Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems

Yukitaka Kimura, Kazuhiro Nakanishi, Ryuichi Matsuno

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The precursor of Leu-enkephalin, Z-l-TyrGlyGly-l-Phe-l-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of l-Tyr. First, Z-GlyGlyOBut and Z-l-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and α-chymotrypsin, respectively. Then, Z-l-Phe-l-LeuOEt was synthesized by thermolysin from Z-l-Phe and l-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-l-TyrGlyGly-l-Phe-l-LeuOEt from Z-l-TyrGlyGly and l-Phe-l-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of l-Phe-l-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-l-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.

Original languageEnglish
Pages (from-to)272-280
Number of pages9
JournalEnzyme and Microbial Technology
Volume12
Issue number4
DOIs
Publication statusPublished - 1990
Externally publishedYes

Fingerprint

Thermolysin
Leucine Enkephalin
Organic solvents
Water
Enzymes
Immobilized Enzymes
Buffers
Papain
Amino acids
Hydrolysis
Derivatives
Chymotrypsin
Peptide Hydrolases
Amino Acids

Keywords

  • aqueous/organic biphasic systems
  • Enzymatic peptide synthesis
  • immobilized enzymes
  • Leu-enkephalin
  • papain
  • reactions in organic solvent
  • thermolysin
  • α-chymotrypsin

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems. / Kimura, Yukitaka; Nakanishi, Kazuhiro; Matsuno, Ryuichi.

In: Enzyme and Microbial Technology, Vol. 12, No. 4, 1990, p. 272-280.

Research output: Contribution to journalArticle

@article{02d78abffacf4ee286ada92c90ddbe3c,
title = "Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems",
abstract = "The precursor of Leu-enkephalin, Z-l-TyrGlyGly-l-Phe-l-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of l-Tyr. First, Z-GlyGlyOBut and Z-l-TyrGlyGlyOBut were synthesized in quite a high yield, 83{\%} and 99{\%}, in an aqueous/organic biphasic system by papain and α-chymotrypsin, respectively. Then, Z-l-Phe-l-LeuOEt was synthesized by thermolysin from Z-l-Phe and l-LeuOEt either in buffer or in a biphasic system; the yields were 95{\%} and 100{\%}, respectively. The synthesis of Z-l-TyrGlyGly-l-Phe-l-LeuOEt from Z-l-TyrGlyGly and l-Phe-l-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68{\%}. The reasons for effective synthesis with IME are: (1) higher concentration of l-Phe-l-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-l-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.",
keywords = "aqueous/organic biphasic systems, Enzymatic peptide synthesis, immobilized enzymes, Leu-enkephalin, papain, reactions in organic solvent, thermolysin, α-chymotrypsin",
author = "Yukitaka Kimura and Kazuhiro Nakanishi and Ryuichi Matsuno",
year = "1990",
doi = "10.1016/0141-0229(90)90099-C",
language = "English",
volume = "12",
pages = "272--280",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier Inc.",
number = "4",

}

TY - JOUR

T1 - Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems

AU - Kimura, Yukitaka

AU - Nakanishi, Kazuhiro

AU - Matsuno, Ryuichi

PY - 1990

Y1 - 1990

N2 - The precursor of Leu-enkephalin, Z-l-TyrGlyGly-l-Phe-l-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of l-Tyr. First, Z-GlyGlyOBut and Z-l-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and α-chymotrypsin, respectively. Then, Z-l-Phe-l-LeuOEt was synthesized by thermolysin from Z-l-Phe and l-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-l-TyrGlyGly-l-Phe-l-LeuOEt from Z-l-TyrGlyGly and l-Phe-l-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of l-Phe-l-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-l-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.

AB - The precursor of Leu-enkephalin, Z-l-TyrGlyGly-l-Phe-l-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of l-Tyr. First, Z-GlyGlyOBut and Z-l-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and α-chymotrypsin, respectively. Then, Z-l-Phe-l-LeuOEt was synthesized by thermolysin from Z-l-Phe and l-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-l-TyrGlyGly-l-Phe-l-LeuOEt from Z-l-TyrGlyGly and l-Phe-l-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of l-Phe-l-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-l-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.

KW - aqueous/organic biphasic systems

KW - Enzymatic peptide synthesis

KW - immobilized enzymes

KW - Leu-enkephalin

KW - papain

KW - reactions in organic solvent

KW - thermolysin

KW - α-chymotrypsin

UR - http://www.scopus.com/inward/record.url?scp=0025420072&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025420072&partnerID=8YFLogxK

U2 - 10.1016/0141-0229(90)90099-C

DO - 10.1016/0141-0229(90)90099-C

M3 - Article

VL - 12

SP - 272

EP - 280

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

IS - 4

ER -