Optimization of α-Chymotrypsin-Catalyzed Dipeptide Derivative (N-Ac-Phe-Gly-NH2) Synthesis

Autor: Hen-Yi Ju, 朱恒儀
Rok vydání: 2010
Druh dokumentu: 學位論文 ; thesis
Popis: 98
The dipeptide derivative, N-Ac-Phe-Gly-NH2, has a heat-resistant character to prevent protein agglutination due to heat and makes inflammation or pathological changes for normal cells. This study focused on the immobilization of α-chymotrypsin and its catalysis to synthesize the dipeptide derivative under optimal conditions. Three sections are included, and they are: (1) optimal synthesis of N-Ac-Phe-Gly-NH2 catalyzed by α-chymotrypsin, (2) optimal immobilization of α-chymotrypsin on Fe3O4-chitosan nanoparticles by covalent-binding and (3) optimal synthesis of N-Ac-Phe-Gly-NH2 catalyzed by the immobilized enzyme in a mixing reactor furnished with a magnet. In the first section (Chapter 3), the dipeptide derivative, N-acetyl-phenylalanine- glycinamide (N-Ac-Phe-Gly-NH2), was synthesized from N-acetyl phenylalanine ethyl ester (N-Ac-Phe-OEt) and glycinamide hydrochloride (Gly-NH2•HCl) and catalyzed by α-chymotrypsin (a protease) in a biphasic system. Response surface methodology with a four-factor-five-level central composite rotatable design was employed to evaluate the effects of selected variables that included the incubation time, reaction temperature, enzyme activity, and pH level on the yield of the dipeptide derivative. The results indicated that pH significantly affected the yield of N-Ac-Phe-Gly-NH2. From a ridge max analysis, the optimum conditions for this synthesis included an incubation time of 30.9 min, a reaction temperature of 35.8°C, an enzyme activity of 159.2 U, and a pH of 8.98. The predicted and the actual (experimental) yields were 98.0% and 95.1%, respectively. The second section (Chapter 4) investigated the immobilization of α-chymotrypsin onto magnetic Fe3O4-chitosan nanoparticles by covalent binding. The RSM with a 3-factor-3-level Box-Behnken experimental design was employed to evaluate the effects of the manipulated variables, including the incubation time, reaction temperature and pH level, on the activity of the immobilized enzyme. The results indicated that the temperature significantly affected the enzyme activity. The binding sites of functional groups were identified by FTIR. By a ridge max analysis, the optimum condition for α-chymotrypsin immobilization was obtained to include a reaction temperature of 21.7°C, a pH level of 7.6, and an incubation time of 1.1 h. The predicted and the actual (experimental) enzyme activities were 353.51 and 346.76±46.51 U/g-support, respectively, under the optimum condition. The immobilized enzyme (Fe3O4-chitosan-chymotrypsin nanoparticles) has a good acid-resisting character and can reduce the operating time to separate the product from the enzyme by a magnetic field. Therefore, the immobilized enzyme could be used to produce N-Ac-Phe-Gly-NH2 in a large-scale. In the third section (Chapter 5), N-Ac-Phe-Gly-NH2 synthesis was catalyzed by the immobilized enzyme in a mixing reactor furnished with a magnet. The RSM with a 3-factor-3-level Box-Behnken experimental design was employed to evaluate the effects of the manipulated variables, including the incubation time, reaction temperature and pH level, on the N-Ac-Phe-Gly-NH2 yield. The results indicated that the reaction temperature and pH level significantly affected the yield. The yield increased with the increase of the reaction temperature or pH. By ridge max and canonical analyses, the optimal condition for N-Ac-Phe-Gly-NH2 synthesis was obtained to include an incubation time of 92.3 min, a reaction temperature of 36.2°C, and a pH level of 8.7. The predicted and the actual (experimental) yields under the optimal condition were 84.22% and 82.26±0.39%, respectively. In summary, this study first obtained an optimal condition for N-Ac-Phe-Gly-NH2 synthesis catalyzed by α-chymotrypsin using CCRD. Then, α-chymotrypsin was immobilized onto Fe3O4-chitosan nanoparticles by covalent binding. The optimal condition for enzyme immobilization was obtained by a Box-Behnken experimental design. The immobilized enzyme has a good acid-resisting character and its catalytic capability is about the same as the free-enzyme. Moreover, the immobilized enzyme can be reused up to more than ten times and therefore could be used to produce N-Ac-Phe-Gly-NH2 in a large-scale. The last part of this study investigated the optimal condition for N-Ac-Phe-Gly-NH2 synthesis in a mixing reactor (with a rotational speed of 150 rpm) furnished with a magnet by a Box-Behnken design. Although the yields obtained in Chapter 5 was 10%-15% lower than those obtained in Chapter 3, the immobilized enzyme can be used up to more than ten times. Furthermore, the immobilized enzyme can be easily separated from the product by a magnetic field and therefore can reduce the operating cost substantially.
Databáze: Networked Digital Library of Theses & Dissertations
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