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

作　　者：Yuyao Shan Wei Qi Mengfan Wang Rongxin Su Zhimin He 

机构地区：[1]State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China [2]Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin 300350, China [3]Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300350, China

出　　处：《Transactions of Tianjin University》2018年第6期513-521,共9页天津大学学报（英文版）

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)

摘　　要：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.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_m^(app) and the apparent maximum reaction rate r_(max)^(app) to be 2.9946 × 10^(-2)mol/L and 2.0406 × 10^(-2)mmol/(mL h), respectively.

关 键 词：Antioxidant dipeptide precursor Kinetic control Reaction kinetic model Enzymatic peptide synthesis 

分 类 号：TQ464.7[化学工程—制药化工]