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.
(Less)
- author
- Gololobov, Mikhail Yu
; Stepanov, Valentin M.
; Voyushina, Tatjana L.
; Morozova, Irina P.
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 1994-01-01
- 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
-
- pmid:7764892
- scopus:0028243065
- 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
- 2024-01-01 12:16:42
@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}},
keywords = {{Chymotrypsin; low-water systems; polymerization in low-water systems; subtilisin}},
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}},
}