Lipase catalyzed synthesis of dipeptide derivative (N-Ac-Phe-Tyr-NH2) with blood pressure-lowering activity

• Main Authors: Chang-Han Tsai, 蔡昌翰
• Other Authors: Yung-Chuan Liu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/27620307808910856381

碩士 === 國立中興大學 === 化學工程學系所 === 101 === 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.