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研究生:林玉蕙
研究生(外文):Yu-Hui Lin
論文名稱:衣康酸生合成相關之研究
論文名稱(外文):Studies on itaconic acid production in Aspergillus terreus
指導教授:蔡英傑蔡英傑引用關係
指導教授(外文):Ying-Chieh Tsai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:106
中文關鍵詞:衣康酸土黴菌
外文關鍵詞:itaconic acidAspergillus terreus
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衣康酸又稱為甲烯基琥珀酸,可應用於合成樹脂、纖維、塑料及橡膠的原料,廣泛應用於化學、高分子工業上。衣康酸的合成,目前主要以生物醱酵法為主,本論文研究的主要菌株Aspergillus terreus M8為衣康酸生產菌株。我們的研究目的有二:一、利用產酸及不產酸菌株,探討衣康酸生產過程中,菌體內衣康酸生合成相關酵素的變化。二、選殖外來基因,改善衣康酸生產菌株對澱粉的利用及克服氧氣供應不穩對衣康酸生合成之影響。
比對衣康酸生產菌株M8及其不產酸突變株17N,兩者於產酸前後,菌體內與衣康酸合成相關的酵素活性變化,我們發現當衣康酸生產菌株M8進入產酸期時,其上游酵素:烏頭酸酶及順-烏頭酸去羧酶的活性上升,而下游酵素:NADP-異檸檬酸脫氫酶活性則受抑制,使得整體代謝反應往衣康酸合成的方向蓄積。而不產酸的菌株17N,相關的酵素活性則無明顯變化;同樣地利用半定量聚合酶鏈鎖反應(semi-quantitative PCR),從RNA層次亦觀察到產酸期相關酵素表現量消長的現象。由實驗結果我們推論衣康酸生產菌株,於產酸期因菌體內酵素活性的改變,使得產酸率較其他菌株高。
為了增加衣康酸生產菌株M8,利用澱粉的能力,我們將Aspergillus kawachii 耐酸性的α-澱粉酶基因(asaA)轉殖入衣康酸生產菌株M8中。我們成功的得到α-澱粉酶基因的轉形株,以6%澱粉為碳源,轉形株(T7)衣康酸的產率為M8株的四倍。由於分泌出的α-澱粉酶酵素活性,會受其分解產物葡萄糖的抑制(carbon catabolite repression),因此將轉形株經UV照射突變後,以2-去氧葡萄糖篩選可抗碳代謝物抑制的突變株,所得到之突變株以澱粉為碳源,其衣康酸產率最高可達至16.7%,為M8株的八倍。
衣康酸生產菌株,培養過程中維持持續的供氧是影響衣康酸生成重要的因素之一。當生產菌株進入產酸期,只要短暫地停止震盪培養3分鐘,其衣康酸的產率會降為持續震盪培養的50%。利用共轉形的方法,將Vitreoscilla菌株攜氧的血紅蛋白質基因轉殖於M8中,以減少培養過程中短暫停止震盪對衣康酸生成的影響。轉形株與M8相較之下,轉形株於培養的早期,對氧的吸收速率、培養基中葡萄糖消耗速率及菌體生長速度均較M8株來得快,且其衣康酸的產率較M8高出17%;而且表現血紅蛋白質於M8中,確實可以減少培養過程中短暫停止震盪對衣康酸生成之影響。
Itaconate, also called methylene succinic acid, is an important monomer for the manufacturing of polyester resins or other polymeric materials and has been widely utilized in chemical industries. A mutant strain named M8 was obtained by chemical mutation, acid domestication and single spore isolation from Aspergillus terreus NRRL 1960.
Itaconate is an abundant metabolite that is generated from cis-aconitate, part of the tricarboxylic acid cycle, in certain strains of filamentous fungi, such as Aspergillus terreus. It is synthesised by the conversion of citrate to cis-aconitate by aconitase, followed by the decarboxylation of cis-aconitate by cis-aconitate decarboxylase. The activities of aconitase, cis-aconitate decarboxylase and NADP-isocitrate dehydrogenase were determined in cell extracts of high and none itaconate-producing strains. A high differentiation in the activities of the enzymes studied was found to occur at various accumulation stages of itaconate. During intensive itaconate synthesis, the activity of cis-aconitate decarboxylase and that of NADP-isocitrate dehydrogenase decreased significantly compared to their initial activities during the growth period of the mycelium.
The filamentous fungus Aspergillus terreus is the microorganism most commonly used for itaconate production. At present, however, direct production of itaconate from starch has not yet succeeded. Moreover, there have been some reports concerning itaconate production from corn starch hydrolyzed by nitric acid or amylotic enzyme. To enable hyper-production of itaconate from starch materials, the acid-stable a-amylase was expressing in M8 and the 2-deoxyglucose resistant mutant strains was selected. The best mutant strain TU13 showed 8 times itaconate production than the parental strain M8.
Continuous aeration is an important factor for successful itaconic production in Aspergillus terreus. During the growth of the itaconate producing strain M8, an interruption of culture aeration for 3 min resulted in an approximately 50% fall in itaconate production, compared to continuous aeration. To overcome this effect, M8 was transformed with the Vitreoscilla hemoglobin gene (vgb). Southern hybridization indicated that the vgb gene was successful integrated into the recipient chromosome, and the biological activity of the Vitreoscilla hemoglobin (VHb) was demonstrated by a carbon monoxide difference spectrum. The VHb in M8 not only promoted cell growth and oxygen uptake, but also increased itaconate production during cultivation. When aeration was interrupted up to 150 min after culturing for 3 days, the vgb-expressing transformant showed only a 38.8% reduction in itaconate production, compared to a 51.4% reduction in M8. This work confirms that a short break in aeration can result in a significant reduction in itaconate production in the A. terreus strain M8 and that this effect was alleviated by the expression of VHb in M8.
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