非水相中固定化脂肪酶拆分（±）-薄荷醇的研究
|
摘要
建立了以纳米级磁性粒子为载体固定化脂肪酶的方法，优化了脂肪酶的固定化条件，考察了固定化酶的性质。制备的磁性载体平均粒径20nm，具有超顺磁性，分散和再分散效果好。固定化酶的最适吸附时间为60min，酶用量为100mg酶/100mg载体，固定化酶的酶活达到718U/g载体。结果表明，经纳米磁性粒子固定化后，脂肪酶得到活化，固定化酶比活为游离酶的1.8倍。同时，固定化脂肪酶的pH稳定性得到显著提高。
脂肪酶在非水相系统中具有水相反应所不具备的很多优点，利用固定化的脂肪酶在有机溶剂中的催化特性，对手性物质外消旋薄荷醇进行不对称的酯化反应，达到两种对映体的拆分。利用具备手性固定相的气相色谱对酯化结果进行分析，确定了适宜的分析条件，从而为研究酶催化反应提供了有力的依据。考察了非水相介质中外消旋薄荷醇不对称酯化反应的特点，从反应时间、反应温度、酶用量、体系水活度、溶剂以及酰基供体选择等方面对反应进行了全面研究。自制固定化酶催化的外消旋薄荷醇酯化反应最佳时间为2.5h，最适加酶量为200U，最适底物浓度为0.2mmol/ml，最适温度为30℃，正己烷和丙酸酐分别为适合实验条件的溶剂和酰基供体，酶的催化活性随反应体系的水活度的降低而升高。在实验条件下，外消旋薄荷醇得到了有效的分离，e.e.%为84.5%，E值为11.9，薄荷醇转化率为37.8%。
Lauric acid-stabilized magnetic nanoparticles were prepared and used for the lipase immobilization. The nanoparticles were characterized by transmission electron microscopy and magnetic measurements. The observations indicated that the particles were superparamagnetic. Magnetic sedimentation of the nanoparticles was achieved within 0.6 min in organic media, 200 times faster than the gravitational sedimentation. The lauric acid on the particles provided a hydrophobic interface for the lipase immobilization, and the conformational changes of the immobilized lipase induced by interaction with the hydrophobic interface yielded an “open structure”. Candida rugosa lipase(CRL) was immobilized to the magnetic carrier at up to 717 U/g carrier, 1.8 times higher than the free lipase containing the same protein. The activity amelioration indicated the occurrence of “interface activation”. The pH dependencies of the immobilized and free lipases were also investigated, of which the optimum pH were determined. Moreover, better stability of the immobilized lipase for the hydrolysis of olive oil was observed.
There are numerous advantages of conducting enzymatic conversions in organic solvents over in water. Optically active menthyl propionate of (-)-menthol was synthesized by enantioselective esterification of (±)-menthol using immobilized CRL as a biocatalylst. Substrate conversion, enantiomeric ratio(E), and enatiomeric excess percentage (e.e.%) were estimated by the analysis of gas chromatography equipped with a chiral column. Effects of various reaction parameters, such as water activity, enzyme load, type of acyl donor, solvent specie, time and temperature, on the conversion as well as enantioselectivity were studied. n-Hexane was found to be the most suitable solvent. Propionic acid anhydride gave better conversion compared to other acyl donor used in the present study. It is experimentally verified that lower water activity in organic solvent provided higher substrate conversion, E and e.e.%. They are 37.8%, 11.9 and 84.9%, respectively.
脂肪酶在非水相系统中具有水相反应所不具备的很多优点，利用固定化的脂肪酶在有机溶剂中的催化特性，对手性物质外消旋薄荷醇进行不对称的酯化反应，达到两种对映体的拆分。利用具备手性固定相的气相色谱对酯化结果进行分析，确定了适宜的分析条件，从而为研究酶催化反应提供了有力的依据。考察了非水相介质中外消旋薄荷醇不对称酯化反应的特点，从反应时间、反应温度、酶用量、体系水活度、溶剂以及酰基供体选择等方面对反应进行了全面研究。自制固定化酶催化的外消旋薄荷醇酯化反应最佳时间为2.5h，最适加酶量为200U，最适底物浓度为0.2mmol/ml，最适温度为30℃，正己烷和丙酸酐分别为适合实验条件的溶剂和酰基供体，酶的催化活性随反应体系的水活度的降低而升高。在实验条件下，外消旋薄荷醇得到了有效的分离，e.e.%为84.5%，E值为11.9，薄荷醇转化率为37.8%。
Lauric acid-stabilized magnetic nanoparticles were prepared and used for the lipase immobilization. The nanoparticles were characterized by transmission electron microscopy and magnetic measurements. The observations indicated that the particles were superparamagnetic. Magnetic sedimentation of the nanoparticles was achieved within 0.6 min in organic media, 200 times faster than the gravitational sedimentation. The lauric acid on the particles provided a hydrophobic interface for the lipase immobilization, and the conformational changes of the immobilized lipase induced by interaction with the hydrophobic interface yielded an “open structure”. Candida rugosa lipase(CRL) was immobilized to the magnetic carrier at up to 717 U/g carrier, 1.8 times higher than the free lipase containing the same protein. The activity amelioration indicated the occurrence of “interface activation”. The pH dependencies of the immobilized and free lipases were also investigated, of which the optimum pH were determined. Moreover, better stability of the immobilized lipase for the hydrolysis of olive oil was observed.
There are numerous advantages of conducting enzymatic conversions in organic solvents over in water. Optically active menthyl propionate of (-)-menthol was synthesized by enantioselective esterification of (±)-menthol using immobilized CRL as a biocatalylst. Substrate conversion, enantiomeric ratio(E), and enatiomeric excess percentage (e.e.%) were estimated by the analysis of gas chromatography equipped with a chiral column. Effects of various reaction parameters, such as water activity, enzyme load, type of acyl donor, solvent specie, time and temperature, on the conversion as well as enantioselectivity were studied. n-Hexane was found to be the most suitable solvent. Propionic acid anhydride gave better conversion compared to other acyl donor used in the present study. It is experimentally verified that lower water activity in organic solvent provided higher substrate conversion, E and e.e.%. They are 37.8%, 11.9 and 84.9%, respectively.
引文
Zaks A, Klibanov, A.M. Enzymatic catalysis in organic media at 100℃，Science. 1984, 224:1249~1251
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases, Enzyme Microb. Technol, 1992, 14: 426~446
Saiz-Díaz C I, Wohlfahrt G, Nogoceke E, Hernández-Laguna A, Smeyers Y G, Menge U., Molecular structure and conformational analysis of chiral alcohols application to the enantioselectivity study of lipases, Mol. Structure(Theochem), 1997, 390:225~237
俞俊棠, 唐孝宣, 生物工艺学, 上海: 华东理工大学出版社, 1991, 188~189
郭勇, 现代生化技术, 广州: 华南理工大学出版社, 1996, 170~173
伦世仪, 生化工程, 中国轻工业出版社 1993, 84~85
Winkler F K, D’Arcy A, Hunzuker W., Structure of human pancreatic lipase, Nature, 1990, 343: 771~774.
Gotz F, Poop F, and Schleifer K H. Complete nucleotide sequence of the lipase gene from staphylococcus hyicus cloned in staphylococcus hyicus, Nucleic Acid Res., 1985, 13: 5895~5906
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases. Enzyme Microb. Technol. 1992, 14: 426~446
Koeller, K.M.; Wong, C.H., Enzymes for chemical synthesis, Nature, 2001, 409: 232~240
Klibanov, A. M., Improving enzymes by using them in organic solvents, Nature, 2001, 409: 241~246
Athawale, V.; Manjrekar N., Mild chemo-enzymatic synthesis of polymer-supported cinchona alkaloids and their application in asymmetric Michael addition, Tetrahedron Lett, 2001, 42: 4541~4543
Kamal A., Khanna, R. G. B., Ramu R., Chemoenzymatic synthesis 2 of both enantiomers of fluoxetine, tomoxetine and nisoxetine: lipase-catalyzed resolution of 3-aryl-3-hydroxypropanenitriles, Tetrahedron: Asymmetry, 2002, 13: 2039~2051
Reetz M.T., Lipases as practical biocatalysts, Curr. Opin. Chem. Biol,. 2002, 6:
145~150
Talon R., Montel M. C., Berdague J. L., Production of flavour ester by lipases from Staphylococcus warneri and S. xylosus. Enzyme Microb. Technol., 1996, 19: 620~622
Zaks A, Dodds D R., Application of biocatalysis and biotransformation to the synthesis of pharmaceuticals, Drug Disc. Today, 1997, 2:513~531
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases. Enzyme Microb. Technol. 1992, 14: 426~446.
Cardenas J, Alvarez E, de Castro-Alvarez M-S, Sanchez-Montero J-M, Valmaseda M, Elson SW, Sinisterra J-V. Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases, Mol Catal B: Enzymatic, 2001, 14: 111~123
Osada K, Takahashi K, Hatano M., Polyunsaturated fatty acid glyceride syntheses by microbial lipase, Am. Chem. Soc., 1990, 67: 921~922
Undurraga D, Markovits A, Erazo S., Cocoa butter equivalent through enzymic interesterification of palm oil midfraction, Process Biochem., 2001, 36: 933~939
Chopineau J, Mccafferty F D, Therisod M, Kilibanov A M., Production of biosurfactants from sugar alcohols and vegetable oils catalyzed by lipases in a nonaqueous media, Biotechnol. Bioengin, 1988, 31: 208~214
Zhang LQ, Zhang YD, Xu L, Li XL, Yang XC, Xu GL, Wu XX, Gao HY, Du WB, Zhang XT, Zhang XZ., Lipase-catalyzed synthesis of RGD diamide in aqueous water-miscible organic solvents, Enzyme Microb Technol., 2001, 29: 129~135
Margolin A L, Fitzpatrick P A, Dubin P L, Kilibanov A M., Chemenzymatic synthesis of optically active (meth)acrylic polymers, Am. Chem. Soc., 1991, 113: 4693~4694
Gill I, Valivety R., Polyunsaturated fatty acids: Part 1. Occurrence biological activities and applications. Trends Biotechnol., 1997a, 15:401~409
Van Dyck S M O, Lemiere G L F, Jonckers T H M, Dommisse R, Pieters L, Buss V., Kinetic resolution of a dihydrobenzofuran-type neolignan by lipase-catalysed acetylation, Tetrahedron Asymmetry, 2001, 12:785~789
Margolin A L., Enzymes in the synthesis of chiral drugs, Enzyme microb. Technol., 1993, 15: 266~280
杨节非, 固定化酶回顾与展望, 国外医药-合成药、生化药、制剂分册, 1994,15,(4): 209~212
ALEKSEY ZAKS AND ALEXANDER M. KLIBANOV, Enzyme-catalyzed processes in organic solvents, biochemistry, 1985,82:3192~3196
朱祥瑞, 徐俊良, 家蚕丝素固定化α-淀粉酶的制备及其理化特性, 浙江大学学报（农业与生命科学版）, 2002,28（1）: 64~69
徐岩, 李建波，微生物脂肪酶的固定化及其在非水相催化中的应用研究, 工业微生物, 2001,31(1): 46~48
曹国民, 盛梅, 高广达, 脂肪酶的固定化及其性质的研究, 生物技术, 1997,7（3）：14~17
ALAN FERSHT, 酶的结构和作用机制(杜锦珠 茹炳根 卫新成),北京: 北京大学出版社, 1991,64~67
魏远安, 姚评佳, 谢庆武, 梁锦添, 游离及固定化果糖基转移酶部分酶学性质的比较研究, 中国生物化学与分子生物学报, 2001,17（4）：501~505
Geluk M.A., Norde W., Van H.K., Kalsbeek A.l., K.Van’s Riet, Adsorption of lipase from Candida rugosa on cellulose and its influence on lipolytic activity, Enzyme microb.Technol.,1992,14: 748~754
朱国辉, 黄卓烈, 丘泰球, 刘石生, 功率超声对酶促反应的影响, 应用声学, 2001, 20(4): 45~48
Khmelnitsky Y L, Levashov A V, Klyachkn N L, et al., Engineering biocatalytic systems in organic media with low water content, Enzyme Microb Technol., 1998. 10(12): 710~721
Marie-Pierre B. et al., Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications, Biotechnology and Bioengineering, 1999, 63(6):730~738
Ruple J A, Gratton E, Careri G, Trend Biochem., Sci., 1983, 8:18~19
Miller, C.; Austin, H.; Posorske, L.; Gonzlez, J., Characteristics of an immobilized lipase for the commericial synthesis of esters, Am. Oil Chem. Soc,. 1988, 65: 927~931
Bornscheuer U, Herar A, Kreye L, Wendel V, Capewell A, Meyer H H, Scheper T, Kolisis F N., Factors affecting the lipase catalyzed transesterification reaction of 3-hydroxy in organic solvents, Tetrahedron: Asymmetry, 1993, 4:1007~1016
Han J J, Iwasaki Y, Yamane T., Monitoring of lipase-catalyzed transesterification between eicosapentaenoic acid ethyl ester and tricaprylin by silver ion
high-performance liquid chromatography and high-temperature gas chromatography, Am. Oil Chem. Soc., 1999, 76:31~39
Sophie C, et al., Water activity control: A way to improve the efficient of continuous lipase esterification, Biotechnology and Bioengineeing, 1998, 60(3):362~367
Kwon S J. et al., Removal of water produced from lipase-catalyzed esterification in organic solvent by pervaporation, Biotechnology and Bioengineering, 1995, 46(4):393~399
Guo X G, et al., Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated pseudomonas strain, Enzyme and Microbial Technology, 2000,27:74~77
Jae EK, et al., Effect of salt hydrate pair on lipase catalyzed regioselective monoacylation of sucrose, Biotechnology and Bioengineering, 1998,57(1): 121~124
Laane C, Boeren S, Vos K, Veeger C., Rules for optimization of biocatalysis in organic solvents, Biotechnol. Bioeng., 1987, 30:81~87
Valivety R H, Halling P J, Peilow A D, Macrae A R., Reaction rate with suspended lipase catalyst shows similar dependence on activity on water activity in different organic solvents, Biochim. Biophys. Acta 1992, 1118:218~222
Bornscheuer U T, Yamane T., Activity and stability of lipase in the solid-phase glycerolysis of triolein, Enzyme Microb. Technol. 1994, 16:864~869
Huge-Jensen B, Galluzzo D R, Jensen R G., Partial purification and characterization of free and immobilized lipases from Mucor miehei, Lipids, 1987, 22: 559~565
Dixon M, Webb E C., Enzyme kinetics. In Enzymes; 3nd ed. Academic Press, New York, 47~206
Carrea G, Riva S., Properties and synthetic applications of enzymes in organic solvents, Angew. Chem. Int. Ed. 2000, 39:2226~2254
Cosrantino H R, Griebenow K, Langer R, Klibanov A M, Biotech. Bioeng.1997. 53(3): 345~348
Jensen R G, Galluzzo D R, Bush V J., Selectivity is an important characteristic of lipases (acylglycerol hydrolases), Biocatalysis 1990, 3:307~317
Villeneuve P, Foglia T A., Lipase selectivities: potential application in lipid bioconversions, Inform 1997, 8:640~650
Yamaguchi S, Mase T., Purification and characterization of monoglycerol and diacylglycerol lipase isolated from Penicillium canenbertii U-150, Appl. Microbiol. Biotechnol, 1991, 34: 720~725
Okumura S, Iwai M, Tsujisaka Y., Purification and properities of partial glyceride hydrolase of Penicillium cyclopium M1. J. Biochem., 1980, 87:205~211
Tornquist H, Belfrage P., Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue, Biol. Chem., 1976, 251: 813~819
Galliard T., The enzyme deacylation of phospholipids and galactolipids in plants: purification and properties of a lipolytic acyl-hydrolase from potato tubers, Biochem, 1971, 121:379~390
Matori M, Asahara T, Ota Y., Positional specificity of microbial lipases. J. Ferment Bioeng. 1991, 72:397~398
Rogalska E, Cudrey C, Ferrato F, Verger R., Stereoselective hydrolysis of triglycerides by animal and microbial lipases, Chirality 1993, 5: 24~30
Sugihara A, Shimada Y, Nakamura M, Nagao T, Tominaga Y., Positional and fatty acid specificities of Geotrichum Candidum Lipase, Protein Eng. 1994. 7: 585~588
Sonnet P E., Lipase selectivities, Am. Oil Chem. Soc., 1988, 65:900~904
Piazza G J, Bilyk A, Brower D P, Haas M J., The positional and fatty acid selectivity of oat seed lipase in aqueous emulsions, Am. Oil Chem. Soc. 1992, 69: 978~981
漆红兰, 磁性微粒的制备方法和研究进展, 生命的化学, 2002, 22(6): 586~589
刘会洲, 何鸣鸿, 绿色化学与生态化工的研究内涵, 化工冶金, 1999, 20(4): 405~409
Robinson PJ, Dunnill P, Lilly MD., Properties of magnetic supports in relation to immobilized enzyme reactors, Biotechnol. Bioeng., 1973, 15:603~607
Arica MY, Yavuz H, Patir S, et al., Immobilization of glucoamylase onto spacer-arm attached magnetic poly (methylmethacrylate) microspheres: characterization and application to a continuous flow reactor, Mol. Catal. B: Enzyme, 2000, 11: 127~138
Liu C, Honda H, Ohshima A, et al., Development of chitosan-magnetite aggregates containing Nitrosomonas europaea cells for nitrification enhancement, Biosci. Bioeng., 2000, 89: 420~425
佟晓冬, 孙彦, 核壳型钕铁硼琼脂糖载体的制备及其膨胀床特性, 过程工程
学报, 2002, 2: 199~203
谢渝春, 刘会洲, 陈家镛, 有机相中的酶促拆分过程, 化学进展, 1997, 9: 292~299
徐小妹, 许孙曲, 用磁性脂质体使酶固定,.生命的化学, 1997, 17(1): 34~35
宫月平，刘会洲，安振涛等, 超顺磁性聚合物微球及其制造方法，CN 1253147，2000-5-17
孙彦, 生物分离工程, 北京: 化学工业出版社, 1998, 183~185
Liu HZ, Yang W, Chen J., Effects of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions, Biochem. Eng. J., 1998, 2: 187~196
Guo Z, Bai S, Sun Y., Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres, Enzyme Microb. Technol., 2003, 32: 776~782
Pawinee K, Suree P., Simple assay method for lipase activity and analysis of its catalytic hydrolysis product in water-poor media, Indian J. Chem., 1993, 32B: 88~89
Bradford MM., A Rapid and Sensitive Method for the Quantition of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 1976, 72: 248~254
朱祥瑞, 徐俊良, 家蚕丝素固定化α-淀粉酶的制备及其理化特性, 浙江大学学报, 2002, 28: 64~69
Brady L, Brzozowski AM, Derewenda ZS, et al., A serine protease triad forms the catalytic center of a triacylglycerol lipase, Nature, 1990, 343: 767~770
Caldwell J., Chiral pharmacology and regulation of drugs, Chemistry & Industry, 1995,5: 176~179
张玉彬, 生物催化的手性合成, 北京: 化学工业出版社, 2002, 67~70
胡伟, 唐世平, 黄荣斌等, 薄荷醇和乙酸薄荷醇酯的气相色谱分离测定, 中国食品添加剂, 2002, 4: 78~80
何汉平, 杨立荣, 朱自强, 在有机相酶催化反应中用盐/盐水合物控制水活度,.有机化学, 2001, (21): 376~379
R.Phillips, Enzyme Microb, Technol, 1992, 14: 417~418
彭立凤, 有机溶剂对酶催化活性和选择性的影响, 化学进展, 2000, 12(3): 296～304
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases, Enzyme Microb. Technol, 1992, 14: 426~446
Saiz-Díaz C I, Wohlfahrt G, Nogoceke E, Hernández-Laguna A, Smeyers Y G, Menge U., Molecular structure and conformational analysis of chiral alcohols application to the enantioselectivity study of lipases, Mol. Structure(Theochem), 1997, 390:225~237
俞俊棠, 唐孝宣, 生物工艺学, 上海: 华东理工大学出版社, 1991, 188~189
郭勇, 现代生化技术, 广州: 华南理工大学出版社, 1996, 170~173
伦世仪, 生化工程, 中国轻工业出版社 1993, 84~85
Winkler F K, D’Arcy A, Hunzuker W., Structure of human pancreatic lipase, Nature, 1990, 343: 771~774.
Gotz F, Poop F, and Schleifer K H. Complete nucleotide sequence of the lipase gene from staphylococcus hyicus cloned in staphylococcus hyicus, Nucleic Acid Res., 1985, 13: 5895~5906
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases. Enzyme Microb. Technol. 1992, 14: 426~446
Koeller, K.M.; Wong, C.H., Enzymes for chemical synthesis, Nature, 2001, 409: 232~240
Klibanov, A. M., Improving enzymes by using them in organic solvents, Nature, 2001, 409: 241~246
Athawale, V.; Manjrekar N., Mild chemo-enzymatic synthesis of polymer-supported cinchona alkaloids and their application in asymmetric Michael addition, Tetrahedron Lett, 2001, 42: 4541~4543
Kamal A., Khanna, R. G. B., Ramu R., Chemoenzymatic synthesis 2 of both enantiomers of fluoxetine, tomoxetine and nisoxetine: lipase-catalyzed resolution of 3-aryl-3-hydroxypropanenitriles, Tetrahedron: Asymmetry, 2002, 13: 2039~2051
Reetz M.T., Lipases as practical biocatalysts, Curr. Opin. Chem. Biol,. 2002, 6:
145~150
Talon R., Montel M. C., Berdague J. L., Production of flavour ester by lipases from Staphylococcus warneri and S. xylosus. Enzyme Microb. Technol., 1996, 19: 620~622
Zaks A, Dodds D R., Application of biocatalysis and biotransformation to the synthesis of pharmaceuticals, Drug Disc. Today, 1997, 2:513~531
Malcata, F.X., Hector R. Reyes, Hugo S. Garcia, Hill C. G. and Amundson C. H. Kinetics and mechanism of reactions catalysed by immobilized lipases. Enzyme Microb. Technol. 1992, 14: 426~446.
Cardenas J, Alvarez E, de Castro-Alvarez M-S, Sanchez-Montero J-M, Valmaseda M, Elson SW, Sinisterra J-V. Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases, Mol Catal B: Enzymatic, 2001, 14: 111~123
Osada K, Takahashi K, Hatano M., Polyunsaturated fatty acid glyceride syntheses by microbial lipase, Am. Chem. Soc., 1990, 67: 921~922
Undurraga D, Markovits A, Erazo S., Cocoa butter equivalent through enzymic interesterification of palm oil midfraction, Process Biochem., 2001, 36: 933~939
Chopineau J, Mccafferty F D, Therisod M, Kilibanov A M., Production of biosurfactants from sugar alcohols and vegetable oils catalyzed by lipases in a nonaqueous media, Biotechnol. Bioengin, 1988, 31: 208~214
Zhang LQ, Zhang YD, Xu L, Li XL, Yang XC, Xu GL, Wu XX, Gao HY, Du WB, Zhang XT, Zhang XZ., Lipase-catalyzed synthesis of RGD diamide in aqueous water-miscible organic solvents, Enzyme Microb Technol., 2001, 29: 129~135
Margolin A L, Fitzpatrick P A, Dubin P L, Kilibanov A M., Chemenzymatic synthesis of optically active (meth)acrylic polymers, Am. Chem. Soc., 1991, 113: 4693~4694
Gill I, Valivety R., Polyunsaturated fatty acids: Part 1. Occurrence biological activities and applications. Trends Biotechnol., 1997a, 15:401~409
Van Dyck S M O, Lemiere G L F, Jonckers T H M, Dommisse R, Pieters L, Buss V., Kinetic resolution of a dihydrobenzofuran-type neolignan by lipase-catalysed acetylation, Tetrahedron Asymmetry, 2001, 12:785~789
Margolin A L., Enzymes in the synthesis of chiral drugs, Enzyme microb. Technol., 1993, 15: 266~280
杨节非, 固定化酶回顾与展望, 国外医药-合成药、生化药、制剂分册, 1994,15,(4): 209~212
ALEKSEY ZAKS AND ALEXANDER M. KLIBANOV, Enzyme-catalyzed processes in organic solvents, biochemistry, 1985,82:3192~3196
朱祥瑞, 徐俊良, 家蚕丝素固定化α-淀粉酶的制备及其理化特性, 浙江大学学报（农业与生命科学版）, 2002,28（1）: 64~69
徐岩, 李建波，微生物脂肪酶的固定化及其在非水相催化中的应用研究, 工业微生物, 2001,31(1): 46~48
曹国民, 盛梅, 高广达, 脂肪酶的固定化及其性质的研究, 生物技术, 1997,7（3）：14~17
ALAN FERSHT, 酶的结构和作用机制(杜锦珠 茹炳根 卫新成),北京: 北京大学出版社, 1991,64~67
魏远安, 姚评佳, 谢庆武, 梁锦添, 游离及固定化果糖基转移酶部分酶学性质的比较研究, 中国生物化学与分子生物学报, 2001,17（4）：501~505
Geluk M.A., Norde W., Van H.K., Kalsbeek A.l., K.Van’s Riet, Adsorption of lipase from Candida rugosa on cellulose and its influence on lipolytic activity, Enzyme microb.Technol.,1992,14: 748~754
朱国辉, 黄卓烈, 丘泰球, 刘石生, 功率超声对酶促反应的影响, 应用声学, 2001, 20(4): 45~48
Khmelnitsky Y L, Levashov A V, Klyachkn N L, et al., Engineering biocatalytic systems in organic media with low water content, Enzyme Microb Technol., 1998. 10(12): 710~721
Marie-Pierre B. et al., Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications, Biotechnology and Bioengineering, 1999, 63(6):730~738
Ruple J A, Gratton E, Careri G, Trend Biochem., Sci., 1983, 8:18~19
Miller, C.; Austin, H.; Posorske, L.; Gonzlez, J., Characteristics of an immobilized lipase for the commericial synthesis of esters, Am. Oil Chem. Soc,. 1988, 65: 927~931
Bornscheuer U, Herar A, Kreye L, Wendel V, Capewell A, Meyer H H, Scheper T, Kolisis F N., Factors affecting the lipase catalyzed transesterification reaction of 3-hydroxy in organic solvents, Tetrahedron: Asymmetry, 1993, 4:1007~1016
Han J J, Iwasaki Y, Yamane T., Monitoring of lipase-catalyzed transesterification between eicosapentaenoic acid ethyl ester and tricaprylin by silver ion
high-performance liquid chromatography and high-temperature gas chromatography, Am. Oil Chem. Soc., 1999, 76:31~39
Sophie C, et al., Water activity control: A way to improve the efficient of continuous lipase esterification, Biotechnology and Bioengineeing, 1998, 60(3):362~367
Kwon S J. et al., Removal of water produced from lipase-catalyzed esterification in organic solvent by pervaporation, Biotechnology and Bioengineering, 1995, 46(4):393~399
Guo X G, et al., Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated pseudomonas strain, Enzyme and Microbial Technology, 2000,27:74~77
Jae EK, et al., Effect of salt hydrate pair on lipase catalyzed regioselective monoacylation of sucrose, Biotechnology and Bioengineering, 1998,57(1): 121~124
Laane C, Boeren S, Vos K, Veeger C., Rules for optimization of biocatalysis in organic solvents, Biotechnol. Bioeng., 1987, 30:81~87
Valivety R H, Halling P J, Peilow A D, Macrae A R., Reaction rate with suspended lipase catalyst shows similar dependence on activity on water activity in different organic solvents, Biochim. Biophys. Acta 1992, 1118:218~222
Bornscheuer U T, Yamane T., Activity and stability of lipase in the solid-phase glycerolysis of triolein, Enzyme Microb. Technol. 1994, 16:864~869
Huge-Jensen B, Galluzzo D R, Jensen R G., Partial purification and characterization of free and immobilized lipases from Mucor miehei, Lipids, 1987, 22: 559~565
Dixon M, Webb E C., Enzyme kinetics. In Enzymes; 3nd ed. Academic Press, New York, 47~206
Carrea G, Riva S., Properties and synthetic applications of enzymes in organic solvents, Angew. Chem. Int. Ed. 2000, 39:2226~2254
Cosrantino H R, Griebenow K, Langer R, Klibanov A M, Biotech. Bioeng.1997. 53(3): 345~348
Jensen R G, Galluzzo D R, Bush V J., Selectivity is an important characteristic of lipases (acylglycerol hydrolases), Biocatalysis 1990, 3:307~317
Villeneuve P, Foglia T A., Lipase selectivities: potential application in lipid bioconversions, Inform 1997, 8:640~650
Yamaguchi S, Mase T., Purification and characterization of monoglycerol and diacylglycerol lipase isolated from Penicillium canenbertii U-150, Appl. Microbiol. Biotechnol, 1991, 34: 720~725
Okumura S, Iwai M, Tsujisaka Y., Purification and properities of partial glyceride hydrolase of Penicillium cyclopium M1. J. Biochem., 1980, 87:205~211
Tornquist H, Belfrage P., Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue, Biol. Chem., 1976, 251: 813~819
Galliard T., The enzyme deacylation of phospholipids and galactolipids in plants: purification and properties of a lipolytic acyl-hydrolase from potato tubers, Biochem, 1971, 121:379~390
Matori M, Asahara T, Ota Y., Positional specificity of microbial lipases. J. Ferment Bioeng. 1991, 72:397~398
Rogalska E, Cudrey C, Ferrato F, Verger R., Stereoselective hydrolysis of triglycerides by animal and microbial lipases, Chirality 1993, 5: 24~30
Sugihara A, Shimada Y, Nakamura M, Nagao T, Tominaga Y., Positional and fatty acid specificities of Geotrichum Candidum Lipase, Protein Eng. 1994. 7: 585~588
Sonnet P E., Lipase selectivities, Am. Oil Chem. Soc., 1988, 65:900~904
Piazza G J, Bilyk A, Brower D P, Haas M J., The positional and fatty acid selectivity of oat seed lipase in aqueous emulsions, Am. Oil Chem. Soc. 1992, 69: 978~981
漆红兰, 磁性微粒的制备方法和研究进展, 生命的化学, 2002, 22(6): 586~589
刘会洲, 何鸣鸿, 绿色化学与生态化工的研究内涵, 化工冶金, 1999, 20(4): 405~409
Robinson PJ, Dunnill P, Lilly MD., Properties of magnetic supports in relation to immobilized enzyme reactors, Biotechnol. Bioeng., 1973, 15:603~607
Arica MY, Yavuz H, Patir S, et al., Immobilization of glucoamylase onto spacer-arm attached magnetic poly (methylmethacrylate) microspheres: characterization and application to a continuous flow reactor, Mol. Catal. B: Enzyme, 2000, 11: 127~138
Liu C, Honda H, Ohshima A, et al., Development of chitosan-magnetite aggregates containing Nitrosomonas europaea cells for nitrification enhancement, Biosci. Bioeng., 2000, 89: 420~425
佟晓冬, 孙彦, 核壳型钕铁硼琼脂糖载体的制备及其膨胀床特性, 过程工程
学报, 2002, 2: 199~203
谢渝春, 刘会洲, 陈家镛, 有机相中的酶促拆分过程, 化学进展, 1997, 9: 292~299
徐小妹, 许孙曲, 用磁性脂质体使酶固定,.生命的化学, 1997, 17(1): 34~35
宫月平，刘会洲，安振涛等, 超顺磁性聚合物微球及其制造方法，CN 1253147，2000-5-17
孙彦, 生物分离工程, 北京: 化学工业出版社, 1998, 183~185
Liu HZ, Yang W, Chen J., Effects of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions, Biochem. Eng. J., 1998, 2: 187~196
Guo Z, Bai S, Sun Y., Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres, Enzyme Microb. Technol., 2003, 32: 776~782
Pawinee K, Suree P., Simple assay method for lipase activity and analysis of its catalytic hydrolysis product in water-poor media, Indian J. Chem., 1993, 32B: 88~89
Bradford MM., A Rapid and Sensitive Method for the Quantition of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 1976, 72: 248~254
朱祥瑞, 徐俊良, 家蚕丝素固定化α-淀粉酶的制备及其理化特性, 浙江大学学报, 2002, 28: 64~69
Brady L, Brzozowski AM, Derewenda ZS, et al., A serine protease triad forms the catalytic center of a triacylglycerol lipase, Nature, 1990, 343: 767~770
Caldwell J., Chiral pharmacology and regulation of drugs, Chemistry & Industry, 1995,5: 176~179
张玉彬, 生物催化的手性合成, 北京: 化学工业出版社, 2002, 67~70
胡伟, 唐世平, 黄荣斌等, 薄荷醇和乙酸薄荷醇酯的气相色谱分离测定, 中国食品添加剂, 2002, 4: 78~80
何汉平, 杨立荣, 朱自强, 在有机相酶催化反应中用盐/盐水合物控制水活度,.有机化学, 2001, (21): 376~379
R.Phillips, Enzyme Microb, Technol, 1992, 14: 417~418
彭立凤, 有机溶剂对酶催化活性和选择性的影响, 化学进展, 2000, 12(3): 296～304