从酵母细胞中分离纯化S-腺苷-L-蛋氨酸和谷胱甘肽的研究
|
摘要
S-腺苷-L-蛋氨酸(S-adenosyl-L-methionine，SAM-e或AdoMet，SAM)和谷胱甘肽(Glutathione，GSH)是生物体内普遍存在的两种重要的生物活性小分子，本文主要对从酵母细胞中分离纯化这两种物质的工艺以及SAM稳定性盐的制备进行了研究。
首先，选定了一种对谷胱甘肽的吸附容量可达8.1mg／ml湿树脂的强酸型阳离子交换树脂HZ001从再培养的废酵母细胞处理液中分离谷胱甘肽，对谷胱甘肽在HZ001树脂离子交换柱上的动态离子交换行为进行了研究，优化了上柱和洗脱条件，使得本步离子交换谷胱甘肽的回收率达到了91％。
然后，筛选了一种高选择性、高吸附容量的弱酸型阳离子交换树脂JK110，考察了SAM在JK110树脂上的离子交换动力学，确定了相关的参数，研究了SAM在JK110树脂上的静态离子交换平衡行为，拟合了吸附等温线。还对SAM在JK110树脂柱上的动态离子交换进行了研究，确立了优化的工艺条件，使得JK110树脂对SAM的吸附容量达到172mg／g干树脂，本步离子交换SAM回收率达93％，SAM纯度95％。并且由于谷胱甘肽在JK110树脂上没有吸附，上柱流出液可以重新进入谷胱甘肽的分离工艺。最后还对高稳定性的SAM硫酸对甲苯磺酸双盐的制备进行了研究，获得了纯度达98％的SAM硫酸对甲苯磺酸双盐。
本论文开发了一条从酵母细胞中同时分离提纯SAM和GSH的工艺路线，为SAM和谷胱甘肽的工业化生产奠定了良好的基础。
S-adenosyl-L-methionine (SAM-e, AdoMet or SAM) and glutathione (GSH) were two important molecules widely existed in various cells. The separation and purification of them as well as the preparation of stable SAM salt were studied.
Firstly, a strong acid anion ion-exchange resin HZ001 was chosen to isolate GSH from the extract of spent yeast cell, and its capacity for GSH was 8.1mg/ml (wet resin). The separation process of SAM was performed in a fixed-bed ion-exchange column, and the operation conditions were optimized for the separation of GSH from the extract. The recovery of GSH reached 91%.
Secondly, the ion-exchange separation process of SAM from the extract of yeast cell was investigated. Among the resins used, JK110 resin was the best one and its capacity for SAM was 172.8mg/g (dry resin) while GSH was hardly adsorbed. So GSH could be separated and purified by HZ001 resin consequently. The ion-exchange equilibrium, exchange velocity and dynamic column process of SAM on JK110 resin were investigated. The equilibrium isotherms were obtained at various pH values and temperatures and the related equilibrium models were examined. The ion-exchange kinetics of SAM+/H+ system based on JK110 resin was also discussed. And the rate-limiting step of the ion-exchange process was determined. A model of particle diffusion was proposed to describe the effects of pH, temperature and SAM concentration in the solution on the exchange velocity. By simulating experimental data with model equation, parameters were determined. In addition, the separation conditions of SAM in a fixed-bed ion-exchange column were optimized, and a recovery of 93% is obtained. After desorption, the eluate was concentrated and then crystallized with methanol. The white powder obtained was analyzed by HPLC and the purity was 95%. At the end, the preparation of stable SAM salt was studied and SAMe disulphate-p-toluenesulphonate was obtained. The purity of SAM product was over 98%.
首先，选定了一种对谷胱甘肽的吸附容量可达8.1mg／ml湿树脂的强酸型阳离子交换树脂HZ001从再培养的废酵母细胞处理液中分离谷胱甘肽，对谷胱甘肽在HZ001树脂离子交换柱上的动态离子交换行为进行了研究，优化了上柱和洗脱条件，使得本步离子交换谷胱甘肽的回收率达到了91％。
然后，筛选了一种高选择性、高吸附容量的弱酸型阳离子交换树脂JK110，考察了SAM在JK110树脂上的离子交换动力学，确定了相关的参数，研究了SAM在JK110树脂上的静态离子交换平衡行为，拟合了吸附等温线。还对SAM在JK110树脂柱上的动态离子交换进行了研究，确立了优化的工艺条件，使得JK110树脂对SAM的吸附容量达到172mg／g干树脂，本步离子交换SAM回收率达93％，SAM纯度95％。并且由于谷胱甘肽在JK110树脂上没有吸附，上柱流出液可以重新进入谷胱甘肽的分离工艺。最后还对高稳定性的SAM硫酸对甲苯磺酸双盐的制备进行了研究，获得了纯度达98％的SAM硫酸对甲苯磺酸双盐。
本论文开发了一条从酵母细胞中同时分离提纯SAM和GSH的工艺路线，为SAM和谷胱甘肽的工业化生产奠定了良好的基础。
S-adenosyl-L-methionine (SAM-e, AdoMet or SAM) and glutathione (GSH) were two important molecules widely existed in various cells. The separation and purification of them as well as the preparation of stable SAM salt were studied.
Firstly, a strong acid anion ion-exchange resin HZ001 was chosen to isolate GSH from the extract of spent yeast cell, and its capacity for GSH was 8.1mg/ml (wet resin). The separation process of SAM was performed in a fixed-bed ion-exchange column, and the operation conditions were optimized for the separation of GSH from the extract. The recovery of GSH reached 91%.
Secondly, the ion-exchange separation process of SAM from the extract of yeast cell was investigated. Among the resins used, JK110 resin was the best one and its capacity for SAM was 172.8mg/g (dry resin) while GSH was hardly adsorbed. So GSH could be separated and purified by HZ001 resin consequently. The ion-exchange equilibrium, exchange velocity and dynamic column process of SAM on JK110 resin were investigated. The equilibrium isotherms were obtained at various pH values and temperatures and the related equilibrium models were examined. The ion-exchange kinetics of SAM+/H+ system based on JK110 resin was also discussed. And the rate-limiting step of the ion-exchange process was determined. A model of particle diffusion was proposed to describe the effects of pH, temperature and SAM concentration in the solution on the exchange velocity. By simulating experimental data with model equation, parameters were determined. In addition, the separation conditions of SAM in a fixed-bed ion-exchange column were optimized, and a recovery of 93% is obtained. After desorption, the eluate was concentrated and then crystallized with methanol. The white powder obtained was analyzed by HPLC and the purity was 95%. At the end, the preparation of stable SAM salt was studied and SAMe disulphate-p-toluenesulphonate was obtained. The purity of SAM product was over 98%.
引文
[1] Kramer D L, Sufrin J R, Porter c W. Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis[J], Biochem J, 1987, 247, 259-265.
[2] Giulidori P, Galli-Kienle M, Catto E, Stramentinoli G. Transmethylation, transsulfuration, and aminopropylation reactions of S-adenosyl-L-methionine in vivo[J]. J Biol Chem, 1984, 259, 4205-4449.
[3] Borchardt R. T., "Enzymatic Basis of Detoxication", Vol. Ⅱ, pp. 43, Academic Press, 1980.
[4] Kersten H., "Biochemistry of S-adenosylmethionine and Related Compounds", pp. 357, Usdin E. et al. Eds., MacMillan Press L. t. D., 1982.
[5] Lu S C. Review article: S-Adenosylmethionine[J], Inter J Biochem cell Bio, 2000, 32: 391～395
[6] Barcelo H A, Wiemeyer J C, Sagasta C L, Macias M, Barreira J C. Experimental osteoarthritis and its course wthen treated with S-adenosyl-L-methionine[J]. Rev Clin Esp, 1990, 187, 74-78.
[7] Cimino M, Vantini G, Algeri S, Curatola G, Pezzoli C, Stramentinoli G. Age-related modification of dopamineric and beta-adrenergic receptor system: restoration to normal activity by modifying membrane fluidity with S-adenosyl-L-methionine[J]. Life Sci 1984, 34, 2029-2039.
[8] Torta R, Cicolin A, Keller R. Transmethylation and affective disorders[J]. Arch Gerontol Geriatr suppl, 1998, 6, 499-506.
[9] Osman E O, Owen J S, Burroughs A K. S-adenosyl-L-methionine. A new therapeutic agent in liver disease[J]? Aliment Pharmacol Ther, 1993, 7, 21-28.
[10] Charles S L. Role of S-adenosyl-L-methionine in the treatment of liver diseases [J]. J Hepatology, 1999, 30, 1155-1159.
[11] Vendemiale G, Altomare E, Trizio T, et al. Effects of oral S-adenosyl-L-methionine on hepatic glutathione in patients with liver disease. Scand J Gastroenterol, 1989, 24 (4): 407-415.
[12] Osman E, Owen J S, Burroughs A K. S-adenosyl-L-methionine-a new therapeutic agent in liver disease? Aliment Pharmcol Ther, 1993, 7: 21-28.
[13] Angelico M, Gandin C, Nistri A, et al. Oral (SAMe) administration enhances bile salt conjugation with taurine in patients with liver cirrhosis. Scand.J.Clin.Lab Invesr. 1993,54: 459-464.
[14] Nakano H, Yamaguchi M, Kaneshiro Y, et al. Transplant Proceed, 1998, 30: 3735-3736..
[15] Benedetto P Di, Iona LG, Zidarich V. Clinical. evaluation of S-adenosyl-L-methionine versus transcutaneous electrical nerve stimulation in primary fibromyalgia. Italy Curr Ther Res, 1993, 53 (2): 222.
[16] Piacentino R, Malara D, Zaccheo F, et al. Preliminary study of the ues of S-adenosyl-L-methionine in the management of male sterility. Minerva.Ginecologica, 1991,43:191-193 [in Italian]..
[17] Gatto G, Caleri D, Michelacci S, Sicuteri F. Analgesizing effect of a methyldodor (S-adenosyl-L-methionine)in migraine: an open clinical trial. Int.J.Clin. Pharmacol.Res.,1986,6:15-17.
[18] Ballerini F B, Anguera A L, Alcaraz P, et al. SAM in the management of postconcussional syndrome. Med.Clin.(Barc)1983,80:161-164
[19] Baddiley J & Jamieson G A. ibid[J], 1954, 4280
[20] Jose R Matos, Frank M Raushel, and Chi-huey Wong. S-Adenosylmethionine: studies on chemical and enzymatic syntesis[J]. Biotech Appl Biochem, 1987, 9, 39-52.
[21] Mudd S. H. & Cantoni G. L. (1958) J. Bio. Chem. 231, 481.
[22] Markham G. D. et al. (1984) J. Biol. Chem. 259, 14505-14507
[23] Gross A, Geresh S, and Whitesides G M. Enzymatic synthesis of S-adenosyl-L-methionine from L-methionine and ATP. Appl Biochem Biotech[J]. 1983, 8, 415-422.
[24] Jeongho Park, Junzhe Tai, Charles A Roessner, A Ian Scott. Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale[J], Bioorgan & Medic chem, 1996, 4(12): 2179～2185
[25] Reed B R, PATTERSONIIH D, Enzymatic synthesis of S-adenosyl-L-methionine, 1986, EP0189322
[26] Thomas D. et al. (1988) Mol. Cell Biol. 8, 5132-5139.
[27] Thomas D, Surdin-Kerjan Y. The synthesis of the two S-adenosyl-L-methionine synthetases is differently regulated in Saccharomyces cerevisiae, Mol Gen Genet, 1991, 226:224-232.
[28] Farooqui J Z, Woo Lee H, Kim S, Park W. Studies on aompartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hepatocytes. Biochim Biophys Acta, 1983, 747:342-351.
[29] Schlenk F, Zydek C R, Ehninger D J, et al. The Production of S-Adenosyl-L-methionine and S-Adenosyl-L-ethionine by yeast[J]. Enzymologia, 1965, 29, 283.
[30] Shiomi, N. et al. U. S. Pat. No. 5, 100, 786, 1992.
[31] Shiomi N, Fukuda H, Murata K, Kimura, A. Improvement of S-Adenosylmethionine production by integration of the ethionine-resistance gene into chromosomes of the yeast Saccharomyces cerevisiae[J], Appl microbial biotechnol, 1995, 42:730～733
[32] Shiozaki, S, Shimizu, S, Yamada, H. Agric Biol Chem, 1989, 53, 3296
[33] Matos J R and Wong Chi-H. S-adenosylmethionine: stability and stabilization. Bioorg Chem[J], 1987, 15, 71-80.
[34] Hoffman J L. Chromatographic anaysis of the chiral and covalent instability of S-adenosyl-L-methionine[J]. Biochemistry, 1986, 25, 4444-4449.
[35] De La Haba, G., Jamieson, G. A., Mudd, S. H. & Richards H. H.(1959), J. Am. Chem. Soc. 81, 3975-3980.
[36] Cornforth J. W., Reichard S. A., Talalay P., Carrell H. L. & Glusher, J. R (1977) J. Am. Chem. Soc. 99, 7292-7300.
[37] Stolowitz, M. L. & Minch, J. J. (1981) J. Am. Chem. Soc. 103, 6015-6019.
[38] Shiozaki, et al. Yeast culture containing S-adenosyl methionine in high concentrations, and process for production of S-adenosyl methionine.U.S.Pat.No. 4, 562, 149
[39] Gennari F., U. S. Pat. No. 4,990,606, 1991.
[40] Zappia V. et al., U. S. Pat. No. 5,073,546, 1991
[41] Hamedani et al. (1993) J. Chromatography. 619, 191-198.
[42] Fiecchi, Alberto. Process of preparing double salts of S-adenosyl-L-methionine. U. S. Pat. No. 4,028,183, 1977
[43] Gennari. Stable sulpho-adenosyl-L-methionine (SAMe) salts, particularly suitable for oral pharmaceutical use.U.S.Pat.No. 5,128,249, July 7, 1992
[44] Farooqui J, Kim S, Paik W K, Measurement of isoelectric point of S-adenosyl-L-methionine and its metabolic products by isoelectric focusing technique. Electrophoresis, 1983, 4: 261～265.
[45] Kawahara et al., U. S. Pat. No. 4,109,079, 1978.
[46] Gennari. Process for producing stable sulpho-adenosyl-L-methionine salts.U.S.Pat.No. 4,621,056, November 4, 1986
[47] Gennari. Stable sulpho-adendoyl-L-methionine (SAMe) salts, particlarly suitable for parenteral use. U. S. Pat. No. 5,102,791, April 7, 1992
[48] Deshpande, et al. Stable salts of S-adenosyl-L-methionine (SAMe) and the process for their preparation. U. S. Pat. No. 6,649,753, November 18, 2003
[49] Hebert. Stable salts of S-adenosyl-l-methionine. U. S. Pat. No. 6,635,615, October 21, 2003
[50] Meister, A., Anderson, M., E., Glutathione, Annu. Rev. Biochem., 52: 711-760, 1983.
[51] 矶和义员，酸发酵，40(7)：631—637，1982
[52] 大竹康之，奥村康，高生产酵母育种，50 (10) 989—994，1992
[53] Harington, C.,H.,Mead, T., H., Biochem.J., 29.. 1602—1611, 1935
[54] du, Vigneaud, V., Miller, G, L., J.Biol.Chem., 116: 469—476, 1936
[55] 袁尔东，郑建仙，功能性食品基料—谷胱甘肽的研究进展，食品与发酵工业，25，5，1999
[56] 郑建仙，功能型食品，中国轻工业出版社，北京，1995
[57] 张洪渊主编，生物化学教程，四川大学出版社，成都，1988
[58] Kosower, E., M., Chemical Properties of Glutathione, Kroc.Foundation Series, Volume 6, Glutathione: Metabolism and Function., Editors: Arias, I., M., Jakoby, W., B., Raven Press, New York, 1976
[59] Jung, G., Breitmaier, E., and Voelter, W., Dissoziationsgleichgewichte von Glutathion. Eine Fourier—Transform—13C—nmr spektroskopische Untersuchung, Eur.J.Biochem., 24: 438—444, 1972
[60] Murata, K., Kimura, A., Agric. Biol. Chem., 50: 1055—1056, 1986
[61] 张平，孟宪钧，宋旭华，李新建，多器官功能衰竭时谷胱甘肽代谢变化与细胞损伤的实验研究，中华医学杂志，69(12)：713—714，1989
[62] Knox, W., E., "The Enzymes", Vol.Ⅱ, pp.253, Academic Press, New York, 1960
[63] Ziegler, D., M., Role of Reversible Oxidation-Reduction of Enzyme
Thiols-Disulfides in Metabolic Regulation, Ann. Rev. Biochem., 54: 305—329, 1985
[64] Kosower, N., S., Kosower, E., M., The Glutathione Status of Cells, Int. Rev. Cytol., 54: 109—160, 1978
[65] 程元恺，谷胱甘肽的解毒作用与毒性代谢物，生物化学与生物物理进展，21(5)：395—399，1994
[66] 赵克健编，中国化学药品大全，pp351，新时代出版社，北京，1990
[67] 中国药物大辞典，下册，pp250，中国医药科技出版社，北京，1991
[68] 尹永祥，陈盛新，魏水易，刘吉祥，由品英主编，常用进口药物手册，pp525，天津科技翻译出版公司，天津，1994
[69] 胡文铎，崔乃杰，高仲阳主编，国家基本药物及新特药临床指南，pp537，天津科技翻译出版公司，天津，1996
[70] 王荣民，曹新志，萧刚，檀亦兵，谷胱甘肽测定方法初探，食品与发酵工业，6：34—38，1992
[71] 刘振玉，谷胱甘肽的研究和应用，生命的化学，15(1)：19—21，1995
[72] 郑建仙编，功能型食品，中国轻工业出版社，北京，1997
[73] 曹新志，广州食品工业科技，1996，12(2)：44—45
[74] 曹新志，四川食品工业科技，1996(2)：17—20
[75] Wieland, T., "Chemistry and Properties of Glutathione" in Glutathione, Colowich, S., et al., Eds, pp45—57, Academic Press, New York, 1954
[76] Jeschkeit, H., S., Jandl, J., H., J.Biol.Chem., 4241—4243, 1966
[77] Perkone, I., Vegners, R., Latv.PSR.Zinat.Akad.Vestis, 12: 77, 1984; through Chem. Abstr, 102: 79290, 1985
[78] Douglas, K., T., Chemical Synthesis of glutathione and Analogs, Coenzumes Cofactors(Glutathione, Chem., Biochem., Med., Aspects, Pt.A), 3: 243—279, 1989
[79] 张琴，“利用啤酒废酵母生物合成谷胱甘肽及其分离过程的研究”，2001，12，浙江大学硕士学位论文
[80] Miyamoto, S., Mitsuba, N., Takemoto, H., Glutathione by Cultivating Yeast on Methanol, 公开特许公报77—51089, 1977
[81] Kumagai, H., Nakayama, R., Tochikura, T., Agric.Boil.Chem., 46: 1301—1309, 1982
[82] Miwa, N., Glutathione,公开特许公报76—144789，1976
[83] Ajinomoto Co., Prodution of Glutathione, 公开特许公报82—05699，1982(21)
[84] Kumagai, H., Suuki, H., Shimizu, M., and Tochikura, T., Utilization of the γ—Glutamyltranspeptidase Reation for Glutathione Synthesis, J.Biotechnol, 9: 129—138, 1989
[85] Snoke, J., E., Block, K., J.Biol.Chem., 199.. 407, 1952
[86] Snoke, J., E., Yanari, S., Block, K., J.Biol.Chem., 201: 573, 1953
[87] 詹谷宇，田萍，刘卫东，叶明，酵母菌生物合成谷胱甘肽，药学学报，25(7)：494—499，1990
[88] Murata, K., Immobilization, Chemical and Industrial Application, and Biosynthetic Preparation of Glutathione and Related Compounds, Coenzymes Cofactors(Glutathione, Chem., Biochem., Med., Aspects, Pt.A), 3: 187
—242，1989
[89] 李寅，陈坚，伦世仪，高密度培养工程菌生产谷胱甘肽，中国医药王业杂志，30(1)：1—4，1999
[90] Miyanoto, I., Miwa, N., Production of Glutathione by Immobilized Glutathione Synthetase, 公开特许公报77—51089, 1977
[91] Alfafara, C., G., Kanda, A., Shioi, T., Shimizu, H., Shioya, S., and Suga, K., Effect of Amino acid on Glutathione Production by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol., 36: 538—540, 1992
[92] Issel, R., Nagele, A., Eckert, K., G. and Wilmanns, W., Pronotion of Cystine Uptake and its Utilization for Glutathione Biosynthesis Induced by Cysteamine and N-Acetylcysteine, Biochem. Pharmacol, 37: 881—888, 1988
[93] Modig, H., Reversz, L., Release of Thiols from Cellular Mixed Disulfides and its Possible Role in Radiation Protection, Int. J. Radiat. Biol., 22: 257—268, 1971
[94] Alfafara, C., G., Miura, K., Shimizu, H., Shioya, S., and Suga, K., Cysteine Addition Strategy for Maximum Glutathione Production in Fed-Batch Culture of Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol, 37: 141—146, 1992
[95] Yin Li, Jian Chen, Yingying Mao and Shiyi Lun, Fermentation Conditions and High-cell Density Culture of Recombinant Escherichia coli for Enhancement of Glutathione Production, 4th Asia-Pacific Biochemical Engineering Conference, pp431—434, 1997
[96] Alfafara, C., G., Miura, K., Shimizu, H., and Shioya, S., Fuzzy Control of Ethanol Concentration and Its Application to Maximum Glutathione Production in Yeast Fed-Batch Culture, Biotechnol. Bioeng., 41(4): 493—501, 1993
[97] Sakato, K., Tanaka, H., Advanced Control of Glutathione Fermentation Process, Biotechnol. Bioeng., 40(8): 904—912, 1992
[98] 周楠迪，李寅，陈坚，阮文权，伦世仪，从酵母中提取谷胱甘肽的初步研究，生物技术7(4)：30—33，1997
[99] 卓肇文，周金鑫，张孝慈，刘树魁，用改进的方法从酵母制取还原型谷胱甘肽(GSH)结晶，氨基酸杂志，(3)，6—9，1988
[100] 陶锐，吴梧桐，许激扬，酶法制备谷胱甘肽工艺的研究，药物生物技术，6(4)，1999
[101] Kimura, Akira, Murata, Kousaku, Mutant strain of Escherichia coli and its use for the preparation of glutathione, United States Patent 4, 598, 046, July 1, 1986
[102] 王辉，冯万祥，几种国产树脂对谷胱甘肽的分离性能的测定，离子交换与吸附，13(3)，318—321，1997
[103] 王辉，冯万祥，含汞树脂分离提纯谷胱甘肽，华东理工大学学报，22(6)，717—721，1996
[104] 苑小林，乐科易，王旭，高馨，鲜酵母中谷胱甘肽(GSH)分离纯化的初步研究，药物生物技术，5(2)，89—91，1998
[105] Takashi, Gotoh, Ken—ichi Kikuchi, Contamination of anion—exchange membrane by glutathione, bioseparation, 9: 37—41, 2000
[106] 梅乐和，林东强，朱自强，双水相分配结合温度诱导相分离从酵母中提取谷胱甘肽，化工学报，49(4)，1998，8
[107] Boyed GE, Adaman AW, Myers LS, J Am Chem Soc, 1947, 69: 2836
[108] Zappia V. et al., U. S. Pat. No. 4, 764, 603, 1988.
[2] Giulidori P, Galli-Kienle M, Catto E, Stramentinoli G. Transmethylation, transsulfuration, and aminopropylation reactions of S-adenosyl-L-methionine in vivo[J]. J Biol Chem, 1984, 259, 4205-4449.
[3] Borchardt R. T., "Enzymatic Basis of Detoxication", Vol. Ⅱ, pp. 43, Academic Press, 1980.
[4] Kersten H., "Biochemistry of S-adenosylmethionine and Related Compounds", pp. 357, Usdin E. et al. Eds., MacMillan Press L. t. D., 1982.
[5] Lu S C. Review article: S-Adenosylmethionine[J], Inter J Biochem cell Bio, 2000, 32: 391～395
[6] Barcelo H A, Wiemeyer J C, Sagasta C L, Macias M, Barreira J C. Experimental osteoarthritis and its course wthen treated with S-adenosyl-L-methionine[J]. Rev Clin Esp, 1990, 187, 74-78.
[7] Cimino M, Vantini G, Algeri S, Curatola G, Pezzoli C, Stramentinoli G. Age-related modification of dopamineric and beta-adrenergic receptor system: restoration to normal activity by modifying membrane fluidity with S-adenosyl-L-methionine[J]. Life Sci 1984, 34, 2029-2039.
[8] Torta R, Cicolin A, Keller R. Transmethylation and affective disorders[J]. Arch Gerontol Geriatr suppl, 1998, 6, 499-506.
[9] Osman E O, Owen J S, Burroughs A K. S-adenosyl-L-methionine. A new therapeutic agent in liver disease[J]? Aliment Pharmacol Ther, 1993, 7, 21-28.
[10] Charles S L. Role of S-adenosyl-L-methionine in the treatment of liver diseases [J]. J Hepatology, 1999, 30, 1155-1159.
[11] Vendemiale G, Altomare E, Trizio T, et al. Effects of oral S-adenosyl-L-methionine on hepatic glutathione in patients with liver disease. Scand J Gastroenterol, 1989, 24 (4): 407-415.
[12] Osman E, Owen J S, Burroughs A K. S-adenosyl-L-methionine-a new therapeutic agent in liver disease? Aliment Pharmcol Ther, 1993, 7: 21-28.
[13] Angelico M, Gandin C, Nistri A, et al. Oral (SAMe) administration enhances bile salt conjugation with taurine in patients with liver cirrhosis. Scand.J.Clin.Lab Invesr. 1993,54: 459-464.
[14] Nakano H, Yamaguchi M, Kaneshiro Y, et al. Transplant Proceed, 1998, 30: 3735-3736..
[15] Benedetto P Di, Iona LG, Zidarich V. Clinical. evaluation of S-adenosyl-L-methionine versus transcutaneous electrical nerve stimulation in primary fibromyalgia. Italy Curr Ther Res, 1993, 53 (2): 222.
[16] Piacentino R, Malara D, Zaccheo F, et al. Preliminary study of the ues of S-adenosyl-L-methionine in the management of male sterility. Minerva.Ginecologica, 1991,43:191-193 [in Italian]..
[17] Gatto G, Caleri D, Michelacci S, Sicuteri F. Analgesizing effect of a methyldodor (S-adenosyl-L-methionine)in migraine: an open clinical trial. Int.J.Clin. Pharmacol.Res.,1986,6:15-17.
[18] Ballerini F B, Anguera A L, Alcaraz P, et al. SAM in the management of postconcussional syndrome. Med.Clin.(Barc)1983,80:161-164
[19] Baddiley J & Jamieson G A. ibid[J], 1954, 4280
[20] Jose R Matos, Frank M Raushel, and Chi-huey Wong. S-Adenosylmethionine: studies on chemical and enzymatic syntesis[J]. Biotech Appl Biochem, 1987, 9, 39-52.
[21] Mudd S. H. & Cantoni G. L. (1958) J. Bio. Chem. 231, 481.
[22] Markham G. D. et al. (1984) J. Biol. Chem. 259, 14505-14507
[23] Gross A, Geresh S, and Whitesides G M. Enzymatic synthesis of S-adenosyl-L-methionine from L-methionine and ATP. Appl Biochem Biotech[J]. 1983, 8, 415-422.
[24] Jeongho Park, Junzhe Tai, Charles A Roessner, A Ian Scott. Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale[J], Bioorgan & Medic chem, 1996, 4(12): 2179～2185
[25] Reed B R, PATTERSONIIH D, Enzymatic synthesis of S-adenosyl-L-methionine, 1986, EP0189322
[26] Thomas D. et al. (1988) Mol. Cell Biol. 8, 5132-5139.
[27] Thomas D, Surdin-Kerjan Y. The synthesis of the two S-adenosyl-L-methionine synthetases is differently regulated in Saccharomyces cerevisiae, Mol Gen Genet, 1991, 226:224-232.
[28] Farooqui J Z, Woo Lee H, Kim S, Park W. Studies on aompartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hepatocytes. Biochim Biophys Acta, 1983, 747:342-351.
[29] Schlenk F, Zydek C R, Ehninger D J, et al. The Production of S-Adenosyl-L-methionine and S-Adenosyl-L-ethionine by yeast[J]. Enzymologia, 1965, 29, 283.
[30] Shiomi, N. et al. U. S. Pat. No. 5, 100, 786, 1992.
[31] Shiomi N, Fukuda H, Murata K, Kimura, A. Improvement of S-Adenosylmethionine production by integration of the ethionine-resistance gene into chromosomes of the yeast Saccharomyces cerevisiae[J], Appl microbial biotechnol, 1995, 42:730～733
[32] Shiozaki, S, Shimizu, S, Yamada, H. Agric Biol Chem, 1989, 53, 3296
[33] Matos J R and Wong Chi-H. S-adenosylmethionine: stability and stabilization. Bioorg Chem[J], 1987, 15, 71-80.
[34] Hoffman J L. Chromatographic anaysis of the chiral and covalent instability of S-adenosyl-L-methionine[J]. Biochemistry, 1986, 25, 4444-4449.
[35] De La Haba, G., Jamieson, G. A., Mudd, S. H. & Richards H. H.(1959), J. Am. Chem. Soc. 81, 3975-3980.
[36] Cornforth J. W., Reichard S. A., Talalay P., Carrell H. L. & Glusher, J. R (1977) J. Am. Chem. Soc. 99, 7292-7300.
[37] Stolowitz, M. L. & Minch, J. J. (1981) J. Am. Chem. Soc. 103, 6015-6019.
[38] Shiozaki, et al. Yeast culture containing S-adenosyl methionine in high concentrations, and process for production of S-adenosyl methionine.U.S.Pat.No. 4, 562, 149
[39] Gennari F., U. S. Pat. No. 4,990,606, 1991.
[40] Zappia V. et al., U. S. Pat. No. 5,073,546, 1991
[41] Hamedani et al. (1993) J. Chromatography. 619, 191-198.
[42] Fiecchi, Alberto. Process of preparing double salts of S-adenosyl-L-methionine. U. S. Pat. No. 4,028,183, 1977
[43] Gennari. Stable sulpho-adenosyl-L-methionine (SAMe) salts, particularly suitable for oral pharmaceutical use.U.S.Pat.No. 5,128,249, July 7, 1992
[44] Farooqui J, Kim S, Paik W K, Measurement of isoelectric point of S-adenosyl-L-methionine and its metabolic products by isoelectric focusing technique. Electrophoresis, 1983, 4: 261～265.
[45] Kawahara et al., U. S. Pat. No. 4,109,079, 1978.
[46] Gennari. Process for producing stable sulpho-adenosyl-L-methionine salts.U.S.Pat.No. 4,621,056, November 4, 1986
[47] Gennari. Stable sulpho-adendoyl-L-methionine (SAMe) salts, particlarly suitable for parenteral use. U. S. Pat. No. 5,102,791, April 7, 1992
[48] Deshpande, et al. Stable salts of S-adenosyl-L-methionine (SAMe) and the process for their preparation. U. S. Pat. No. 6,649,753, November 18, 2003
[49] Hebert. Stable salts of S-adenosyl-l-methionine. U. S. Pat. No. 6,635,615, October 21, 2003
[50] Meister, A., Anderson, M., E., Glutathione, Annu. Rev. Biochem., 52: 711-760, 1983.
[51] 矶和义员，酸发酵，40(7)：631—637，1982
[52] 大竹康之，奥村康，高生产酵母育种，50 (10) 989—994，1992
[53] Harington, C.,H.,Mead, T., H., Biochem.J., 29.. 1602—1611, 1935
[54] du, Vigneaud, V., Miller, G, L., J.Biol.Chem., 116: 469—476, 1936
[55] 袁尔东，郑建仙，功能性食品基料—谷胱甘肽的研究进展，食品与发酵工业，25，5，1999
[56] 郑建仙，功能型食品，中国轻工业出版社，北京，1995
[57] 张洪渊主编，生物化学教程，四川大学出版社，成都，1988
[58] Kosower, E., M., Chemical Properties of Glutathione, Kroc.Foundation Series, Volume 6, Glutathione: Metabolism and Function., Editors: Arias, I., M., Jakoby, W., B., Raven Press, New York, 1976
[59] Jung, G., Breitmaier, E., and Voelter, W., Dissoziationsgleichgewichte von Glutathion. Eine Fourier—Transform—13C—nmr spektroskopische Untersuchung, Eur.J.Biochem., 24: 438—444, 1972
[60] Murata, K., Kimura, A., Agric. Biol. Chem., 50: 1055—1056, 1986
[61] 张平，孟宪钧，宋旭华，李新建，多器官功能衰竭时谷胱甘肽代谢变化与细胞损伤的实验研究，中华医学杂志，69(12)：713—714，1989
[62] Knox, W., E., "The Enzymes", Vol.Ⅱ, pp.253, Academic Press, New York, 1960
[63] Ziegler, D., M., Role of Reversible Oxidation-Reduction of Enzyme
Thiols-Disulfides in Metabolic Regulation, Ann. Rev. Biochem., 54: 305—329, 1985
[64] Kosower, N., S., Kosower, E., M., The Glutathione Status of Cells, Int. Rev. Cytol., 54: 109—160, 1978
[65] 程元恺，谷胱甘肽的解毒作用与毒性代谢物，生物化学与生物物理进展，21(5)：395—399，1994
[66] 赵克健编，中国化学药品大全，pp351，新时代出版社，北京，1990
[67] 中国药物大辞典，下册，pp250，中国医药科技出版社，北京，1991
[68] 尹永祥，陈盛新，魏水易，刘吉祥，由品英主编，常用进口药物手册，pp525，天津科技翻译出版公司，天津，1994
[69] 胡文铎，崔乃杰，高仲阳主编，国家基本药物及新特药临床指南，pp537，天津科技翻译出版公司，天津，1996
[70] 王荣民，曹新志，萧刚，檀亦兵，谷胱甘肽测定方法初探，食品与发酵工业，6：34—38，1992
[71] 刘振玉，谷胱甘肽的研究和应用，生命的化学，15(1)：19—21，1995
[72] 郑建仙编，功能型食品，中国轻工业出版社，北京，1997
[73] 曹新志，广州食品工业科技，1996，12(2)：44—45
[74] 曹新志，四川食品工业科技，1996(2)：17—20
[75] Wieland, T., "Chemistry and Properties of Glutathione" in Glutathione, Colowich, S., et al., Eds, pp45—57, Academic Press, New York, 1954
[76] Jeschkeit, H., S., Jandl, J., H., J.Biol.Chem., 4241—4243, 1966
[77] Perkone, I., Vegners, R., Latv.PSR.Zinat.Akad.Vestis, 12: 77, 1984; through Chem. Abstr, 102: 79290, 1985
[78] Douglas, K., T., Chemical Synthesis of glutathione and Analogs, Coenzumes Cofactors(Glutathione, Chem., Biochem., Med., Aspects, Pt.A), 3: 243—279, 1989
[79] 张琴，“利用啤酒废酵母生物合成谷胱甘肽及其分离过程的研究”，2001，12，浙江大学硕士学位论文
[80] Miyamoto, S., Mitsuba, N., Takemoto, H., Glutathione by Cultivating Yeast on Methanol, 公开特许公报77—51089, 1977
[81] Kumagai, H., Nakayama, R., Tochikura, T., Agric.Boil.Chem., 46: 1301—1309, 1982
[82] Miwa, N., Glutathione,公开特许公报76—144789，1976
[83] Ajinomoto Co., Prodution of Glutathione, 公开特许公报82—05699，1982(21)
[84] Kumagai, H., Suuki, H., Shimizu, M., and Tochikura, T., Utilization of the γ—Glutamyltranspeptidase Reation for Glutathione Synthesis, J.Biotechnol, 9: 129—138, 1989
[85] Snoke, J., E., Block, K., J.Biol.Chem., 199.. 407, 1952
[86] Snoke, J., E., Yanari, S., Block, K., J.Biol.Chem., 201: 573, 1953
[87] 詹谷宇，田萍，刘卫东，叶明，酵母菌生物合成谷胱甘肽，药学学报，25(7)：494—499，1990
[88] Murata, K., Immobilization, Chemical and Industrial Application, and Biosynthetic Preparation of Glutathione and Related Compounds, Coenzymes Cofactors(Glutathione, Chem., Biochem., Med., Aspects, Pt.A), 3: 187
—242，1989
[89] 李寅，陈坚，伦世仪，高密度培养工程菌生产谷胱甘肽，中国医药王业杂志，30(1)：1—4，1999
[90] Miyanoto, I., Miwa, N., Production of Glutathione by Immobilized Glutathione Synthetase, 公开特许公报77—51089, 1977
[91] Alfafara, C., G., Kanda, A., Shioi, T., Shimizu, H., Shioya, S., and Suga, K., Effect of Amino acid on Glutathione Production by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol., 36: 538—540, 1992
[92] Issel, R., Nagele, A., Eckert, K., G. and Wilmanns, W., Pronotion of Cystine Uptake and its Utilization for Glutathione Biosynthesis Induced by Cysteamine and N-Acetylcysteine, Biochem. Pharmacol, 37: 881—888, 1988
[93] Modig, H., Reversz, L., Release of Thiols from Cellular Mixed Disulfides and its Possible Role in Radiation Protection, Int. J. Radiat. Biol., 22: 257—268, 1971
[94] Alfafara, C., G., Miura, K., Shimizu, H., Shioya, S., and Suga, K., Cysteine Addition Strategy for Maximum Glutathione Production in Fed-Batch Culture of Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol, 37: 141—146, 1992
[95] Yin Li, Jian Chen, Yingying Mao and Shiyi Lun, Fermentation Conditions and High-cell Density Culture of Recombinant Escherichia coli for Enhancement of Glutathione Production, 4th Asia-Pacific Biochemical Engineering Conference, pp431—434, 1997
[96] Alfafara, C., G., Miura, K., Shimizu, H., and Shioya, S., Fuzzy Control of Ethanol Concentration and Its Application to Maximum Glutathione Production in Yeast Fed-Batch Culture, Biotechnol. Bioeng., 41(4): 493—501, 1993
[97] Sakato, K., Tanaka, H., Advanced Control of Glutathione Fermentation Process, Biotechnol. Bioeng., 40(8): 904—912, 1992
[98] 周楠迪，李寅，陈坚，阮文权，伦世仪，从酵母中提取谷胱甘肽的初步研究，生物技术7(4)：30—33，1997
[99] 卓肇文，周金鑫，张孝慈，刘树魁，用改进的方法从酵母制取还原型谷胱甘肽(GSH)结晶，氨基酸杂志，(3)，6—9，1988
[100] 陶锐，吴梧桐，许激扬，酶法制备谷胱甘肽工艺的研究，药物生物技术，6(4)，1999
[101] Kimura, Akira, Murata, Kousaku, Mutant strain of Escherichia coli and its use for the preparation of glutathione, United States Patent 4, 598, 046, July 1, 1986
[102] 王辉，冯万祥，几种国产树脂对谷胱甘肽的分离性能的测定，离子交换与吸附，13(3)，318—321，1997
[103] 王辉，冯万祥，含汞树脂分离提纯谷胱甘肽，华东理工大学学报，22(6)，717—721，1996
[104] 苑小林，乐科易，王旭，高馨，鲜酵母中谷胱甘肽(GSH)分离纯化的初步研究，药物生物技术，5(2)，89—91，1998
[105] Takashi, Gotoh, Ken—ichi Kikuchi, Contamination of anion—exchange membrane by glutathione, bioseparation, 9: 37—41, 2000
[106] 梅乐和，林东强，朱自强，双水相分配结合温度诱导相分离从酵母中提取谷胱甘肽，化工学报，49(4)，1998，8
[107] Boyed GE, Adaman AW, Myers LS, J Am Chem Soc, 1947, 69: 2836
[108] Zappia V. et al., U. S. Pat. No. 4, 764, 603, 1988.