Biocatalysts and Bioreactor Design

Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology

  1. Shih-Hao Hu1,†,
  2. Chia-Hung Kuo2,†,
  3. Chieh-Ming J. Chang3,
  4. Yung-Chuan Liu3,
  5. Wen-Dee Chiang1,*,
  6. Chwen-Jen Shieh2,*

Article first published online: 18 OCT 2012

DOI: 10.1002/btpr.1623

Issue

Biotechnology Progress

Biotechnology Progress

Volume 28, Issue 6, pages 1443–1449, November/December 2012

Additional Information

How to Cite

Hu, S.-H., Kuo, C.-H., Chang, C.-M. J., Liu, Y.-C., Chiang, W.-D. and Shieh, C.-J. (2012), Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology. Biotechnol Progress, 28: 1443–1449. doi: 10.1002/btpr.1623

Author Information

  1. 1

    Dept. of Food Science, Tunghai University, Taichung 407, Taiwan

  2. 2

    Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan

  3. 3

    Dept. of Chemical Engineering, National Chung Hsing University Taichung, 402, Taiwan

  1. †

    These authors contributed equally to this work

*Dept. of Food Science, Tunghai University, Taichung 407, Taiwan

  1. †

    These authors contributed equally to this work

Publication History

  1. Issue published online: 4 DEC 2012
  2. Article first published online: 18 OCT 2012
  3. Accepted manuscript online: 22 AUG 2012 09:08AM EST
  4. Manuscript Revised: 23 JUL 2012
  5. Manuscript Received: 14 MAY 2012

Funded by

  • National Science Council of Taiwan, ROC. Grant Numbers: 98-2313-B-005-027-MY3, 101-2313-B-005-040-MY3, 100-2811-B-005-050
  • Ministry of Education, Taiwan, ROC, under the ATU plan

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Keywords:

  • N-Ac-Phe-Tyr-NH2;
  • enzymatic peptide synthesis;
  • organic-aqueous solution;
  • chymotrypsin

Abstract

A peptide, N-Ac-Phe-Tyr-NH2, 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-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 = 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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

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