the metabolic pathway can be prevented. The modified BRNA methods were used to investigate the effects of regulations, including split ratios along with intermediates variations, on the critical nodes in the metabolic pathways. As respiratory quotient (RQ) increased, the fluxes into the PPP, TCA and PYR pathway will be enhanced. In addition, the larger the ratio of glucose consumption to CO2 production rate, the larger the fluxes through PEP into PYR (except that RQ equal to 0.5) and fluxes into TCA cycle, yet the smaller the fluxes into PPP pathway. In the simulation, it was also found that varying the production rate of PYR showed no significant effect on flux into TCA cycle, but it will affect the fluxes into PPP pathway and PYR nodes, since the fluxes were increased as PYR production rate increased. In another aspect, when acetic acid formation rate increased, the fluxes into PYR will also increased, while those fluxes into TCA cycle will decrease. In addition, ammonia consumption rate had a reverse effect on fluxes into PPP pathway. From the data shown, it also can be seen that increasing ammonia consumption rate inhibited the PGA production rate. The biomass formation rate was also a critical factor on the fluxes of metabolic pathway. As the biomass increased, the fluxes into TCA cycle and PYR will increase too. In comparisons of ammonia concentration effects on rDNA E.coli producing PGA, it was found that ammonia concentration below 3% had no inhibition effects on both cell growth and PGA activity. Yet adjusting the concentration of ammonia up to 5%, significant inhibition effect was observed. In normal situation, PYR concentration will keep on a constant concentration in the broth, but as the concentration of (NH4)2SO4 increased up to 5%, PYR will quickly accumulated in the broth at the late phase in the fermentation. As ammonia concentration higher than 3%, the growth rate of the cell at the late log phase was larger than that at the early log phase, this phenomena was contrary to that performed at low ammonia concentration. In another experiment, the addition of arsenic acid, which was reported of capability to reduce the fluxes into the TCA cycle, resulting in significant improvement in PGA activity.en_US
dc.description.tableofcontents目 錄 第一章 緒論1 1-1前言1 1-2 青黴素G醯胺酵素5 1-3 代謝通量9 1-3-1 代謝工程模式9 1-3-2 直接量測方法12 1-4 結論13 第二章 代謝網路方程式之建立與理論值計算15 2-1 前言15 2-2 代謝方程式18 2-2-1 文獻回顧18 2-2-2 整體代謝途徑之建立20 2-2-3 青黴素G醯化酵素合成方程式之建立23 2-2-4 大腸桿菌菌體合成方程式之建立25 2-3 代謝計算方法33 2-3-1 方法之建立33 2-3-2 代謝計算需考量之重點與困難34 2-3-2-1代謝計算考量的重點 :34 2-3-2-2初步計算遇到的問題:35 2-3-3 計算方法之改進36 2-3-3-1 原計算方式之缺點36 2-3-3-2初步構想之計算方法:37 2-3-3-3 改進之計算方法 :39 2-3-4 代謝計算策略41 2-4 代謝途徑之理論計算46 2-4-1 節點之分析47 2-4-2 重要代謝物節點對整體代謝通量之影響51 2-4-3 PGA理論產率之計算57 2-5 結論61 第三章 大腸桿菌醱酵生產青黴素G醯化酵素63 3-1 前言63 3-2 實驗器材與方法65 3-2-1 菌種65 3-2-2 藥品66 3-2-3 培養基配方67 3-2-4 實驗方法68 3-2-4-1 錐形瓶菌體培養:69 3-2-4-2醱酵槽菌體培養:69 3-2-4 分析方法73 3-2-4-1 菌量濃度73 3-2-4-2 還原糖之分析77 3-2-4-3 銨離子濃度分析78 3-2-4-4 PGA活性分析79 3-2-4-5 胺基酸之量測82 3-3 結果與討論89 3-3-1 以0.5%(NH4)2SO4為氮源培養89 3-3-2以3%(NH4)2SO4為氮源進行醱酵92 3-3-3 以5%(NH4)2SO4為氮源進行醱酵95 3-3-4 錐形瓶之測試98 3-4結論99 第四章 大腸桿菌醱酵生產盤尼西林G醯胺酵素之代謝工程計算100 4-1 0.5%(NH4)2SO4之氮源培養100 4-2 以3% (NH4)2SO4為氮源之醱酵105 4-3 以5% (NH4)2SO4為氮源之醱酵109 4-4 結論114 第五章 結論與展望115 5-1 結論115 5-2 未來與展望118 參考文獻:120 附錄 1128 附錄 2131 附錄 3133 附錄 4139 附錄 5153zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系zh_TW
dc.subject生物反應網路方法zh_TW
dc.subjectBRNAen_US
dc.subject盤尼西林醯胺酵素zh_TW
dc.subject大腸桿菌zh_TW
dc.subject分流率zh_TW
dc.subject代謝途徑zh_TW
dc.subjectPGAen_US
dc.subjectE. colien_US
dc.subjectsplit ratioen_US
dc.subjectmeatbolic pathwayen_US
dc.titleMetabolic Engineering Study of E. coli Fermentation for Penicillin Acylase Productionen_US
dc.title大腸桿菌醱酵生產盤尼西林G醯胺酵素代謝工程之研究zh_TW
dc.typeThesis and Dissertationzh_TW
item.grantfulltextnone-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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