反胶束体系中维生素E醋酸酯的酶催化水解
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摘要
本工作分别考察了反胶束体系的组成对胆甾醇酯酶催化水解维生素E醋酸酯性能的影响。实验结果表明有机试剂、表面活性剂和体系含水量(W_O)等对反胶束体系中酶的催化性能影响较大。通过实验优选0.014mol／L卵磷脂／0.006mol／L胆固醇／环己烷反胶束体系作为胆甾醇酯酶催化水解维生素E醋酸酯的反应介质。
在0.014mol／L卵磷脂／0.006mol／L胆固醇／环己烷反胶束体系中，分别考察了温度、pH值、离子强度、表面活性剂浓度、酶浓度等反应条件对胆甾醇酯酶催化性能的影响，并优选最佳反应条件。
较系统地研究了胆甾醇酯酶在0.014mol／L卵磷脂／0.006mol／L胆固醇／环己烷反胶束体系中稳定性及催化水解维生素E醋酸酯的动力学特征和光学选择性，与胶束溶液中情况对比结果表明胆甾醇酯酶在反胶束体系中具有更高的稳定性，催化活性和光学选择性。
用磷脂／胆固醇／环己烷反胶束体系模拟体内微环境，研究维生素E醋酸酯被胰脏提取物催化水解规律，考察体内的胰酯酶对d，l-维生素E醋酸酯水解作用的光学选择性。用磷脂和胆固醇混合反胶束体系模拟生物膜，分别考察低浓度的d-维生素E醋酸酯，d，l-维生素E醋酸酯，d-α-生育酚和d，l-α-生育酚的抗氧化活性及维生素E醋酸酯的酶催化水解反应进程对维生素E醋酸酯抗氧化活性影响。实验结果表明引起维生素E醋酸酯在体内的光学活性的原因有(1)在体内胰脂酶等催化水解维生素E醋酸酯时具有光学选择性，d-维生素E醋酸酯水解速度比l-维生素E醋酸酯快得多，导致其在体内能迅速生成α-生育酚，及时发挥抗氧化活性；(2)在体内d-维生素E醋酸酯的水解反应产物(d-α-生育酚)与SOD
沈阳药科大学硕士学位论文 摘要
等抗氧化酶之间的抗氧化协同作用比卜维生素E醋酸酯的水解反应产物
（1—Q一生育酚）与 SOD等抗氧化酶之间的的抗氧化协同作用更强。
The effects of ingredients that constitute reverse micelle systems on stability and activity of cholesterol esterase which catalyze the hydrolysis of vitamin E acetate in reverse micelle systems have been studied. It was found that organic solvent, surfactant, and water content (Wo) have great impact on the catalysis behaviors of enzyme in reverse micelle systems. The reverse micelle system that consists of 0.014mol/L lecithin / 0.006 mol/L cholesterol/ cyclohexane is the optimal reaction system.
The effects of temperature and other reaction conditions on the activity of cholesterol esterase in reverse micelle systems have been studied, and the reaction conditions were optimized.
The stability, behavior of kinetics and optical selectivity of the cholesterol esterase in reverse micelle systems have been systematically studied, and compared with those in micelle solutions. The results showed that the stability, activity and optical selectivity of the enzyme in reverse micelle systems are much higher than in micelle solutions.
Using the reverse micelle systems that consists of 0.014mol/L lecithin / 0.006mol/L cholesterol/ cyclohexane as biomembrane-simulating systems, the optical selectivity in the courses of hydrolysis of vitamin E acetate has been studied. The antioxidation activity of d-vitamin E acetate, d,l-vitamin E
acetate, d-a-tocopherol and d,l-#-tocopherol in the reverse micelle systems have been studied. The result indicated that the in vivo optical activity of vitamin E acetate was caused by (1) the optical selectivity in the courses of hydrolysis of vitamin E acetate in vivo, the velocity of hydrolysis of d -vitamin E acetate is much higher than that of 1- vitamin E acetate, which can produce more a-tocopherol that protect the lipids from oxidation ; (2) the hydrolysis product of d -vitamin E acetate ,d -a-tocopherol, have a higher antioxidation activity in vivo than 1-a-tocopherol which is the hydrolysis product of 1 -vitamin E acetate when they display cooperate antioxidation with SOD.
在0.014mol／L卵磷脂／0.006mol／L胆固醇／环己烷反胶束体系中，分别考察了温度、pH值、离子强度、表面活性剂浓度、酶浓度等反应条件对胆甾醇酯酶催化性能的影响，并优选最佳反应条件。
较系统地研究了胆甾醇酯酶在0.014mol／L卵磷脂／0.006mol／L胆固醇／环己烷反胶束体系中稳定性及催化水解维生素E醋酸酯的动力学特征和光学选择性，与胶束溶液中情况对比结果表明胆甾醇酯酶在反胶束体系中具有更高的稳定性，催化活性和光学选择性。
用磷脂／胆固醇／环己烷反胶束体系模拟体内微环境，研究维生素E醋酸酯被胰脏提取物催化水解规律，考察体内的胰酯酶对d，l-维生素E醋酸酯水解作用的光学选择性。用磷脂和胆固醇混合反胶束体系模拟生物膜，分别考察低浓度的d-维生素E醋酸酯，d，l-维生素E醋酸酯，d-α-生育酚和d，l-α-生育酚的抗氧化活性及维生素E醋酸酯的酶催化水解反应进程对维生素E醋酸酯抗氧化活性影响。实验结果表明引起维生素E醋酸酯在体内的光学活性的原因有(1)在体内胰脂酶等催化水解维生素E醋酸酯时具有光学选择性，d-维生素E醋酸酯水解速度比l-维生素E醋酸酯快得多，导致其在体内能迅速生成α-生育酚，及时发挥抗氧化活性；(2)在体内d-维生素E醋酸酯的水解反应产物(d-α-生育酚)与SOD
沈阳药科大学硕士学位论文 摘要
等抗氧化酶之间的抗氧化协同作用比卜维生素E醋酸酯的水解反应产物
（1—Q一生育酚）与 SOD等抗氧化酶之间的的抗氧化协同作用更强。
The effects of ingredients that constitute reverse micelle systems on stability and activity of cholesterol esterase which catalyze the hydrolysis of vitamin E acetate in reverse micelle systems have been studied. It was found that organic solvent, surfactant, and water content (Wo) have great impact on the catalysis behaviors of enzyme in reverse micelle systems. The reverse micelle system that consists of 0.014mol/L lecithin / 0.006 mol/L cholesterol/ cyclohexane is the optimal reaction system.
The effects of temperature and other reaction conditions on the activity of cholesterol esterase in reverse micelle systems have been studied, and the reaction conditions were optimized.
The stability, behavior of kinetics and optical selectivity of the cholesterol esterase in reverse micelle systems have been systematically studied, and compared with those in micelle solutions. The results showed that the stability, activity and optical selectivity of the enzyme in reverse micelle systems are much higher than in micelle solutions.
Using the reverse micelle systems that consists of 0.014mol/L lecithin / 0.006mol/L cholesterol/ cyclohexane as biomembrane-simulating systems, the optical selectivity in the courses of hydrolysis of vitamin E acetate has been studied. The antioxidation activity of d-vitamin E acetate, d,l-vitamin E
acetate, d-a-tocopherol and d,l-#-tocopherol in the reverse micelle systems have been studied. The result indicated that the in vivo optical activity of vitamin E acetate was caused by (1) the optical selectivity in the courses of hydrolysis of vitamin E acetate in vivo, the velocity of hydrolysis of d -vitamin E acetate is much higher than that of 1- vitamin E acetate, which can produce more a-tocopherol that protect the lipids from oxidation ; (2) the hydrolysis product of d -vitamin E acetate ,d -a-tocopherol, have a higher antioxidation activity in vivo than 1-a-tocopherol which is the hydrolysis product of 1 -vitamin E acetate when they display cooperate antioxidation with SOD.
引文
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[29]Zahalka P.J., Dutton B.O., Doherty T.A., et al., Bile salt modulated stereo selection in the cholesterol esterase catalyzed hydrolysis of α-tocopheryl acetate Am. Chem. Soc., 1991, 113:2797-2812.
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[34]Martinek K.,Levashov A.V, Klyachko V.I.,et al.,The principles of enzyme stabilization Ⅵ.catalysis by water-soluble enzymes entrapped into reversed micelles of suffactant in organic solvents,Biochim et Biophys Acta, 1981,657:277-294.
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[2]Freeman Lee S.S.,Douglas J.,Aires-Barres M.R.,et aL,Increased chymotrypsin activity in AOT/bile salt reverse micelles,J. Colloid Interface Sci, 1998,207(2):344-352.
[3]Phenonae K.L.,Maetomas MJ.,Cleck P,et al.Contrast of activities of alkphosphatacideastase regulating by Mg~(2+) in water and reverse micelles,Pro. Chem., 1997,(8):562-575.
[4]Luisi P.L.,Blandamer M.J.,Burgess J., et al. ,Reverse micelles as hosts for proteins and small molecules, Biochem. Biophys. Acta,1988,947:209-216.
[5]Bru R, Pires MJ., Sebatiao M .J.,et al.,Kinetic models in reverse micelles, Biochem. J.,1995,310:721-729.
[6]周国伟，李干佐，程文君等，W/O型微乳液中的酶促反应动力学，中国科学(B)辑，1995，25(10)：326-331．
[7]朱洁，范映婷，李文杰，反胶束体系中的酶学研究，生物与生物物理进展，1998，25(3):204-210．
[8]Verhart R.M.D.,Hilhorst R,Vermue M.,et al.,Description of enzyme kinetics in reverse micelles,Eur. J. Biochem.,1990,187(1):59-72.
[9]Sanchez-Ferrer A.,Garacia-Carmona,Biocatalysis in reverse self-assembling structure:Reverse micelles and reverse micelles and reverse resides,Enzyme Microbe Technol., 1994,16(5):409-415.
[10]Dordick,Characters of lipase in some organic solvent,J Biochem.,1996,468:327-335.
[11]Wescott ,Klibanov T.D.,Parameters of organic solvent modulated lipase activities,J. Biotech. Bioeng, 1999,75:375-387.
[12]Goto M.,Ono T,Nakashio F.,et al.,Design of surfactants suitable for protein extraction by reverse micelles.,Biotech.Bioeng., 1997,54(1):26.-39.
[13]Bru R,Sanchez-Ferrer A.,Garacia-Carmona,The effect of substrate portioning on the kinetics of enzyme micelles, Biochem J, 1990,268(3):679-684.
[14]Huang W.,Li X.F. Zhou J.M.,et al., Activity and conformation of bovine pancreatic fibonuclease in reverse micelles formed by dodecylammonium butyrate and water in cyclohexane,Colloida Surfaces B, 1996,7(1):23-29.
[15]Pshezesky A.V.,Klyachko N.L.,Modeling of mernbrane environment of enzymes:superactivity of laccase entrapped into surfactant reverse micelles in organic solvents,Biokhimiya, 1988,53(2): 1013-1016.
[16]Leavashov A.V.,Klyachko N.L.,Pshezesky A.V.,et al.,Catalysis by watersoluble enzyme in organic solvents stabilization of enzymes against the denaturation (inactivation) when they are included in reverse micelles of surface-active substance,Dokl Akad Nauk SSSR, 1986,289(3):1271-1274.
[17]Dougles G,Hayes,Erdogan,Fonnation of polyol-fatty acid esters by lipase in reversemicelles media,Biotech. Bioeng.,1992,40:110-118.
[18]Roque Bru.,Alvaro,Review optical kinetic models in reverse rnicelles,Biochem.J, 1995,310:721-729.
[19]Fletcher D.D.L.,Robinson B.H.A theoretical study on the expression of enzymic activity in reverse micelles,J.Chem. Soc. Faraday Trans 1,1986(82):2311-2321.
[20]姚凇年，微较团酶学的研究与进展，生命的化学，1998，18(4)：35-38．
[21]Khmelnitski Y.L.,Neverova N.I.,Polyakov V.I.,et al.,Kinetics of enzymatic reaction in reversed micellar systems,Eur. J. Biochem.,1990,190:155-159.
[22]陈琼华．生物化学：第三版[M]．北京：人民卫生出版社，1995．130-131．
[23]Pedraz T.L.,CalvoB,Bortolotti A.,Pharmacody of α-tocopherol and its ramifications,J. Pharm. Pharmacol.,1989,41 (3):415-417.
[24]Takata J.,Karube Y.,Nagata Y.,Hydrolysis of α-tocopheryl acetate,J. Pharm. Sci.,1995,84(1): 96-100.
[25]李正化．药物化学：第三版[M]．北京：人民卫生出版社，1995．330-331．
[26]Nakamura T.,Reicher H.,Sattler.,Comparison of d-α-and 1-α-tocopherol uptake by permanent rat skeletal muscle myoblasts (L6 cells): effects of exogenous lipoprotein lipase,Lipids, 1998,33(10):1001-1008.
[27]Yasuhito Nakagawa, Shoshichi Nojima,Keizo Inoue. Transfer of steroids and α-tocopherol between Liposomal Membrances.J. Biochem. 1980,87:497-502.
[28]Lombardo Dominique,Odette guy. Studys on the substrate specificity of a carboxyl easter hydrolase from human pancreatic juice,Biochemica et Biophysica Acta, 1980,611:147-152.
[29]Zahalka P.J., Dutton B.O., Doherty T.A., et al., Bile salt modulated stereo selection in the cholesterol esterase catalyzed hydrolysis of α-tocopheryl acetate Am. Chem. Soc., 1991, 113:2797-2812.
[30]Chang G.G,Shiao S.L.,Possible kinetics mechanism of human placemental alkaline phosphates in vivo as implemented in reverse micelles,Eur. J. Biochem.,1994, 220:861-870.
[31]Hung H.C., Huang T.M,Chang G.G.,Reverse micelles as model system to study leaving group effects on alkaline phosphates-catalyzed hydralysis,J. Chem. Soc. Perkin Trans, 1997,2:2757-2760.
[32]Hung H.C., Huang T.M.,Chang G.G.,Inhibitory effects of magnesium ion on the human placemental alkaline phosphates catalyzed reaction in reverse micellar system, J. Protein Chem.,1998,17:99-105.
[33]蒋太交，吉鑫忪，袁中一，反向胶束体系对辣根过氧化物酶结构与功能的影响，生物化学与生物物理学报，1998，30(2)：132-136．
[34]Martinek K.,Levashov A.V, Klyachko V.I.,et al.,The principles of enzyme stabilization Ⅵ.catalysis by water-soluble enzymes entrapped into reversed micelles of suffactant in organic solvents,Biochim et Biophys Acta, 1981,657:277-294.
[35]Pileni M.P.,Structural studies ofmicroemultion,J. Phys. Chem.,1993,97(27):3961-3973.
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