Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology.
A peptide, N-Ac-Phe-Tyr-NH(2) , with angiotensin I-converting enzyme (ACE) inhibitor activity was synthesized by an α-chymotrypsin-catalyzed condensation reaction of N-acetyl phenylalanine ethyl ester (N-Ac-Phe-OEt) and tyrosinamide (Tyr-NH(2) ). 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 = 2:1:1) in which 73.55% of the yield of N-Ac-Phe-Tyr-NH(2)  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-NH(2) , so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012.
Dept. of Food Science, Tunghai University, Taichung, Taiwan.
Shih-Hao
SH
Hu
Chia-Hung
CH
Kuo
Chieh-Ming J
CM
Chang
Yung-Chuan
YC
Liu
Wen-Dee
WD
Chiang
Chwen-Jen
CJ
Shieh
D000097:Acetonitriles / Q000737:chemistry; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism; D019149:Bioreactors; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D009842:Oligopeptides / Q000096:biosynthesis / Q000138:chemical synthesis*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D012044:Regression Analysis; D012107:Research Design; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014325:Tromethamine / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000097:Acetonitriles; D000806:Angiotensin-Converting Enzyme Inhibitors; C505757:L-phenylalanine ethylester; D009842:Oligopeptides; D012997:Solvents; D014325:Tromethamine; C036387:tyrosinamide; D014443:Tyrosine; D010649:Phenylalanine; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D004121:Dimethyl Sulfoxide; C032159:acetonitrile