Synthesis of phosphatidylethanolamine by enzymatic catalysis and its substrate inhibition kinetics

doi:10.16085/j.issn.1000-6613.2016-2298

Abstract

Abstract: 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 K_iB=1.50mmol/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.

Key words: phosphatidylethanolamine, phospholipase D, transphosphatidylation, kinetics, substrate inhibition

摘要： 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K_iB=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。

关键词: 磷脂酰乙醇胺, 磷脂酶D, 磷脂酰基转移反应, 动力学, 底物抑制效应

CLC Number:

TQ033

DENG Yangmin, LI Binglin, DONG Wenbo, YANG Kaili, ZHANG Xiaoli, ZHAO Binxia. Synthesis of phosphatidylethanolamine by enzymatic catalysis and its substrate inhibition kinetics[J]. Chemical Industry and Engineering Progress, 2017, 36(07): 2601-2606.

邓杨敏, 李冰麟, 董文博, 杨开利, 张小里, 赵彬侠. 磷脂酰乙醇胺的酶促合成及底物抑制动力学[J]. 化工进展, 2017, 36(07): 2601-2606.

References

[1] 曹栋,裘爱泳,王兴国.磷脂及磷脂酰胆碱生物学功能[J].粮食与油脂,2002(11):23-24. CAO D,QIU A Y,WANG X G.Biology function of phospholipids and phosphatidylcholine[J]. Cereals & Oils,2002(11):23-24.
[2] MODICANAPOLITANO J S,RENSHAW P F.Ethanolamine and phosphoethanolamine inhibit mitochondrial function in vitro:implications for mitochondrial dysfunction hypothesis in depression and bipolar disorder[J].Biological Psychiatry,2004,55(3):273-277.
[3] 封娟娟,朱南南,邓杨敏,等.反复分批式反应提高磷脂酰丝氨酸的生产能力[J].化工进展,2016,35(4):1180-1183. FENG J J,ZHU N N,DENG Y M,et al.Increasing productivity of phosphatidyserine by repeated batch reaction[J].Chemical Industry and Engineering Progress,2016,35(4):1180-1183.
[4] SHIELD J W,FERGUSON H D,BOMMARIUS,et al.Enzymes in reversed micelles as catalysts for organic-phase synthesis reactions[J]. Industrial & Engineering Chemistry Fundamentals,1986,25(4):603-612.
[5] JUNEJA L R,KAZUOKA T,YAMANE T,et al.Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources[J].Biochimica et Biophysica Acta,1988,960(960):334-41.
[6] MATSUMOTO Y,SUGIMORI D.Substrate recognition mechanism of Streptomyces,phospholipase D and enzymatic measurement of plasmalogen[J].Journal of Bioscience & Bioengineering,2015,120(4):372-379.
[7] DAMNJANOVI? J,IWASAKI Y.Phospholipase D as a catalyst:application in phospholipid synthesis,molecular structure and protein engineering[J].Journal of Bioscience & Bioengineering,2013,116(3):271-280.
[8] BRUZIK K,MING D T.Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D[J].Biochemistry,1984,23(8):1656-61.
[9] 姚娜,张小里,赵彬侠,等.磷脂酶D催化大豆磷脂合成磷脂酰丝氨酸工艺[J]. 化工进展,2011,30(s1):281-284. YAO N,ZHANG X L,ZHAO B X,et al.Research on the synthetic process of phosphatidylserine from soybean lecithin catalyzed by phospholipase D[J].ChemicalIndustry and Engineering Progress,2011,30(s1):281-284.
[10] 温正.MATLAB 8.0从入门到精通[M].北京:清华大学出版社,2013:226. WEN Z.Mastering MATLAB 8.0[M].Beijing:Tsinghua University Press,2013:226.
[11] UESUGI Y,HATANAKA T.Phospholipase D mechanism using Streptomyces PLD[J].Biochimica et Biophysica Acta,2009,1791(9):962-969.
[12] RAETZ C R,CARMAN G M,DOWHAN W,et al.Phospholipids chiral at phosphorus.Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast[J]. Biochemistry,1987,26(13):4022-4027.

[1]	GU Yongzheng, ZHANG Yongsheng. Dynamic behavior and kinetic model of Hg⁰ adsorption by HBr-modified fly ash [J]. Chemical Industry and Engineering Progress, 2023, 42(S1): 498-509.
[2]	WANG Peng, ZHANG Yang, FAN Bingqiang, HE Dengbo, SHEN Changshuai, ZHANG Hedong, ZHENG Shili, ZOU Xing. Process and kinetics of hydrochloric acid leaching of high-carbon ferrochromium [J]. Chemical Industry and Engineering Progress, 2023, 42(S1): 510-517.
[3]	WANG Junjie, PAN Yanqiu, NIU Yabin, YU Lu. Molecular level catalytic reforming model construction and application [J]. Chemical Industry and Engineering Progress, 2023, 42(7): 3404-3412.
[4]	GE Weitong, LIAO Yalong, LI Mingyuan, JI Guangxiong, XI Jiajun. Preparation and dechlorination kinetics of Pd-Fe/MWCNTs bimetallic catalyst [J]. Chemical Industry and Engineering Progress, 2023, 42(4): 1885-1894.
[5]	LI Yun, CUI Nan, XIONG Xingxing, HUANG Zhiyuan, WANG Dongliang, XU Dan, LI Jun, LI Zebing. Influence of rare earth element Er(Ⅲ) on performance of short-cut nitrification and its inhibition kinetics [J]. Chemical Industry and Engineering Progress, 2023, 42(3): 1659-1668.
[6]	SONG Ye, CHEN Yuzhuo, SONG Yuncai, FENG Jie. Catalyst design and reactor analysis for in-situ purification of organic solid waste syngas [J]. Chemical Industry and Engineering Progress, 2023, 42(3): 1383-1396.
[7]	WANG Wei, ZHANG Dongxu, LI Zunzhao, WANG Xiaolin, HUANG Qiyu. Research progress on the growth behavior of hydrates in water-in-oil emulsion systems [J]. Chemical Industry and Engineering Progress, 2023, 42(3): 1155-1166.
[8]	DUAN Yihang, GAO Ningbo, QUAN Cui. Effect of hydrothermal treatment on pyrolysis characteristics and kinetics of oily sludge [J]. Chemical Industry and Engineering Progress, 2023, 42(2): 603-613.
[9]	KANG Yu, GOU Zenian. Kinetics studies of carbon gas hydrate separation in the presence of amino acids and DTAC [J]. Chemical Industry and Engineering Progress, 2023, 42(10): 5067-5075.
[10]	FAN Jiahao, ZHANG Yang, FAN Binqiang, ZHANG Hedong, ZHENG Shili, ZOU Xing. Crystallization kinetics of (NH₄)₂SO₄ in mixed solution of (NH₄)₂SO₄ and Na₂SO₄ and the influence of Fe/Al/Mn/Cr ions on crystallization [J]. Chemical Industry and Engineering Progress, 2023, 42(1): 488-496.
[11]	CHEN Qiushi, ZHENG Chen, ZHANG Mindi. Numerical simulation of the esterification between chlorophosphate and n-butyl alcohol in microchannel reactor [J]. Chemical Industry and Engineering Progress, 2022, 41(S1): 29-35.
[12]	TIAN Yazhou, HU Yujing, LI Jiyou, REN Jiangyan, WANG Liwei, WANG Xiuli, DING Ying, CHENG Jue, ZHANG Junying. Synthesis, curing kinetics and properties of vanilla alcohol-based epoxy resin [J]. Chemical Industry and Engineering Progress, 2022, 41(S1): 477-484.
[13]	FU Chunlong, WANG Songjiang, LI Guozhi. Research progress on combustion technology of coal gasification fine slag [J]. Chemical Industry and Engineering Progress, 2022, 41(S1): 516-523.
[14]	MA Yunfei, WANG Jianbing, JIA Chaomin, XING Yixin, KE Shu, ZHANG Xian. Recent progress of kinetics model and reactor modeling of ozonation [J]. Chemical Industry and Engineering Progress, 2022, 41(S1): 556-570.
[15]	LUO Zhenmin, LIU Lu, SU Bin, SONG Fangzhi. Effect of inert gas on ethylene explosion limit parameters and kinetic characteristics [J]. Chemical Industry and Engineering Progress, 2022, 41(9): 4653-4661.