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dc.contributorYung-Chuan Liuen_US
dc.contributor.authorTsai, Chang-Hanen_US
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dc.description.abstract近年來高血壓一直是國內十大死因之一,會引起中風、心臟病、血管瘤、腎衰竭等疾病,許多研究指出從蛋白水解物分離之二肽Phe-Tyr具有抑制血管收縮素轉化酵素(Angiotensin Converting Enzyme, ACE)的功能,可達到降血壓的效果。由於水解產物分離與純化不易,本研究用脂解酵素(Porcine pancreatic lipase, PPL)催化合成具有降血壓活性二胜肽Phe-Tyr,所使用的基質為N-乙醯-苯丙胺酸乙酯(N-Ac-Phe-OEt)與L-酪胺酸胺(L-Tyr-NH2)。 實驗首先挑選三種不同溶劑包括二甲基亞碸(DMSO)、三羥甲基鹽酸緩衝溶液(Tris-HCl buffer)、乙腈(acetonitrile)進行混合實驗設計法的探討,找出反應最佳溶劑比例,結果以100% Tris-HCl buffer有最大產率為60.7%。因此以Tris-HCl buffer為溶劑來配合五階層四變數之中心混成設計及反應曲面法,探討反應時間(2~10min)、反應溫度(20~40℃)、酵素用量(2~10U)及基質莫耳數比值(Tyr/Phe=1~3)對二肽產率之影響,結果顯示出反應時間、酵素用量、和基質莫耳數比值對於二肽的產率皆有顯著影響,其中以反應溫度與基質莫耳數比影響最大;藉由脊型分析得知,最適化合成反應條件為反應時間3.8min,反應溫度為20.9℃,酵素用量6.5U及基質莫耳數比值2.53(Tyr/Phe),所得到之預測產率與實際產率分別為85%與84.4%。 在酵素動力學方面,本研究以Ping-Pong Bi-Bi的動力學模式,比較PPL與α-chymotrypsin催化合成二肽之動力學參數差異。PPL合成系統中Vmax=13.14 mM/min和Km=7.74 mM,Vmax/Km=1.69 min-1,而α-chymotrypsin合成系統中Vmax=20.24 mM/min和Km=11.56 mM,Vmax/Km=1.75 min-1,結果顯示PPL的合成胜肽的催化能力與α-chymotrypsin相當。本研究證實用脂解酵素合成胜肽的可行性,提供了酵素合成胜肽一個嶄新的途徑。zh_TW
dc.description.abstractHypertension, which can cause strokes, heart attacks, vascular tumors, kidney failure and other diseases, is one of the top ten causes of death in Taiwan. Phe-Tyr dipeptide, isolated from the hydrolyzation of proteins, inhibits angiotensin converting enzyme (ACE) activity which can subsequently lower blood pressure. Because the hydrolysate is not easily separated or purified, this study uses porcine pancreatic lipase (PPL) to catalyze the synthesis of N-Ac-Phe-Tyr-NH2, a dipeptide derivative with blood pressure lowering capabilities, from the two substrates, N-acetyl-phenylalanine ethyl ester (N-Ac-Phe-OEt) and L-tyrosinamide (L-Tyr-NH2). At first three different solvents were tested: Tris-HCl buffer (80 mM, pH9.0), dimethyl sulfoxide (DMSO), and acetonitrile (ACN). The experiment showed that the solvent containing 100 % Tris -HCl buffer gave the optimum molar production of N-Ac-Phe-Tyr-NH2 at 60.7 %. Therefore, 100 % Tris-HCl buffer was selected as the solvent used for the synthesis of N-Ac-Phe-Tyr-NH2 in this experiment. Response surface methodology (RSM) and five-level-four-factor central composite rotatable design (CCRD) were employed to evaluate the effect of synthesis parameters, such as reaction time (2~10 min), temperature (20~40 ℃), enzyme amount (2~10 U) and substrate molar ratio (Tyr/Phe=1~3) on molar conversion percentage of N-Ac-Phe-Tyr-NH2. Based on a ridge max analysis, the optimum synthesis condition included an incubation time of 3.8 min, a reaction temperature of 20.9 ℃, an enzyme amount of 6.5 U and a substrate molar ratio (Tyr/Phe) of 2.53. The predicted and the actual (experimental) yields were 85 % and 84.4 %, respectively. The reaction kinetic followed the Ping-Pong mechanism of enzyme kinetics was used and the reaction rate and kinetic constant for PPL and α-chymotrypsin were obtained. The Vmax, Km, and Vmax/Km, for PPL were 13.14 mM/min, 7.74 mM, and 1.69 min-1 respectively. The Vmax, Km, and Vmax/Km, for α-chymotrypsin were 20.24 mM/min, 11.56 mM, and 1.75 min-1 respectively. These results show the similarity between PPL’s and α-chymotrypsin’s synthetic peptide catalytic abilities. Therefore, this study demonstrates the feasibility of using lipase, specifically PPL, for the enzymatic synthesis of peptides.en_US
dc.description.tableofcontents第一章、緒論 1 1.1前言 1 1.2研究動機 2 第二章、文獻回顧 3 2.1胜肽 3 2.2胜肽合成 5 2.2.1化學合成 5 2.2.2酵素合成 5 2.3有機溶劑中之酵素反應 8 表2-1常用有機溶劑之log P值 10 2.4 Porcine pancreatic lipase (PPL)及其相關研究 11 2.4.1 Porcine pancreatic lipase (PPL)介紹 11 2.4.2PPL合成胜肽相關研究 13 2.5高血壓 14 2.5.1高血壓簡介 14 2.5.2高血壓之成因 15 2.5.3高血壓的治療 17 2.6ACE與ACE-I 18 2.6.1ACE生化特性 18 2.6.2ACE-I抑制原理 18 2.7實驗設計 19 2.7.1單體形心設計(Simplex Centorid Design) 19 2.7.2反應曲面法(Response Surface Methodology ,RSM) 22 2.7.3中心混成設計(Central Composite Design, CCD) 22 2.8酵素動力學 23 2.8.1乒乓機制(Ping-Pong Bi-Bi mechanism) 24 2.8.2Alberty速率方程式之通式 24 第三章、材料與方法 28 3.1實驗流程 28 3.2實驗藥品 30 3.3儀器設備 31 3.4實驗方法 33 3.4.1酵素選擇 33 3.4.2最適化混合溶劑實驗 33 3.4.3最適化生產條件實驗 37 3.5酵素動力學 40 第四章、結果與討論 41 4.1酵素選取 41 4.2 LC/MS分析 41 4.3最適化溶劑的探討 44 4.4最適化生產條件探討 49 4.4.1單一因子探討 49 4.4.2多因子及其交互作用探討 55 4.5動力學模式探討 66 第五章、結論與未來展望 69 5.1結論 69 5.2未來展望 70 參考文獻 71 附錄 76zh_TW
dc.subjectresponse surface methodologyen_US
dc.subjectpeptide synthesisen_US
dc.titleLipase catalyzed synthesis of dipeptide derivative (N-Ac-Phe-Tyr-NH2) with blood pressure-lowering activityen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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