Phosphatidylethanolamine (PE) was synthesized by phospholipase D (PLD) catalyzed transphosphatidylation from phosphatidylcholine (PC) and ethanolamine (EA) in bi-phasic system.The conditions of enzyme-catalyzed reaction were investigated,and the optimum conditions were as follows:reaction temperature 28℃,pH 5.5,and the mole ratio of EA to PC 20 ∶ 1.The highest yield of PE can reach 87.2％.In addition,the kinetic study indicated that phospholipids were catalyzed by PLD in a Ping-Pang manner.The kinetic model of PLD-catalyzed reaction considering the substrate inhibition was built,which based on the Ping-Pang Bi-Bi mechanism.The parameters of kinetic model were determined by unconstrained nonlinear regression of the Fminsearch function in Matlab software.It showed that the results from this method were identical with that from the double reciprocal Lineweaver-Burk plot,the inhibition constant for substrate EA was KiB=l.50mmol/L.Inhibition mechanism is a phosphatidyl-enzyme complex associating with two nucleophile molecules EA to form phosphatidyl-PLD-(EA)2 dead-end complex,and the inhibition mechanism complied with the competitive inhibition of enzyme by EA during the transphosphatidylation reaction.The simulation results were well fitted to the experimental data for different conditions.Therefore,it is speculated that this model supports the hypothesis described above and can provide guidance for the phospholipid synthesis process and design of the devices.%对磷脂酶D (phospholipase D,PLD)在有机溶媒-水两相体系中催化磷脂酰胆碱(phosphatidylcholine,PC)和乙醇胺(ethanolamine,EA)合成磷脂酰乙醇胺(phosphatidylethanolamine,PE)的反应条件进行优化,并对反应动力学机制进行了研究.确定最佳反应条件为:温度28℃,pH 5.5,底物摩尔比(EA/PC)20∶1,在此条件下PE产率达87.2％.通过研究底物浓度对反应速率的影响,确定反应符合单底物抑制的Ping-Pang机制.分别以Matlab软件的Fminsearch函数与Lineweaver-Burk双倒数作图法求取动力学参数,结果基本一致,底物乙醇胺EA的抑制常数为KiB=1.50mmol/L.抑制机理为两分子EA同时结合到一分子磷脂酰基-PLD中间体上,生成磷脂酰基-PLD-(EA)2死端复合物,符合竞争性抑制反应动力学规律.该动力学模型预测与实验数据吻合良好,对PLD催化合成PE反应过程的操作及反应器选型设计具有指导作用.
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