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

查看全文 作　　者：Yuyao [1]Shan;Wei [1,2,3]Qi;Mengfan [1,2]Wang;Rongxin [1,2,3]Su;Zhimin [1]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》索引2018年第24卷第6期,共9页 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 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：运动模型 抗氧化剂 肽合成 催化 控制 PASTORIS 合成反应 应用软件