Protease-Catalyzed Peptide Formation under High Pressure

Abstract

Abstract The effect of high pressure on peptide formation by the catalysis of carboxypeptidase Y (substitution of ester or peptide by amino acid derivative) or by thermolysin (condensation of N-acylamino acid and amino acid amide) was studied. The carboxypeptidase Y-catalyzed substitution reaction of N-[3-(2-furyl)acryloyl]phenylalanine ethyl ester with glycinamide or phenylalaninamide showed a six-fold higher total peptide yield at 200 MPa than at atmospheric pressure. In the case of the reaction of N-acyldipeptide and amino acid amide, both the peptide yield and substitution efficiency were improved at elevated pressure and the wasteful hydrolysis of the substrate was highly depressed by increasing pressure. The pressure was also effective to get rid of the substrate inhibition by the amino acid ester in the reaction between the N-acylamino acid ester and the amino acid ester and to yield much dipeptide ester at high pressure. An improvement of the peptide yield by pressure for the reaction of thermolysin was observed in a combination of less specific substrates, N-benzyloxycarbonyl-l-aspartic acid and phenylalanine methyl ester, since the high catalytic activity of this enzyme under elevated pressure was significant only in the case that the peptide yield was kinetic-controlled.