Kinetic study of thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester in an ethyl acetate saturated aqueous system

K. Nam, Cheol-Koo Lee, S. W. Jeong, Young Min Chi

Research output: Contribution to journalArticle

Abstract

The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the kcat value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and 45°C, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 and 40°C. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, the affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups of Z-Phe was more important than that of the amino groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and 30°C was 8% higher than that at pH 7.0 and 40°C, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.

Original languageEnglish
Pages (from-to)649-655
Number of pages7
JournalJournal of Microbiology and Biotechnology
Volume11
Issue number4
Publication statusPublished - 2001 Jan 1

Fingerprint

phenylalanylleucine
Thermolysin
Esters
Kinetics
Condensation reactions
Temperature
Protonation
Newton-Raphson method
Peptides
Enzymes
carbobenzoxyphenylalanine
ethyl acetate
Phenylalanine
Leucine
Substrates

Keywords

  • Enzymatic peptide synthesis
  • Ethyl acetate saturated aqueous system
  • Thermolysin

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

@article{c217b3923fa34f00baea095940c80bb4,
title = "Kinetic study of thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester in an ethyl acetate saturated aqueous system",
abstract = "The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the kcat value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and 45°C, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 and 40°C. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, the affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups of Z-Phe was more important than that of the amino groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and 30°C was 8{\%} higher than that at pH 7.0 and 40°C, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.",
keywords = "Enzymatic peptide synthesis, Ethyl acetate saturated aqueous system, Thermolysin",
author = "K. Nam and Cheol-Koo Lee and Jeong, {S. W.} and Chi, {Young Min}",
year = "2001",
month = "1",
day = "1",
language = "English",
volume = "11",
pages = "649--655",
journal = "Journal of Microbiology and Biotechnology",
issn = "1017-7825",
publisher = "Korean Society for Microbiolog and Biotechnology",
number = "4",

}

TY - JOUR

T1 - Kinetic study of thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester in an ethyl acetate saturated aqueous system

AU - Nam, K.

AU - Lee, Cheol-Koo

AU - Jeong, S. W.

AU - Chi, Young Min

PY - 2001/1/1

Y1 - 2001/1/1

N2 - The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the kcat value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and 45°C, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 and 40°C. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, the affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups of Z-Phe was more important than that of the amino groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and 30°C was 8% higher than that at pH 7.0 and 40°C, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.

AB - The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the kcat value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and 45°C, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 and 40°C. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, the affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups of Z-Phe was more important than that of the amino groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and 30°C was 8% higher than that at pH 7.0 and 40°C, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.

KW - Enzymatic peptide synthesis

KW - Ethyl acetate saturated aqueous system

KW - Thermolysin

UR - http://www.scopus.com/inward/record.url?scp=0034802308&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034802308&partnerID=8YFLogxK

M3 - Article

VL - 11

SP - 649

EP - 655

JO - Journal of Microbiology and Biotechnology

JF - Journal of Microbiology and Biotechnology

SN - 1017-7825

IS - 4

ER -