Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced...Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant car- boxypeptidase Y （CPY） expressed in P. pastoris GS 115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t-butyloxycarbonyl-L-tyrosine-methyl ester （BOC-Tyr-OMe） as the acyl donor and L-alanine （L-Ala） was the amino donor. We optimized the reaction conditions to be： 30 ℃, pH 9.5, organic phase （methanol）/aqueous phase = 1：20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis-Menten equation according to the con-centration-time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant K^（app）mand the apparent maximum reaction rate r^（app）max to be 2.9946 x 10^-2 mol/L and 2.0406 x 10.2 mmol/（mL h）, respectively.展开更多
A process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was re-ported. Enzymes were used for the formation of peptide bonds and the removal of protecting group. Starting with p...A process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was re-ported. Enzymes were used for the formation of peptide bonds and the removal of protecting group. Starting with phenylacetyl (PhAc) glycin, N-protected dipeptide PhAc-Gly-Trp-OMe was obtained by coupling PhAc-protected glycine carboxamidomethyl ester (OCam) with Trp-OMe catalyzed by immobilized papain in buffered ethyl acetate. Then the condensation between PhAc-Gly-Trp-OMe and Met-OEtHCl was carried out by immobilized -chy-motrypsin catalysis in solvent free system. Basic hydrolysis was followed getting PhAc-Gly-Trp-Met-OH. The PhAc-group was removed with penicillin G amidase and H-Gly-Trp-Met-OH was obtained in an overall yield of 43.9%. The reaction conversion of tripeptide in solvent free system was strongly affected by the system of basic salts added. The influence of the support materials used to deposit enzymes and structures of acyl donor and nu-cleophile on the reaction was also investigated.展开更多
基金supported by Ministry of Science and Technology of China(No.2012YQ090194 and No.2013AA102204)the National Natural Science Foundation of China(No.21676191,No.21476165,and No.21621004)
文摘Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant car- boxypeptidase Y （CPY） expressed in P. pastoris GS 115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t-butyloxycarbonyl-L-tyrosine-methyl ester （BOC-Tyr-OMe） as the acyl donor and L-alanine （L-Ala） was the amino donor. We optimized the reaction conditions to be： 30 ℃, pH 9.5, organic phase （methanol）/aqueous phase = 1：20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis-Menten equation according to the con-centration-time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant K^（app）mand the apparent maximum reaction rate r^（app）max to be 2.9946 x 10^-2 mol/L and 2.0406 x 10.2 mmol/（mL h）, respectively.
文摘A process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was re-ported. Enzymes were used for the formation of peptide bonds and the removal of protecting group. Starting with phenylacetyl (PhAc) glycin, N-protected dipeptide PhAc-Gly-Trp-OMe was obtained by coupling PhAc-protected glycine carboxamidomethyl ester (OCam) with Trp-OMe catalyzed by immobilized papain in buffered ethyl acetate. Then the condensation between PhAc-Gly-Trp-OMe and Met-OEtHCl was carried out by immobilized -chy-motrypsin catalysis in solvent free system. Basic hydrolysis was followed getting PhAc-Gly-Trp-Met-OH. The PhAc-group was removed with penicillin G amidase and H-Gly-Trp-Met-OH was obtained in an overall yield of 43.9%. The reaction conversion of tripeptide in solvent free system was strongly affected by the system of basic salts added. The influence of the support materials used to deposit enzymes and structures of acyl donor and nu-cleophile on the reaction was also investigated.
