透明质酸的生物合成及其纯化
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摘要
本文分别对种子培养基和发酵培养基进行优化，确定了以淀粉为碳源的培养基培养兽疫链球菌NJUST01。最佳种子培养基成分为：可溶性淀粉1.7％；酵母浸出粉2.0％；KH_2PO_41.0％；K_2HPO_41.0％；MgSO_4·7H_2O1.0％；pH7.0(自然)，35℃培养18h。并用此培养基进行了60代传代培养，实验证明传代后菌株及其生产HA的能力没有明显改变。分别对发酵培养基的C／N、无机盐、添加剂、接种量、时间、溶氧量、pH等方面进行优化。最终确定最佳发酵培养基：可溶性淀粉4％；酵母浸出粉1.5％；KH_2PO_41.0％；K_2HPO_41.0％；MgSO_4·7H_2O1.0％；CaCO_31.0％；MnSO_40.06％；(NH_4)_2SO_40.06％；pH7.0(自然)，35℃培养36h，接种量为10％，转速250转／分。
本文比较了不同分离纯化发酵产HA的方法，最终确立了以冷藏去蛋白质，CPC选择性沉淀得到HA产品的提取纯化工艺。最终得到HA产量为3.5g／L，分子量为2.047×10~5Da。
本论文测试了外源HA对抗生素抑制链球菌的效果，结果表明在培养基中加HA后，抗生素的抑菌效果有一定程度的提高。
The paper related to a novel medium using core starch as primary carbon source, in which the strain of hyaluronic acid-producing Streptococcus zooepidemicus NJUST01 reproduced 60 generations continuously at 35癈. A comparison was made between the first and the 60th generation. The 60th colonies had no obvious difference with the first one in morphology as well as the yield level and the molecular weight of the hyaluronic acid produced by both. The optimal starch medium comprises starch 1.7% (wt/vol, the same as follow), yeast extract 2.0%, K2HPO4 1.0%, KH2PO4 1.0%, MgSO4 0.5%, pH 7.0, which was shaking at 35癈 for 18 hours until exponential growth phase was reached. The paper tested the effect of different factors to the final fermentation, such as MgSO4, inorganic compound, C/N, additive, inoculum volume, fermentation time, oxygen demand. The optimal final fermentation comprises starch 4.0%, yeast extract 1.0%, K2HPO4 1.0%, KH2PO4 1.0%, MgS04 0.5%, CaCO3 1.0%, MnSO4 0.06%, (NH4)2SO4 0.06%, pH 7.0, which was shaking at 37 C for 36 hours in the speed of 250 rpm. Automatic temperature and pH control were used.
Hyaluronic acid was isolated from fermentation suspension. The process involved extraction with water, precipitation by ethanol, purification with cetyl-pyridinium salt. The yields were 3.5g/L, the molecular weight were 2.047 X 105Da.
The paper observed the effect of antibiotic to Streptococcus zooepidemicus, when hyaluronic acid was added into the medium. As a result, the action of antibiotic to the tested bacteria was increase 1 - 1.5 times compared with the common medium without hyaluronic acid.
本文比较了不同分离纯化发酵产HA的方法，最终确立了以冷藏去蛋白质，CPC选择性沉淀得到HA产品的提取纯化工艺。最终得到HA产量为3.5g／L，分子量为2.047×10~5Da。
本论文测试了外源HA对抗生素抑制链球菌的效果，结果表明在培养基中加HA后，抗生素的抑菌效果有一定程度的提高。
The paper related to a novel medium using core starch as primary carbon source, in which the strain of hyaluronic acid-producing Streptococcus zooepidemicus NJUST01 reproduced 60 generations continuously at 35癈. A comparison was made between the first and the 60th generation. The 60th colonies had no obvious difference with the first one in morphology as well as the yield level and the molecular weight of the hyaluronic acid produced by both. The optimal starch medium comprises starch 1.7% (wt/vol, the same as follow), yeast extract 2.0%, K2HPO4 1.0%, KH2PO4 1.0%, MgSO4 0.5%, pH 7.0, which was shaking at 35癈 for 18 hours until exponential growth phase was reached. The paper tested the effect of different factors to the final fermentation, such as MgSO4, inorganic compound, C/N, additive, inoculum volume, fermentation time, oxygen demand. The optimal final fermentation comprises starch 4.0%, yeast extract 1.0%, K2HPO4 1.0%, KH2PO4 1.0%, MgS04 0.5%, CaCO3 1.0%, MnSO4 0.06%, (NH4)2SO4 0.06%, pH 7.0, which was shaking at 37 C for 36 hours in the speed of 250 rpm. Automatic temperature and pH control were used.
Hyaluronic acid was isolated from fermentation suspension. The process involved extraction with water, precipitation by ethanol, purification with cetyl-pyridinium salt. The yields were 3.5g/L, the molecular weight were 2.047 X 105Da.
The paper observed the effect of antibiotic to Streptococcus zooepidemicus, when hyaluronic acid was added into the medium. As a result, the action of antibiotic to the tested bacteria was increase 1 - 1.5 times compared with the common medium without hyaluronic acid.
引文
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[2] Meyer K.The biological significance of hyaluronic acid and hyaluronidase.Physiol Rev. 1947,27:335-359
[3] Kendall FE,Heidelberger M,Dawson MH.A serologically inactive polysaccharide streptococcus.J Biol Chem.1937,118:61-69
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Biol,1990,2:839-844
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[30] Kendall K, Palmer JW.The polysaccharide of the vitreous houmor.J Biol
Chem. 1934,107:629-633
[31] 潘华，汪玉松．透明质酸(HA)应用研究的进展．黑龙江医药．1997，10(4)：226-227
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[33] Prehm P.Synthesis of hyaluronate in differentiated teratocarcinoma cells. Ⅱ. Characterization of the synthase.Biochem J.1983,211:191-198
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[37] Laurent TC, Ryan M, Pietruszkiewicz A. Fraction of hyaluronic acid.The polydispersity of hyaluronic acid from the bovine vitreous body. Biochim Biophys. 1960,42:476-485
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[48] Romeo Aurelio, Lorenzi Silvana. Procedure for the purification of hyaluronic acid and fraction of pure hyaluronic acid for ophthalmic use.WO9218543. 1992-10-29
[49] Ruiz Linda L Clem, Brown Karen K, Vanderijn Ivo.Ultrapure hyaluronic acid and method of making it. EP0144019.1985-06-12
[50] Akasaka Hidemichi, Komasaki Hisayuki, Arai Takayuki. Fermentation method for producing hyaluronic acid.US4801539.1989-01-31
[51] Ellwood Derek C,Evans Charles GT,Dunn Geoffrey M,Mcinnes Neil,Eo Richard G,Smith Keith J.Production of hyaluronic acid.US5411874.1995-05-02
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[66] Nimrod Abraham, Greenman Benjamin, Kanner Dov, Landsberg Moshe, Beck Yaffa. Method of producing high molecular weight sodium hyallronate by fermentation of streptococcus.US4780414.1988-10-25
[67] Ellwood Derek C, Evans Charles GT, Dunn Geoffrey M, Mcinnes Neil,Yeo Richard G,Smith Keith J.Production of hyaluronic acid.US5411874,1995-05-02
[68] Magnette Francois, Carlino Stefano. Process for purifying high molecular weight hyaluronic acid. WO0044925,2000-08-03
[69] 殷晓煌，李洪．透明质酸的性质及制备方法．中国生化药物杂志．1993(3)：58-61
[2] Meyer K.The biological significance of hyaluronic acid and hyaluronidase.Physiol Rev. 1947,27:335-359
[3] Kendall FE,Heidelberger M,Dawson MH.A serologically inactive polysaccharide streptococcus.J Biol Chem.1937,118:61-69
[4] 沈渤江，张天民．公鸡冠透明质酸的制备及其理化性质．医药工业，1986，17：291-294
[5] 张天民，凌沛学，沈渤江．透明质酸的研制及应用研究．药学通报．1987，22：556-559
[6] Laurent TC．Biochemistry of Hyaluronan．Acta Otolaryngol(Stockh)，1987(suppl)，442：7-12
[7] Pogrel MA,Lowe MA,Stern R.Hyaluronan(hyaluronic acid)in human saliva. Arch Oral Biol,1996,41: 667-671
[8] 凌沛学，贺艳丽．玻璃酸钠的临床应用进展．中国生化药物杂志．1998，19(4)：200-204
[9] 罗瑞明．透明质酸(HA)的国内外研究现状．宁夏农学院学报．2001，22(1)：62-64
[10] 顾其胜，杨春涛．透明质酸研究新进展．国外医学．诊断．治疗用生物制品分册．1991，14(1)：20-23
[11] 鲁念慈，谭天伟．透明质酸的制备及其应用．功能高分子学报．2001，14(3)：370-376
[12] 朱广华等．透明质酸制备方法及应用研究的进展．中国医药工业杂志．1996，2(8)：382-384
[13] Akasaka Hidemichi,Komasaki Hisayuki,Arai Takayuki.Fermentation method for producing hyaluronic acid.US4801539,1989-01-31
[14] Kaufmann J, Mohle K, Hofmann HJ, Arnold K. Molecular dynamics study of hyaluronic acid in water.Theochem(Netherlands), 12 Jan. 1998, 422:109-121
[15] Prehm P.Hyaluronate is synthesized at plasma membranes.Biochem J,1984,220: 597-600
[16] Prehm P.Synthesis of hyaluronate in differentiated teratocarcinoma cells. Ⅰ. Characterization of the synthase.Biochem J. 1983,211:181-189
[17] Toole BP.hyaluronan and its binding proteins,the hyaladherins.Curr Opin Cell
Biol,1990,2:839-844
[18] Main N.Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex.Biochem J. 1986,237:343-357
[19] Main N.Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fraction of culture human skin fibroblasts.Biochem J. 1986,237:333-342
[20] Spicer A. P.,McDonald J. A. Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family. J Biol Chem, 1998, 273: 1923～1932
[21] Dougherty B A.,Van de Rijn I. Molecular characterization of hasA from an operon for hyaluronic acid synthesis in group A streptococci. Journal of Biological Chemistry.1994, 269, 169-175
[22] Crater D L, Dougherty B A, van de Rijn I. Molecular characterization of hasC from an operon for hyaluronic acid synthesis in group A streptococci demonstration of UDP-glucose pyrophosphorylase activity. Journal of Biological Chemistry.1995, 270:28676-28680
[23] DeAngelis PL, Achyuthan AM.Yeast-derived recombinant DG42 protein of Xenopus can synthesize hyaluronan in vitro. J Biol Chem.1996, 271: 23657-23660
[24] 凌沛学．透明质酸．北京：中国轻工业出版社(第一版)，2000：100-102
[25] Hadler NM.Enhanced diffusivity of glucose in a matrix of hyaluronic acid. J Biol Chem.1980,255(8):3532-3535
[26] Kitchen J R, Cysyk R L. Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts.Biochemical Journal. 1995,309:649-656
[27] Armstrong D C,Johns M R. Effect of Culture Conditions on Molecular Weight of Hyaluronic Acid Produced by Streptococcus zooepidemicus.Applied and Environmental Microbiology. 1997,63:2759-2764
[28] Kumari K,Weigel PH.Molecular cloning,expression and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis.Journal of Biological Chemistry.1997,272:32539-32546
[29] Oregan M, Martini I, Crescenzi F, Deluca S, Lansing M. Molecular mechanisms and genetics of hyaluronan biosynthesis.Int J Biol Macromol,1994,16:283-284
[30] Kendall K, Palmer JW.The polysaccharide of the vitreous houmor.J Biol
Chem. 1934,107:629-633
[31] 潘华，汪玉松．透明质酸(HA)应用研究的进展．黑龙江医药．1997，10(4)：226-227
[32] 冯文帅．透明质酸及其发展应用前景．四川食品与发酵．2001，1：4-6
[33] Prehm P.Synthesis of hyaluronate in differentiated teratocarcinoma cells. Ⅱ. Characterization of the synthase.Biochem J.1983,211:191-198
[34] 包定元，方志平．药理学．四川：四川大学出版社(第一版)，2001
[35] 诸葛健，王正详．工业微生物实验技术手册．北京：中国轻工业出版社(第一版)，1994
[36] Bitter T, Muir HM. A modified uronic acid carbazole reaction.Anal Biochem. 1962,4:330-333
[37] Laurent TC, Ryan M, Pietruszkiewicz A. Fraction of hyaluronic acid.The polydispersity of hyaluronic acid from the bovine vitreous body. Biochim Biophys. 1960,42:476-485
[38] 拉普洛夫主编．胶体化学实验．陈宗琪等译．济南：山东大学出版社(第一版)，1998，138-139
[39] 边藏丽，王兵，边丽梅．乙醇对常见病原菌最低杀菌浓度的试验研究．中华临床医药．2002，3(6)：41-42
[40] 李影林主编．临床微生物及检验．北京：人民卫生出版社(第一版)，1995：85
[41] 江红霞，郑怡．8种微藻抗菌活性研究．福建师范大学学报(自然科学版)．2002，18(2)：117-121
[42] Mazela D, Daviesb J. Antibiotic resistance in microbes.CMLS,Cell.Mol.Life. Sci.1999, 56: 742-754
[43] 陈默蕊，曹锡标，蔡意和，吴教仁，李贵才，宋锦煌．临床感染标本中常见革兰氏阳性球菌的分离及其耐药性分析．实用医技杂志．2002，9(12)：905-907
[44] 马红霞，邓旭明，欧阳红生．细菌多重耐药分子机制的研究进展．国外医药抗生素分册．2002，23(3)：102-109
[45] 贺小贤．生物工艺原理．北京：化学工业出版社(第一版)，2003
[46] Magnette Francois, Carlino Stefano. Process for purifying high molecular weight hyaluronic acid. WO0044925.2000-08-03
[47] Staahl Sten. Method and means for the production of hyaluronic acid.WO9533067.2000-07-18
[48] Romeo Aurelio, Lorenzi Silvana. Procedure for the purification of hyaluronic acid and fraction of pure hyaluronic acid for ophthalmic use.WO9218543. 1992-10-29
[49] Ruiz Linda L Clem, Brown Karen K, Vanderijn Ivo.Ultrapure hyaluronic acid and method of making it. EP0144019.1985-06-12
[50] Akasaka Hidemichi, Komasaki Hisayuki, Arai Takayuki. Fermentation method for producing hyaluronic acid.US4801539.1989-01-31
[51] Ellwood Derek C,Evans Charles GT,Dunn Geoffrey M,Mcinnes Neil,Eo Richard G,Smith Keith J.Production of hyaluronic acid.US5411874.1995-05-02
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