Kinetically Controlled Carboxypeptidase-Catalyzed Synthesis of Novel Antioxidant Dipeptide Precursor BOC-Tyr-Ala

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 carboxypeptidase Y (CPY) expressed in P. pastoris GS115. 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 °C, 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 concentration–time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant $$K_{\text{m}}^{\text{app}}$$ and the apparent maximum reaction rate $$r_{\hbox{max} }^{\text{app}}$$ to be $$2.9946 \times 10^{{^{ - 2} }} {\text{ mol/L}}$$ and $$2.0406 \times 10^{ - 2} {\text{ mmol/(mL}}\,{\text{h)}}$$ , respectively.