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

Yukitaka Kimura, Kazuhiro Nakanishi, Ryuichi Matsuno

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35 Citations (Scopus)


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
Issue number4
Publication statusPublished - Apr 1990
Externally publishedYes


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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology


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