Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3251
標題: 脂解酵素催化合成具降血壓活性之二肽衍生物(N-Ac-Phe-Tyr-NH2)
Lipase catalyzed synthesis of dipeptide derivative (N-Ac-Phe-Tyr-NH2) with blood pressure-lowering activity
作者: 蔡昌翰
Tsai, Chang-Han
關鍵字: 反應曲面法;Lipase;最適化;胜肽合成;N-Ac-Phe-Tyr-NH2;response surface methodology;optimization;peptide synthesis;N-Ac-Phe-Tyr-NH2
出版社: 化學工程學系所
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摘要: 
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。

Hypertension, 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.
URI: http://hdl.handle.net/11455/3251
其他識別: U0005-0108201322413400
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