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

• 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.

Subject headings

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)

Keyword

Chymotrypsin

low-water systems

polymerization in low-water systems

subtilisin

Publication and Content Type

art (subject category)

ref (subject category)