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研究生:陳林琤
研究生(外文):Lin-Cheng Chen
論文名稱:利用Clonostachycompactiuscula生產羅瓦斯達汀酯化酶以轉化莫那克林K(羅瓦斯達汀)成為莫那克林J
論文名稱(外文):Production of Lovastatin Esterase by Clonostachy compactiuscula to Convert Monacolin K (Lovastatin) to Monacolin J
指導教授:黎耀基黎耀基引用關係
指導教授(外文):Yiu-Kay Lai
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:42
中文關鍵詞:高膽固醇血症莫那可林反應曲面法酵素純化核磁共振
外文關鍵詞:hypercholesterolemiamonacolinresponse surface methodologyenzyme purificationNMR
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斯達汀系列 (Statins series) 為控制高膽固醇血症之處方用藥,其中,羅瓦斯達汀(lovastatin;又稱為莫那可林K;monacolin K;也稱為美芬諾林;mevinolin)是這類斯達汀系列的一種。羅瓦斯達汀能夠被一個叫羅瓦斯達汀酯化酶的酵素水解,切去其側鏈,轉化成莫那可林J (又稱為三酸醇;triol acid),莫那可林J 是這類斯達汀的核心結構,因此具有合成其他斯達汀的潛力與價值。本實驗結果顯示,Clonostachys compactiuscula可以讓羅瓦斯達汀轉化生成莫那可林J,因此進一步使用二階多項式模型 (second-order polynomial model) 實驗設計的反應曲面方法,以五個變因當成轉化作用的影響因子,探討轉化作用的最適條件。實驗結果顯示,使用C. compactiuscula菌絲抽出液將羅瓦斯達汀完全轉化成為莫那可林J的最適條件為:培養基葡萄糖含量0.59 %、培養基最初pH 8.5、菌絲培養時間4天、與轉化作用反應時間15小時,在這樣的條件下,最適的受質羅瓦斯達汀的濃度為1 mg/ml。本實驗也從C. compactiuscula的菌絲將羅瓦斯達汀酯化酶以硫酸銨沉澱法、分子篩濾層析法及離子交換層析法進行分離與純化,而這局部純化的酵素經由SDS-PAGE分析,顯示為均一聚合胜肽鏈,其分子量約為28-kDa。此外,經轉化形成的莫那可林J亦被分離與純化,並藉由核磁共振光譜儀的分析,進行產物莫那可林J結構之確認。
Statins series were often prescribed as a therapeutic medicine for controlling hypercholesterolemia. Lovastatin, also called monacolin K and mevinolin, is one of the statins series. Lovastatin can be hydrolyzed by an enzyme named lovastatin esterase to cleave its side chain to form monacolin J, also called triol acid, which is the core structure of various statins for synthesis of other statins. Using Clonostachys compactiuscula, we were able to demonstrate the conversion of lovastatin to form monacolin J. Response surface methodology was used to achieve the optimization of the conversion process. To study the proposed second-order polynomial model, we used a central composite experimental design with multiple linear regression to estimate the model coefficients of the five selected factors believed to influence the conversion process. The experimental results indicated that the optimal conditions for complete conversion of lovastatin to form monacolin J by using C. compactiuscula mycelium lysate were obtained as follows: glucose content of the medium 0.59%, the initial pH of the medium 8.5, mycelium incubation time 4 days, and the conversion time 15 hours. Under such a condition, the optimal concentration of substrate lovastatin was 1 mg/ml. The lovastatin esterase was isolated and purified from the mycelium of C. compactiuscula by a series of ammonium sulfate precipitation, size-exclusion chromatography, and ion-exchange chromatography. The partially purified enzyme showed a homopolymer peptide with a molecular mass of 28-kDa found by SDS-PAGE. The converted product, monacolin J, was isolated and purified, and its structure was verified by NMR.
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