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標題: | 氧氣對液態發酵牛樟芝時4-AAQB生合成之影響 Influence of oxygen on 4-AAQB biosynthesis of Antrodia cinnamomea during submerged fermentation |
作者: | Yu-Ying Wei 魏瑜瑩 |
指導教授: | 蔣丙煌(Been-Huang Chiang) |
關鍵字: | 牛樟芝,4-AAQB,AQ,液態發酵,氧氣載體,n-hexadecane,n-dodecane, Antrodia cinnamomea,4-AAQB,AQ,submerged fermentation,oxygen vector,n-hexadecane,n-dodecane, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 牛樟芝(Antrodia cinnamomea)為台灣特有真菌,野生的樟芝生長於海拔1300至2800公尺間之牛樟樹,具有強烈的宿主專一性。樟芝具有許多泛醌類化合物,而菌絲體中之4-Acetylamtroquinonol B (4-AAQB)被證實對於HepG2具有極佳的抑制生長能力,其IC50之值僅為0.1ppm,較目前由國鼎生技公司所生產之泛醌類Antroquinonol (AQ)之效果高出許多。本實驗室過去的研究已經初步建構出4-AAQB及AQ之可能產生路徑,並指出其合成路徑是由mevalonate pathway及polyketide pathway共構而成。本研究擬於液態發酵系統中加入氧氣載體並探討液態培養樟芝時培養基中氧氣含量對4-AAQB生產路徑之影響。搖瓶發酵之結果顯示,n-hexadecane及n-dodecane皆能顯著提升4-AAQB 及AQ之產量,且兩種載體皆在5%之濃度達到其最大效果,分別提升了約4倍及2倍之4-AAQB。相反的,n-decane幾乎完全抑制了4-AAQB之生產。此外,也發現不同種類的載體添加顯著影響了樟芝發酵時菌絲體的大小、pH值及發酵液之顏色。添加n-hexadecane所產生的菌絲體顆粒最大、pH 值最低,且發酵液顏色最紅。在後續研究中選擇5% 之n-hexadecane 於5 L 之攪拌式發酵槽進行樟芝之液態培養。HPLC分析結果顯示,氧氣添加之組別所產生之4-AAQB 較對照組高約10倍,而AQ則較對照組高30倍。此外,本實驗以LC-MS-MS進一步分析上游化合物之間的消長,發現在氧氣載體組別中,生合成路徑中benzoquinone ring precursors之farnesylation 反應有加速之現象。初步推斷是由於進行farnesylation之酵素prenyltransferase (PTase) 在溶氧提升之狀況下,催化了其金屬離子cofactor 之游離,進而提升prenyltransferase之活性。除此之外,上游化合物CoQ3B及CoQ3 之keto group還原反應也顯現加速之現象,推斷是由於此反應需要氫離子參與並將電子釋出,而氧氣具有較高的電子親和力故加速了此反應。總體而言,本研究證實溶氧的提升確實會對於4-AAQB及AQ之生合成產生正面效應。 Antrodia cinnamomea (AC) is a traditional pharmaceutical fungus native to Taiwan, which is well-known for its diverse bioactivities. A wide variety of bioactive compounds have been isolated from both fruiting body and mycelium of AC. 4-acetylantroquinonol B (4-AAQB) is a kind benzoquinone derivatives isolated from mycelium of AC, showing great inhibition activity on HepG2 cell with only 0.1 ppm of IC50 value. The biosynthetic pathway of 4-AAQB was constructed and found to be highly relevant to another bioactive benzoquinone compound, antroquinonol (AQ), by Chou et al 1. According to the structures of intermediates in the constructed pathway, the conversion of several upstream compounds seems to be relevant to redox reaction. Therefore, this study aims to investigate the influence of oxygen on biosynthetic pathway of 4-AAQB and AQ during submerged fermentation of AC. Addition of oxygen vectors, including 5% of n-hexadecane and n-dodecane, induced approximately 4-folds and 2-folds increase of 4-AAQB respectively in shake flask submerged fermentation. Different oxygen vectors (hydrocarbons) would result in different particle size of mycelium, color of fermentation broth, and the yields of target secondary metabolites. Submerged cultivation of ac in 5 L stirred bioreactor using 5% of n-hexadecane as oxygen vector, the yields of 4-AAQB and AQ in the vector treated group was about 8 –folds and 21-folds higher than the control group. Further analyses of upstream compounds by LC-MS-MS revealed that oxygen vector accelerated farnesylation of benzoquinone ring precursors and reduction of CoQ3B and CoQ3 in the biosynthetic pathway. We suspect that during submerged cultivation of AC, the prenesyltransferase (PTase) and ketoreductase dependent chemical reactions require electrophilic molecular for accepting the release of electrons from ionized cofactors, and the elevated DO concentration by oxygen vector may facilitate these chemical reactions, which in turn increased the yields of AQ and 4-AAQB. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70501 |
DOI: | 10.6342/NTU201803027 |
全文授權: | 有償授權 |
顯示於系所單位: | 食品科技研究所 |
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