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標題: Solvent Selection and Optimization of a-Chymotrypsin-Catalyzed Synthesis of N-Ac-Phe-Tyr-NH2 Using Mixture Design and Response Surface Methodology
作者: Hu, Shih-Hao
Kuo, Chia-Hung
Chieh-Ming, J.Chang
Liu, Yung-Chuan
Chiang, Wen-Dee
Shieh, Chwen-Jen
關鍵字: N-Ac-Phe-Tyr-NH2;enzymatic peptide synthesis;organic-aqueous solution;chymotrypsin
Project: Biotechnol. Prog., Volume 28, Page(s) 1443–1449.
A peptide, N-Ac-Phe-Tyr-NH2, with angiotensin I-converting enzyme (ACE) inhibitor activity
was synthesized by an a-chymotrypsin-catalyzed condensation reaction of N-acetyl phenylalanine
ethyl ester (N-Ac-Phe-OEt) and tyrosinamide (Tyr-NH2). Three kinds of solvents:
a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were
employed in this study. The optimum reaction solvent component was determined by simplex
centroid mixture design. The synthesis efficiency was enhanced in an organic-aqueous solvent
(Tris-HCl buffer: DMSO: acetonitrile ¼ 2:1:1) in which 73.55% of the yield of N-Ac-
Phe-Tyr-NH2 could be achieved. Furthermore, the effect of reaction parameters on the yield
was evaluated by response surface methodology (RSM) using a central composite rotatable
design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis
included a reaction time of 7.4 min, a reaction temperature of 28.1 C, an enzyme activity
of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the
actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and
RSM performed well in the optimization of synthesis of N-Ac-Phe-Tyr-NH2, so it is expected
to be an effective method for obtaining a good yield of enzymatic peptide.
DOI: 10.1002/btpr.1623
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