微波耦合酶催化反应中酶的光谱特性研究
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摘要
微波辐射可以用来消解蛋白质,也可以用来加速酶催化反应,而后者是在不损伤酶的一级结构的低功率辐射下进行的。过度的微波辐射可以用来消解,也会使酶蛋白失活或消解。将微波辐射适度应用于酶催化反应中,会产生单独使用这两种方法所不能达到的效果。本文以脂肪酶催化辛酸和丁醇、辛酸和甘油的酯化反应体系为研究对象,将反应体系中各组分分别在低功率(200 w)微波辐射和常规加热下对LRI、Novozym 435作用后,用荧光发射光谱分级逐步地研究LRI、Novozym 435的光谱变化。另外,研究了微波辐射对洗涤剂用脂肪酶和蛋白酶活力的影响以及两者的光谱变化。主要内容和结果如下:
1.以溶剂相脂肪酶LRI催化辛酸和丁醇的酯化反应体系为对象,将反应体系中各组分分别在低功率微波辐射和常规加热下对LRI作用后,用荧光发射光谱和紫外吸收光谱分级逐步地研究LRI在有机溶剂和水环境中的光谱变化。
实验发现,微波辐射和常规加热在所有实验条件下均增强LRI的荧光强度而没有导致最大发射波长位移。在微波辐射能够提高反应初速度的范围内,当LRI与有机分子共热后,微波辐射更有利于LRI蛋白质分子在水中的裸露。不同log P溶剂的反应体系对酶构象的影响主要表现为溶剂对其的影响。反应初速度对log P的变化规律与水相LRI蛋白质的荧光强度对log P的变化接近,而与溶剂相LRI蛋白质的荧光强度对log P的变化规律之间基本无共同之处。
2.以脂肪酶Novozym 435催化辛酸和甘油的酯化反应体系为对象,将反应体系中各组分分别在低功率微波辐射和常规加热下对Novozym 435作用后,用荧光发射光谱和紫外吸收光谱分级逐步地研究Novozym 435在水环境中的光谱变化。
实验结果表明,微波辐射较常规加热在所有实验条件下更能增强Novozym 435的荧光强度而没有导致最大发射波长位移。同时,Novozym 435在水相中的荧光强度分别对应不同温度、加水量以及底物配比的变化规律与反应初速度对应上述条件的变化基本一致。
此外,考察了在不同底物配比或加水量反应条件下的辛酸与甘油酯化反应(辛酸转化率为40%)时的Novozym 435在水环境中的光谱变化。水相Novozym 435的荧光强度对上述条件的变化规律与1-MG/2-MG及1,3-DG/1,2-DG对上述条件的变化基本一致。
3.研究了1000 w微波辐射洗涤用脂肪酶(Lipex 100 T、Lipolase 100 T)、蛋白酶(Properase 1000 E、Purafect 2000 E)后,其酶活随辐射时间的变化规律;并且利用荧光发射、紫外吸收光谱研究其光谱变化。经微波处理后,上述两类酶的酶活均随辐射时间延长呈先提高后降低的趋势。微波辐射并未改变上述两类酶的荧光发射峰位置与紫外吸收峰位置,峰强均随辐射时间的延长呈先增强后降低。此外,放置两个月后的脂肪酶的荧光强度、紫外吸收峰强度随辐射时间的变化规律与刚辐射后的一致,但放置后的荧光强度及紫外吸收峰强度均强于刚辐射后的。
Microwave radiation can be used to dispel protein, and also can be used to accelerate enzymatic catalysis reactions. The proper microwave irradiation doesn’t damage the primary structure of protein, while excessive microwave radiation would dispel, or deactive protein. Applying microwave irradiation-enzyme coupling catalysis (MIECC) can result in many unique findings, which were quite different from those observed from conventional heated enzymatic reactions. In this paper, the enzymatic esterification of caprylic acid and butanol, caprylic acid and glycerol were studied. The fluorescence change of LRI and Novozym 435 were recorded to investigate the formation change of the enzyme protein during the reactions. In addition,the effect of microwave irradiation time on the activity of the lipase and proteinase, along with their UV and fluorescence spectrum change were investigated. The main contents and results are as follows:
1. We studied the fluorescence and ultraviolet spectrum change of immobilized lipase from Mucor miehei in the microwave assisted enzymatic esterification of caprylic acid and butanol in organic medium, by investigating the fluorescence spectra in solvent or aqueous buffer after incubated the lipase with the solvent, caprylic acid and butanol under microwave irradiation, respectively. The comparison was made with the conventional heated enzymatic esterification in the solvents.
It was found that both of the heating modes, the microwave irradiation and conventional heating, can enhance the fluorescence intensity without shifting of the emission wavelength of the lipase. At the circumstance that the irradiation can accelerate the esterification, the irradiation enhanced the exposure of the lipase protein molecules in the aqueous environment after incubating the lipase with solvents or the substrates. The effect of the reaction mixture on the fluorescence intensity was driven by the solvents. The trend of the plot of log P versus the initial reaction rate was similar with that of the log P versus the fluorescence intensity of lipase in aqueous buffer after the esterification; but different with that of the log P versus the fluorescence intensity of lipase in organic medium.
2. We studied the fluorescence and ultraviolet spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol, by investigating the fluorescence spectra in aqueous buffer after incubated the lipase with the caprylic acid and glycerol under microwave irradiation, respectively.
The comparison between the microwave irradiated and conventional heated reactions was made. The microwave irradiation can enhance the fluorescence intensity more without shifting of the emission wavelength of the lipase protein. The trend of the plot of temperature, or the ratio caprylic acid to glycerol, or the water dosage versus the initial reaction rate, all of them were similar with that of the temperature, or the ratio caprylic acid to glycerol, or water dosage versus the fluorescence intensity of lipase in aqueous buffer after the esterification, respectively.
Additionally, the fluorescence spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol under different reaction conditions was studied. The fluorescence spectra in aqueous buffer were recorded after incubating the lipase with the caprylic acid or glycerol or the reaction mixture at the 40% caprylic acid conversion. The trend of the plot of reaction parameters versus the ratio 1- monoglyceride to 2- monoglyceride and the ratio 1,3-diglyceride to 1,2-diglyceride was similar with that of different reaction conditon versus the fluorescence intensity of lipase in aqueous buffer after the esterification,respectively.
3. The activity change of lipase(Lipex 100 T、Lipolase 100 T) and proteinase(Properase 1000 E、Purafect 2000 E) induced by microwave irradiation were investigated. We also studied the fluorescence or ultraviolet spectrum with different irradiation time. The activity of lipase and proteinase increased firstly and then decreased with rise microwave irradiation. The fluorescence and ultraviolet intensity enhanced firstly, and reduced with the rise irradiation time later without shifting of the emission wavelength of the lipase and proteinase. The trend of the plot of irradiation time versus the fluorescence or ultraviolet intensity of the lipase after storaged the lipase for two months was coincident with that of irradiation time versus the fluorescence or ultraviolet intensity of lipase irradiated after 30s. But the fluorescence or ultraviolet intensity of lipase after storged two months stronger than that of lipase irradiated after 30s.
1.以溶剂相脂肪酶LRI催化辛酸和丁醇的酯化反应体系为对象,将反应体系中各组分分别在低功率微波辐射和常规加热下对LRI作用后,用荧光发射光谱和紫外吸收光谱分级逐步地研究LRI在有机溶剂和水环境中的光谱变化。
实验发现,微波辐射和常规加热在所有实验条件下均增强LRI的荧光强度而没有导致最大发射波长位移。在微波辐射能够提高反应初速度的范围内,当LRI与有机分子共热后,微波辐射更有利于LRI蛋白质分子在水中的裸露。不同log P溶剂的反应体系对酶构象的影响主要表现为溶剂对其的影响。反应初速度对log P的变化规律与水相LRI蛋白质的荧光强度对log P的变化接近,而与溶剂相LRI蛋白质的荧光强度对log P的变化规律之间基本无共同之处。
2.以脂肪酶Novozym 435催化辛酸和甘油的酯化反应体系为对象,将反应体系中各组分分别在低功率微波辐射和常规加热下对Novozym 435作用后,用荧光发射光谱和紫外吸收光谱分级逐步地研究Novozym 435在水环境中的光谱变化。
实验结果表明,微波辐射较常规加热在所有实验条件下更能增强Novozym 435的荧光强度而没有导致最大发射波长位移。同时,Novozym 435在水相中的荧光强度分别对应不同温度、加水量以及底物配比的变化规律与反应初速度对应上述条件的变化基本一致。
此外,考察了在不同底物配比或加水量反应条件下的辛酸与甘油酯化反应(辛酸转化率为40%)时的Novozym 435在水环境中的光谱变化。水相Novozym 435的荧光强度对上述条件的变化规律与1-MG/2-MG及1,3-DG/1,2-DG对上述条件的变化基本一致。
3.研究了1000 w微波辐射洗涤用脂肪酶(Lipex 100 T、Lipolase 100 T)、蛋白酶(Properase 1000 E、Purafect 2000 E)后,其酶活随辐射时间的变化规律;并且利用荧光发射、紫外吸收光谱研究其光谱变化。经微波处理后,上述两类酶的酶活均随辐射时间延长呈先提高后降低的趋势。微波辐射并未改变上述两类酶的荧光发射峰位置与紫外吸收峰位置,峰强均随辐射时间的延长呈先增强后降低。此外,放置两个月后的脂肪酶的荧光强度、紫外吸收峰强度随辐射时间的变化规律与刚辐射后的一致,但放置后的荧光强度及紫外吸收峰强度均强于刚辐射后的。
Microwave radiation can be used to dispel protein, and also can be used to accelerate enzymatic catalysis reactions. The proper microwave irradiation doesn’t damage the primary structure of protein, while excessive microwave radiation would dispel, or deactive protein. Applying microwave irradiation-enzyme coupling catalysis (MIECC) can result in many unique findings, which were quite different from those observed from conventional heated enzymatic reactions. In this paper, the enzymatic esterification of caprylic acid and butanol, caprylic acid and glycerol were studied. The fluorescence change of LRI and Novozym 435 were recorded to investigate the formation change of the enzyme protein during the reactions. In addition,the effect of microwave irradiation time on the activity of the lipase and proteinase, along with their UV and fluorescence spectrum change were investigated. The main contents and results are as follows:
1. We studied the fluorescence and ultraviolet spectrum change of immobilized lipase from Mucor miehei in the microwave assisted enzymatic esterification of caprylic acid and butanol in organic medium, by investigating the fluorescence spectra in solvent or aqueous buffer after incubated the lipase with the solvent, caprylic acid and butanol under microwave irradiation, respectively. The comparison was made with the conventional heated enzymatic esterification in the solvents.
It was found that both of the heating modes, the microwave irradiation and conventional heating, can enhance the fluorescence intensity without shifting of the emission wavelength of the lipase. At the circumstance that the irradiation can accelerate the esterification, the irradiation enhanced the exposure of the lipase protein molecules in the aqueous environment after incubating the lipase with solvents or the substrates. The effect of the reaction mixture on the fluorescence intensity was driven by the solvents. The trend of the plot of log P versus the initial reaction rate was similar with that of the log P versus the fluorescence intensity of lipase in aqueous buffer after the esterification; but different with that of the log P versus the fluorescence intensity of lipase in organic medium.
2. We studied the fluorescence and ultraviolet spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol, by investigating the fluorescence spectra in aqueous buffer after incubated the lipase with the caprylic acid and glycerol under microwave irradiation, respectively.
The comparison between the microwave irradiated and conventional heated reactions was made. The microwave irradiation can enhance the fluorescence intensity more without shifting of the emission wavelength of the lipase protein. The trend of the plot of temperature, or the ratio caprylic acid to glycerol, or the water dosage versus the initial reaction rate, all of them were similar with that of the temperature, or the ratio caprylic acid to glycerol, or water dosage versus the fluorescence intensity of lipase in aqueous buffer after the esterification, respectively.
Additionally, the fluorescence spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol under different reaction conditions was studied. The fluorescence spectra in aqueous buffer were recorded after incubating the lipase with the caprylic acid or glycerol or the reaction mixture at the 40% caprylic acid conversion. The trend of the plot of reaction parameters versus the ratio 1- monoglyceride to 2- monoglyceride and the ratio 1,3-diglyceride to 1,2-diglyceride was similar with that of different reaction conditon versus the fluorescence intensity of lipase in aqueous buffer after the esterification,respectively.
3. The activity change of lipase(Lipex 100 T、Lipolase 100 T) and proteinase(Properase 1000 E、Purafect 2000 E) induced by microwave irradiation were investigated. We also studied the fluorescence or ultraviolet spectrum with different irradiation time. The activity of lipase and proteinase increased firstly and then decreased with rise microwave irradiation. The fluorescence and ultraviolet intensity enhanced firstly, and reduced with the rise irradiation time later without shifting of the emission wavelength of the lipase and proteinase. The trend of the plot of irradiation time versus the fluorescence or ultraviolet intensity of the lipase after storaged the lipase for two months was coincident with that of irradiation time versus the fluorescence or ultraviolet intensity of lipase irradiated after 30s. But the fluorescence or ultraviolet intensity of lipase after storged two months stronger than that of lipase irradiated after 30s.
引文
1. Gedye R, Smith F, Westawa K. The use of microwave ovens for rapid organic synthesis.Tetrahedron Letters, 1986, 27(3):279—283
2. Loupy A, Perreux L, Liagre M, et al. Reactivity and selectivity under microwave in organic chemistry. Pure Appl Chem, 2001, 73(1):161—166
3. Favretto L, Nugent W A. Highly regioselective microwave—assisted synthesis of enantiopure C3-symmetric trialkanolamines. Tetrahedron Letters, 2002, 43:2581—2584
4. Marwah P, Marwah A, Lardy H A. Microwave induced selective enolization of steroidal ketones and efficient acetylation of sterols in semisolid stat. Tetrahedron, 2003, 59:2273—2287
5. Hoz A, Moreno A. Microwaves in organic synthesis. Thermal and non-thermal microwave effects. Chemical Society Reviews, 2005, 34(2):164—178
6.金钦汉.微波化学[M].北京:科学出版社,1999.146—147
7. Lidstrom P, Tiemey J, Wathey B, et al. Microwave assisted organic synthesis-a review. Tetrahedron, 2001, 57:9225—9283
8. Mingos D M, Bagurst D R. Application of microwave dielectric heating effects to synthetic problems in chemistry. Chem Soc Rev, 1991, 20:1—47
9.黄卡玛,胡希明,唐敬贤,等.电磁波对化学反应非致热作用的实验研究[J].高等学校化学学报,1996,17(5):764—770
10. Porelli M, Cacciapuoti G, Fusco S, et al. Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Letters, 1997, 402:102—106
11.孙晓娟,苏跃增,金凤明.微波化学非热效应初探[J].江苏石油化工学院学报,2000,12(3):42—45
12. Goswami S. Adak A K. The first microwave-assisted regiospecific synthesis of 6-substituted pterins. Tetrahedron Letter, 2002, 43(46):8371—8373
13. Carrillo-Munoz J R, Bouvet D, Guidbé-Jampel E, et al. Microwave-promoted lipase-catalyzed reactions. resolution of (±) 1-Phenylethanol. J Org Chem, 1996, 61: 7746—7750
14. Bini M, Checcucci A, Millanta L, et al. Analysis of the effects of microwave energy on enzymatic activity of lactate dehydrogenase. J Microw Power, 1978, 13:96—99
15. Vukova T, Atanassov A, Ivanov R, et al. Intensity-dependent effects of microwave electromagnetic fields on acetylcholinesterase activity and protein. Medical Science Monitor, 2005, 11:50–56
16.蔡汉成.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2004
17. Cara F L, Scarfi M R, Sabato D A. Different effects of microwave energy and conventional heat on the activity of a thermophilicβ-galactosidase from bacilus acidocaldarius. Bioelectromagneics, 1999, 20: 172—176
18. Chen Y P, Liu Y J, Wang X L, et al. Effect of microwave and He-Ne laser on enzyme activity and biophoton emission of isatis indigotica fort. Journal of Integrative Plant Biology, 2005, 47 (7): 849—855
19. Soysal C, S?oyleemez Z. Kinetics and inactivation of carrot peroxidase by heat treatment.Journal of Food Engineering, 2005, 68:349—356
20. Réjasse B, Lamare S, Legoy M D. Stability improvement of immobilized Candida Antarctica Lipase B in an organic medium under microwave radiation. Org Biomol Chem, 2004, 2:1086—1089
21. Roy I, Munishwar N. Non-thermal effects of microwaves on protease-catalyzed esterification and transesterification. Tetrahedron, 2003, 59:5431—5436
22. Yadav G D, Lathi P S. Synergism of microwaves and immobilized enzyme catalysis in synthesis of adipic acid esters in nonaqueous media. Synthetic Communications, 2005, 35:1699—1705.
23. Zhu S D, Yu Z N, Wu Y X. Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis. Process biochemistry, 2005, 40:3082—3086
24. Zhu S D, Wu Y X, Yu Z N, et al. Comparison of three microwave/chemical pretreatment processes for enzymatic hydrolysis of rice straw. Biosystems Engineering, 2006, 93(3):279—283
25. Mondal K, Roy I, Gupta M N. Enhancement of catalytic efficiencies of xylanase, pectinaseand cellulaseby microwave pretreatment of their substrates. Biocatlysis and Biotransformation, 2004, 22:9—16
26. Lin S S, Wu C H. Microwave-assisted enzyme-catalyzed reaction in various solvent systems. J. Am Soc Mass Spectrum, 2005, 16,581—588
27. Khobragade C N, Kore G V. Influence of microwave irradiation on enzymatic activity of invertase in aqueous and organic solvents. India Journal of Chemical Technology, 2004, 11:377—381
28.黄伟.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2004
29. Yadav G D, Piyush S. Synergism between microwave and enzyme catalysis in intensification of reactions: transesterification of methyl acetoacetate with alcohols. Journal of molecular Catalysis A: Chemical, 2004, 223:51—56
30. Yadav G D, Lathi P S. Intensityfication of enzymatic synthesis of propylene glycol monolaurate from 1, 2-propanediol and lauric acid under microwave irradiation: Kinetics of forward and reverse reaction. Enzyme and Microbial Technology, 2006, 38: 814–820
31. Bradoo S, Rathi P, Saxena R K. Microwave-assisted rapid characterization of lipase selectivities. J. Biochem. Biophys, 2000, 51:115—120
32. Vacek M, Zarevucka M, Wimmer Z. Selective enzymes esterification of free fatty acids with n-butanol under microwave irradiation and under classical heating. Biotechnology Letters, 2000, 22:1565—1570.
33. Zarevúcka M, Vacek M, Wimmer Z, et al. Models for Glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation. Biotech. Letters, 1999, 21:785—790
34.彭先群,程恭仁.加酶洗涤剂国内外发展概况[J].日用化学工业,1995,25(3):30—31
35. Hoz A, Moreno A. Microwaves in organic synthesis. Thermal and non-thermal microwave effects. Chemical Society Reviews, 2005, 34(2):164—178
36. Lidstrom P, Tiemey J, Wathey B, et al. Microwave assisted organic synthesis-a review. Tetrahedron, 2001, 57:9225—9283
37. Mingos D M, Bagurst D R. Application of microwave dielectric heating effects to synthetic problems in chemistry. Chem Soc Rev, 1991, 20:1—47
38.黄卡玛,胡希明,唐敬贤,等.电磁波对化学反应非致热作用的实验研究[J].高等学校化学学报,1996,17(5):764—770
39.孙晓娟,苏跃增,金凤明.微波化学非热效应初探[J].江苏石油化工学院学报,2000,12(3):42—45
40. Goswami S. Adak A K. The first microwave-assisted regiospecific synthesis of 6-substituted pterins. Tetrahedron Letter, 2002, 43(46):8371—8373
41. Carrillo-Munoz J R, Bouvet D, Guidbé-Jampel E, et al. Microwave-promoted lipase-catalyzed reactions. resolution of (±) 1-Phenylethanol. J Org Chem, 1996, 61: 7746—7750
42. Lever A B P. Inorganic Electronic spectroscopy . Amsterdam ,The Netherlands: Elseveier ,1984 ,211-214
43.柯惟中,余多慰,冯玉英.牛血清白蛋白水溶液的紫外吸收光谱和激光拉曼光谱分析[J].光谱学与光谱分析,1993 ,13(5) :55—58
44.计亮年,莫庭焕.生物无机化学导论[M].广州:中山大学出版社,1992:266—269
45.刘清亮,吴双顶,张祖德,等.蛇毒蛋白质中金属离子及其配位化学的研究[J].化学通报,1997 ,10 :16—22
46.孙献茹,程鹏,廖代正,等.草酸根桥联的钒氧铜配合物的合成,表征及抗肿瘤活性研究[J].化学通报,1997 ,4 :45-49
47. Fu F N. Secondary structure estimation of proteins using the amideⅢregion of fourier transform infrared spectrosocopy: application to analyze calcium-binding-induced structural changes in calsequestrin.Appl.Spectrosc.1994,48:1432—1441
48. Kaiden K, Matsui T, Tanaka S. A study of the amideⅢband by FT-IR spectrometry of the secondary structure of albumin myoglobin andγ-globulin.Appl.Spectrosc.,1987,41:180—184
49. Mendelsohn R, Anderle G. Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amideⅢspectral region. Biophys. J.1987,52:69—73
50. Dev S B, Keller. Secondary structure of globulin in aqueous solution investigated by FT-IR derivative spectroscopy, Biochim.Biophys.Acta,1988,957(2):272—280
51. Yamamoto T,Tasumi M. Infrared studies on thermally-induced conformational changes of ribonuclease a by the methods of difference-spectrum and self-deconvolution, Canadian Journal of Spectroscopy,1988,33(5):133—137
52. Fabian H, Schultz C, Nauman D, et al. Secondary structure and temperature-induced unfolding and refolding of ribonuclease in aqueous solution a fourier transform infrared spectroscopic study.J.Mol.Biol.,1993,232,967—981
53.李国红,李刚,邱阳. pH值及Na+诱导α-辅肌动蛋白构象变化[J],生物物理学报.1999,15(2):283—288
54. Purcell J M, Susi H H. Solvent denaturation of proteins as observed by resolution-enhanced fourier transform infrared spectroscopy. J. Biochem. Biophys. Meth., 1984,9:193—199
55.陈立新,周筠梅,许振华.核糖核酸酶A氘代盐酸胍变性红外光谱的研究[J].科学通报,1992,8:746—749
56.胡朝红,邹承鲁.二硫键完整对蛋白质在盐酸胍中变性时伸展的影响-Fourier红外光谱研究[J].中国科学(B辑),1992:1260—1265
57. Antonine M, Devanathan S, Patonary G., Determination of critical micelle concentration of surfactants using a near-infrared hydrophobicity probe. Spectrochim.Acta,1991,47:510—508
58. Villalain J, Gomez-Fernandez J C. Molecular interactions between sphingomyelin and phosphatidylcholine in phospholipid wesicles.Biochim.Biophys.Acta,1988,941:55—62
59.孙素琴,赵树兵,卢为琴,等.PsПRC光抑制过程中蛋白质二级结构变化的FT-IR光谱法研究[J].光谱学与光谱分析,1996,16(4):25—27
60.沈子威,孙克利,杨钧,等.应用傅立叶红外光谱研究强声波作用下植物壁蛋白质二级结构变化[J].光子学报.1999,28(7):600—602
61. Belasco J G, Knowles J R. Direct observation of substrate distortion by trisephosphate isomerase using fourier transform infrared spectroscopy.Biochemistry,1980,19:472—477
62. Wei J, Lin Y Z, Zhou J M, et al. FTIR studies of secondary structure of bovine insulin and derivatives, Biochim. Biophys. Acta,1991,1080:29—33
63. Wei J, Xie L, Lin Y Z, et al. The pairing of the separated A and B chains of insulin and its derivative-FTIR studies, Biochim. Biophys.Acta.,1992,1120:69—74
64. Susi H, Zell T, Timasheff S N. Investigation of the carboxy ionization of beta-lactoglobulin by differential infrared spectroscopy. Arch Biochem.Biophys.,1959,85:437-442
65. Timasheff S N, Rupley J A, Infrared titration of lysozyme carboxyls. Arch. Biochem. Biophys.,1972,150:318-322
66.阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:11—170
67.赵南明,周海梦.生物物理学[M].北京:中国高等教育出版社,2000:5—344
68.杨频,高飞.生物无机化学原理[M].北京:科学出版社,2002:22—352
69. Brahms S, Brahm J. Determination of protein secondary structure in solution by vacuum ultrovilet circular dichroism. J. Mol. Bio.,1980,138(2):149—178
70. Dobryszycki P, Rymarczuk M, Bulaj G,et al. Biochim. Biophys. Acta, 1999, 1431: 338—350
71.王守业,余华明,陈真龙,等.皖南尖吻蝮蛇蛇毒中纤溶组分的荧光特性[J].生物化学与生物物理学报, 1998, 30 (3) : 303—306
72.陈真龙,张利民,丁兰,等.皖南尖吻蝮蛇蛇毒出血毒素Ⅲ(AaHⅢ)的荧光猝灭研究[J].化学物理学报,1998,11(5) :465—470
73. Lloyd J B F. Synchronized excitation of fluorescence emission spectra: Nature (Phys. Sci.), 1971, 231: 64—65
74. Inman J E, Winefordner J D, Constant energy synchronous fluorescence for analysisi of polynuclear aromatic hydrocarbon mixtures.Anal.Chem.,1982,54:2018—2022
75. Chou J, Qu X, Lu T, et al. The effect of solution pH on synchronous fluorescence spectra of cytochrome c solutions. Microchem. J., 1995, 52: 159—165
76.鄢远,许金钩,陈国珍.三维荧光光谱法研究蛋白质溶液构象[J].中国科学(B辑),1997, 27 (1) : 16—22
77. Chen Z, Liu Q , Wang S, Xu X, et al. Study on hemorrhaginⅢpurified fron the venom of agkistrodon acutus by three-dimensional fluorescence spectrometry. Spectrochim Acta Part A , 1999, 55: 1909—1914
78. Manuel G., Venegas. Powdered detergents[M].New York: Marcel Dekker,1998,285—287
79. Rytom N M. Multiple enzymes in household detergents. Riv Ital Sostanze Grasse,2001,78:425—430
80. Erik G, Peter R, Mads L. Enzymes for laundry products. Proceedings of the 3rd world conference on detergents, AOCS Press,1993:198—203
81. Novozymes A/S. Remove fatty stains first time. Bio. Times,2002,3:3
82. Keith G. The importance of detergent amylases for whiteness maintenance. Riv Ital Sostanze Grasse, 1998,75:207—211
83. Hans S O, Per F. The role of enzymes in modern detergency. J. Surface Deterg., 1998,1(4):555—567
84. Byler D M, Brouillette J N, Susi H. Spectroscopy, 1986,1(3):29—32
85. Liu H, Yang W, Chen J. Effect of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions. Biochemi. Engineering J., 1998,2:187—196
86.邓一兵,杨体强.应用红外光谱研究电场对超氧化物歧化酶二级结构的影响[J].光谱学与光谱分析,2007,27(7):1312—1315
87. Vukova T, Atanassov A, Ivanov R, et al. Intensity-dependent effects of microwave electromagnetic fields on acetylcholinesterase activity and protein. Medical Science Monitor, 2005, 11:50—56
88.范寰,元英进.Ce4+对过氧化氢酶构象的影响[J].中国稀土学报,2006,24(6):734—738
89.秦身钧,李艳延.荧光法研究钍与牛血清蛋白的结合反应[J].河北师范大学学报,2003,27(3):274—277
90.蒋惠亮,余晓斌.洗涤剂用碱性纤维素酶应用研究:碱性纤维素酶在洗涤剂中的应用[J],1997,(3):10—14
91. Carter D.Advances in protein chemistry[M].New York:Academic press,1994:153—203
92.孙诗雨.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2007
93. Porelli M, Cacciapuoti G, Fusco S, et al. Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Letters, 1997, 402:102—106
94. Laane C, Boeren S, Vos K, et al.Rules for optimization of biocatalysis in organic solventsBiotechnology and Bioengineering, 1987,30:81—87
95. Kitaguchi H. Effects of water and water-mimicking solvents on the lipase-catalyzed esterification in an apolar solvent. Chemistry Letter,1990:1203—1207
2. Loupy A, Perreux L, Liagre M, et al. Reactivity and selectivity under microwave in organic chemistry. Pure Appl Chem, 2001, 73(1):161—166
3. Favretto L, Nugent W A. Highly regioselective microwave—assisted synthesis of enantiopure C3-symmetric trialkanolamines. Tetrahedron Letters, 2002, 43:2581—2584
4. Marwah P, Marwah A, Lardy H A. Microwave induced selective enolization of steroidal ketones and efficient acetylation of sterols in semisolid stat. Tetrahedron, 2003, 59:2273—2287
5. Hoz A, Moreno A. Microwaves in organic synthesis. Thermal and non-thermal microwave effects. Chemical Society Reviews, 2005, 34(2):164—178
6.金钦汉.微波化学[M].北京:科学出版社,1999.146—147
7. Lidstrom P, Tiemey J, Wathey B, et al. Microwave assisted organic synthesis-a review. Tetrahedron, 2001, 57:9225—9283
8. Mingos D M, Bagurst D R. Application of microwave dielectric heating effects to synthetic problems in chemistry. Chem Soc Rev, 1991, 20:1—47
9.黄卡玛,胡希明,唐敬贤,等.电磁波对化学反应非致热作用的实验研究[J].高等学校化学学报,1996,17(5):764—770
10. Porelli M, Cacciapuoti G, Fusco S, et al. Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Letters, 1997, 402:102—106
11.孙晓娟,苏跃增,金凤明.微波化学非热效应初探[J].江苏石油化工学院学报,2000,12(3):42—45
12. Goswami S. Adak A K. The first microwave-assisted regiospecific synthesis of 6-substituted pterins. Tetrahedron Letter, 2002, 43(46):8371—8373
13. Carrillo-Munoz J R, Bouvet D, Guidbé-Jampel E, et al. Microwave-promoted lipase-catalyzed reactions. resolution of (±) 1-Phenylethanol. J Org Chem, 1996, 61: 7746—7750
14. Bini M, Checcucci A, Millanta L, et al. Analysis of the effects of microwave energy on enzymatic activity of lactate dehydrogenase. J Microw Power, 1978, 13:96—99
15. Vukova T, Atanassov A, Ivanov R, et al. Intensity-dependent effects of microwave electromagnetic fields on acetylcholinesterase activity and protein. Medical Science Monitor, 2005, 11:50–56
16.蔡汉成.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2004
17. Cara F L, Scarfi M R, Sabato D A. Different effects of microwave energy and conventional heat on the activity of a thermophilicβ-galactosidase from bacilus acidocaldarius. Bioelectromagneics, 1999, 20: 172—176
18. Chen Y P, Liu Y J, Wang X L, et al. Effect of microwave and He-Ne laser on enzyme activity and biophoton emission of isatis indigotica fort. Journal of Integrative Plant Biology, 2005, 47 (7): 849—855
19. Soysal C, S?oyleemez Z. Kinetics and inactivation of carrot peroxidase by heat treatment.Journal of Food Engineering, 2005, 68:349—356
20. Réjasse B, Lamare S, Legoy M D. Stability improvement of immobilized Candida Antarctica Lipase B in an organic medium under microwave radiation. Org Biomol Chem, 2004, 2:1086—1089
21. Roy I, Munishwar N. Non-thermal effects of microwaves on protease-catalyzed esterification and transesterification. Tetrahedron, 2003, 59:5431—5436
22. Yadav G D, Lathi P S. Synergism of microwaves and immobilized enzyme catalysis in synthesis of adipic acid esters in nonaqueous media. Synthetic Communications, 2005, 35:1699—1705.
23. Zhu S D, Yu Z N, Wu Y X. Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis. Process biochemistry, 2005, 40:3082—3086
24. Zhu S D, Wu Y X, Yu Z N, et al. Comparison of three microwave/chemical pretreatment processes for enzymatic hydrolysis of rice straw. Biosystems Engineering, 2006, 93(3):279—283
25. Mondal K, Roy I, Gupta M N. Enhancement of catalytic efficiencies of xylanase, pectinaseand cellulaseby microwave pretreatment of their substrates. Biocatlysis and Biotransformation, 2004, 22:9—16
26. Lin S S, Wu C H. Microwave-assisted enzyme-catalyzed reaction in various solvent systems. J. Am Soc Mass Spectrum, 2005, 16,581—588
27. Khobragade C N, Kore G V. Influence of microwave irradiation on enzymatic activity of invertase in aqueous and organic solvents. India Journal of Chemical Technology, 2004, 11:377—381
28.黄伟.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2004
29. Yadav G D, Piyush S. Synergism between microwave and enzyme catalysis in intensification of reactions: transesterification of methyl acetoacetate with alcohols. Journal of molecular Catalysis A: Chemical, 2004, 223:51—56
30. Yadav G D, Lathi P S. Intensityfication of enzymatic synthesis of propylene glycol monolaurate from 1, 2-propanediol and lauric acid under microwave irradiation: Kinetics of forward and reverse reaction. Enzyme and Microbial Technology, 2006, 38: 814–820
31. Bradoo S, Rathi P, Saxena R K. Microwave-assisted rapid characterization of lipase selectivities. J. Biochem. Biophys, 2000, 51:115—120
32. Vacek M, Zarevucka M, Wimmer Z. Selective enzymes esterification of free fatty acids with n-butanol under microwave irradiation and under classical heating. Biotechnology Letters, 2000, 22:1565—1570.
33. Zarevúcka M, Vacek M, Wimmer Z, et al. Models for Glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation. Biotech. Letters, 1999, 21:785—790
34.彭先群,程恭仁.加酶洗涤剂国内外发展概况[J].日用化学工业,1995,25(3):30—31
35. Hoz A, Moreno A. Microwaves in organic synthesis. Thermal and non-thermal microwave effects. Chemical Society Reviews, 2005, 34(2):164—178
36. Lidstrom P, Tiemey J, Wathey B, et al. Microwave assisted organic synthesis-a review. Tetrahedron, 2001, 57:9225—9283
37. Mingos D M, Bagurst D R. Application of microwave dielectric heating effects to synthetic problems in chemistry. Chem Soc Rev, 1991, 20:1—47
38.黄卡玛,胡希明,唐敬贤,等.电磁波对化学反应非致热作用的实验研究[J].高等学校化学学报,1996,17(5):764—770
39.孙晓娟,苏跃增,金凤明.微波化学非热效应初探[J].江苏石油化工学院学报,2000,12(3):42—45
40. Goswami S. Adak A K. The first microwave-assisted regiospecific synthesis of 6-substituted pterins. Tetrahedron Letter, 2002, 43(46):8371—8373
41. Carrillo-Munoz J R, Bouvet D, Guidbé-Jampel E, et al. Microwave-promoted lipase-catalyzed reactions. resolution of (±) 1-Phenylethanol. J Org Chem, 1996, 61: 7746—7750
42. Lever A B P. Inorganic Electronic spectroscopy . Amsterdam ,The Netherlands: Elseveier ,1984 ,211-214
43.柯惟中,余多慰,冯玉英.牛血清白蛋白水溶液的紫外吸收光谱和激光拉曼光谱分析[J].光谱学与光谱分析,1993 ,13(5) :55—58
44.计亮年,莫庭焕.生物无机化学导论[M].广州:中山大学出版社,1992:266—269
45.刘清亮,吴双顶,张祖德,等.蛇毒蛋白质中金属离子及其配位化学的研究[J].化学通报,1997 ,10 :16—22
46.孙献茹,程鹏,廖代正,等.草酸根桥联的钒氧铜配合物的合成,表征及抗肿瘤活性研究[J].化学通报,1997 ,4 :45-49
47. Fu F N. Secondary structure estimation of proteins using the amideⅢregion of fourier transform infrared spectrosocopy: application to analyze calcium-binding-induced structural changes in calsequestrin.Appl.Spectrosc.1994,48:1432—1441
48. Kaiden K, Matsui T, Tanaka S. A study of the amideⅢband by FT-IR spectrometry of the secondary structure of albumin myoglobin andγ-globulin.Appl.Spectrosc.,1987,41:180—184
49. Mendelsohn R, Anderle G. Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amideⅢspectral region. Biophys. J.1987,52:69—73
50. Dev S B, Keller. Secondary structure of globulin in aqueous solution investigated by FT-IR derivative spectroscopy, Biochim.Biophys.Acta,1988,957(2):272—280
51. Yamamoto T,Tasumi M. Infrared studies on thermally-induced conformational changes of ribonuclease a by the methods of difference-spectrum and self-deconvolution, Canadian Journal of Spectroscopy,1988,33(5):133—137
52. Fabian H, Schultz C, Nauman D, et al. Secondary structure and temperature-induced unfolding and refolding of ribonuclease in aqueous solution a fourier transform infrared spectroscopic study.J.Mol.Biol.,1993,232,967—981
53.李国红,李刚,邱阳. pH值及Na+诱导α-辅肌动蛋白构象变化[J],生物物理学报.1999,15(2):283—288
54. Purcell J M, Susi H H. Solvent denaturation of proteins as observed by resolution-enhanced fourier transform infrared spectroscopy. J. Biochem. Biophys. Meth., 1984,9:193—199
55.陈立新,周筠梅,许振华.核糖核酸酶A氘代盐酸胍变性红外光谱的研究[J].科学通报,1992,8:746—749
56.胡朝红,邹承鲁.二硫键完整对蛋白质在盐酸胍中变性时伸展的影响-Fourier红外光谱研究[J].中国科学(B辑),1992:1260—1265
57. Antonine M, Devanathan S, Patonary G., Determination of critical micelle concentration of surfactants using a near-infrared hydrophobicity probe. Spectrochim.Acta,1991,47:510—508
58. Villalain J, Gomez-Fernandez J C. Molecular interactions between sphingomyelin and phosphatidylcholine in phospholipid wesicles.Biochim.Biophys.Acta,1988,941:55—62
59.孙素琴,赵树兵,卢为琴,等.PsПRC光抑制过程中蛋白质二级结构变化的FT-IR光谱法研究[J].光谱学与光谱分析,1996,16(4):25—27
60.沈子威,孙克利,杨钧,等.应用傅立叶红外光谱研究强声波作用下植物壁蛋白质二级结构变化[J].光子学报.1999,28(7):600—602
61. Belasco J G, Knowles J R. Direct observation of substrate distortion by trisephosphate isomerase using fourier transform infrared spectroscopy.Biochemistry,1980,19:472—477
62. Wei J, Lin Y Z, Zhou J M, et al. FTIR studies of secondary structure of bovine insulin and derivatives, Biochim. Biophys. Acta,1991,1080:29—33
63. Wei J, Xie L, Lin Y Z, et al. The pairing of the separated A and B chains of insulin and its derivative-FTIR studies, Biochim. Biophys.Acta.,1992,1120:69—74
64. Susi H, Zell T, Timasheff S N. Investigation of the carboxy ionization of beta-lactoglobulin by differential infrared spectroscopy. Arch Biochem.Biophys.,1959,85:437-442
65. Timasheff S N, Rupley J A, Infrared titration of lysozyme carboxyls. Arch. Biochem. Biophys.,1972,150:318-322
66.阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:11—170
67.赵南明,周海梦.生物物理学[M].北京:中国高等教育出版社,2000:5—344
68.杨频,高飞.生物无机化学原理[M].北京:科学出版社,2002:22—352
69. Brahms S, Brahm J. Determination of protein secondary structure in solution by vacuum ultrovilet circular dichroism. J. Mol. Bio.,1980,138(2):149—178
70. Dobryszycki P, Rymarczuk M, Bulaj G,et al. Biochim. Biophys. Acta, 1999, 1431: 338—350
71.王守业,余华明,陈真龙,等.皖南尖吻蝮蛇蛇毒中纤溶组分的荧光特性[J].生物化学与生物物理学报, 1998, 30 (3) : 303—306
72.陈真龙,张利民,丁兰,等.皖南尖吻蝮蛇蛇毒出血毒素Ⅲ(AaHⅢ)的荧光猝灭研究[J].化学物理学报,1998,11(5) :465—470
73. Lloyd J B F. Synchronized excitation of fluorescence emission spectra: Nature (Phys. Sci.), 1971, 231: 64—65
74. Inman J E, Winefordner J D, Constant energy synchronous fluorescence for analysisi of polynuclear aromatic hydrocarbon mixtures.Anal.Chem.,1982,54:2018—2022
75. Chou J, Qu X, Lu T, et al. The effect of solution pH on synchronous fluorescence spectra of cytochrome c solutions. Microchem. J., 1995, 52: 159—165
76.鄢远,许金钩,陈国珍.三维荧光光谱法研究蛋白质溶液构象[J].中国科学(B辑),1997, 27 (1) : 16—22
77. Chen Z, Liu Q , Wang S, Xu X, et al. Study on hemorrhaginⅢpurified fron the venom of agkistrodon acutus by three-dimensional fluorescence spectrometry. Spectrochim Acta Part A , 1999, 55: 1909—1914
78. Manuel G., Venegas. Powdered detergents[M].New York: Marcel Dekker,1998,285—287
79. Rytom N M. Multiple enzymes in household detergents. Riv Ital Sostanze Grasse,2001,78:425—430
80. Erik G, Peter R, Mads L. Enzymes for laundry products. Proceedings of the 3rd world conference on detergents, AOCS Press,1993:198—203
81. Novozymes A/S. Remove fatty stains first time. Bio. Times,2002,3:3
82. Keith G. The importance of detergent amylases for whiteness maintenance. Riv Ital Sostanze Grasse, 1998,75:207—211
83. Hans S O, Per F. The role of enzymes in modern detergency. J. Surface Deterg., 1998,1(4):555—567
84. Byler D M, Brouillette J N, Susi H. Spectroscopy, 1986,1(3):29—32
85. Liu H, Yang W, Chen J. Effect of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions. Biochemi. Engineering J., 1998,2:187—196
86.邓一兵,杨体强.应用红外光谱研究电场对超氧化物歧化酶二级结构的影响[J].光谱学与光谱分析,2007,27(7):1312—1315
87. Vukova T, Atanassov A, Ivanov R, et al. Intensity-dependent effects of microwave electromagnetic fields on acetylcholinesterase activity and protein. Medical Science Monitor, 2005, 11:50—56
88.范寰,元英进.Ce4+对过氧化氢酶构象的影响[J].中国稀土学报,2006,24(6):734—738
89.秦身钧,李艳延.荧光法研究钍与牛血清蛋白的结合反应[J].河北师范大学学报,2003,27(3):274—277
90.蒋惠亮,余晓斌.洗涤剂用碱性纤维素酶应用研究:碱性纤维素酶在洗涤剂中的应用[J],1997,(3):10—14
91. Carter D.Advances in protein chemistry[M].New York:Academic press,1994:153—203
92.孙诗雨.微波辐射—酶耦合催化(MIECC)效应的研究[D]:[硕士学位论文].无锡:江南大学化学与材料工程学院,2007
93. Porelli M, Cacciapuoti G, Fusco S, et al. Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Letters, 1997, 402:102—106
94. Laane C, Boeren S, Vos K, et al.Rules for optimization of biocatalysis in organic solventsBiotechnology and Bioengineering, 1987,30:81—87
95. Kitaguchi H. Effects of water and water-mimicking solvents on the lipase-catalyzed esterification in an apolar solvent. Chemistry Letter,1990:1203—1207