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Side reactions in enzymatic peptide synthesis in organic media : Effects of enzyme, solvent, and substrate concentrations

Gololobov, Mikhail Yu ; Stepanov, Valentin M. ; Voyushina, Tatjana L. ; Morozova, Irina P. and Adlercreutz, Patrick LU (1994) In Enzyme and Microbial Technology 16(6). p.522-528
Abstract

The progress of enzymatic peptide synthesis catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the l-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group... (More)

The progress of enzymatic peptide synthesis catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the l-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH2 or ZAlaAlaPheXaaNH2 occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'1-specificity of α-chymotrypsin and the Sl-specificity of subtilisin 72, while the Sl-specificity of α-chymotrypsin and the S'l-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the α-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the α-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed a the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Chymotrypsin, low-water systems, polymerization in low-water systems, subtilisin
in
Enzyme and Microbial Technology
volume
16
issue
6
pages
7 pages
publisher
Elsevier
external identifiers
  • scopus:0028243065
  • pmid:7764892
ISSN
0141-0229
DOI
10.1016/0141-0229(94)90024-8
language
English
LU publication?
yes
id
b6f80e78-f717-4345-9b87-cd00faa8fb82
date added to LUP
2019-06-22 09:13:48
date last changed
2019-09-21 02:18:12
@article{b6f80e78-f717-4345-9b87-cd00faa8fb82,
  abstract     = {<p>The progress of enzymatic peptide synthesis catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH<sub>2</sub> (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the l-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH<sub>2</sub> or ZAlaAlaPheXaaNH<sub>2</sub> occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'<sub>1</sub>-specificity of α-chymotrypsin and the S<sub>l</sub>-specificity of subtilisin 72, while the S<sub>l</sub>-specificity of α-chymotrypsin and the S'<sub>l</sub>-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the α-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the α-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed a the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.</p>},
  author       = {Gololobov, Mikhail Yu and Stepanov, Valentin M. and Voyushina, Tatjana L. and Morozova, Irina P. and Adlercreutz, Patrick},
  issn         = {0141-0229},
  language     = {eng},
  month        = {01},
  number       = {6},
  pages        = {522--528},
  publisher    = {Elsevier},
  series       = {Enzyme and Microbial Technology},
  title        = {Side reactions in enzymatic peptide synthesis in organic media : Effects of enzyme, solvent, and substrate concentrations},
  url          = {http://dx.doi.org/10.1016/0141-0229(94)90024-8},
  doi          = {10.1016/0141-0229(94)90024-8},
  volume       = {16},
  year         = {1994},
}