从发酵液中提取生物防腐剂ε-聚赖氨酸的初步研究
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摘要
ε-聚赖氨酸(ε-Poly-L-Lysine)是由微生物发酵合成的一种由赖氨酸单体通过ε-酰胺键形成的多肽。ε-PL具有优良的抑菌作用，对革兰氏阳性菌和阴性菌、酵母和一些病毒都有抑制作用，而且兼有水溶性好、热稳定性高等特点。目前，ε-PL已广泛用于食品加工和贮藏的防腐保鲜、可降解生物材料、药物包被物、靶向药物载体以及医学研究中。
本文采用D152弱酸性阳离子交换树脂(H型)对发酵法生产的ε-聚赖氨酸进行了分离提取的研究。参考外文文献设计了合理的工艺流程。离心分离发酵液(pH3．2)，调节滤液的pH值到8.5，过滤除去沉淀。滤液通过D152离子交换柱(H型)，用水和0.2N的醋酸洗涤，用0.1N的盐酸洗脱。用1N的NaON调节洗脱液的pH值到6.5，加入活性炭脱色后浓缩到小体积。加入乙醇和乙醚的混合物(体积比2.1)后，析出白色晶体。产品可进一步使用葡聚糖凝胶柱精制。
通过静态交换实验，研究了．D152树脂对ε-聚赖氨酸的静态交换的一些参数，通过动态交换实验，研究了以盐酸为洗脱剂，以盐酸为再生剂的工艺流程，确定了主要工艺参数。
ε -Poly-L-Lysine is comprised of lysine linked by amide bond through microbial fermentation. It has excellent antibacterial activity against Gram-positive and Gram-negative bacteria, yeast and some kinds of viruses. In additional ε -Poly-L-Lysine is soluble and attractive heat stable. At present ε -Poly-L-Lysine have been used as natural food preservative mainly, degradable biological matreria, coat of drug, carrier of garget drug and medical research.
The separation and extraction of ε -Poly-L-Lysine from fermentation liquid by using weak acid anion-exchange resin D152 was studied. The culture broth(pH3.2) was filtered and the filtrate was adjusted to Ph8.5. Then, the precipitate formed was removed by filtration. The filtrate was applied on a column of Amberlite IRC-50(H+-from) and washed with 0.2N acetic acid and water, successively. The substance was eluted with 0.1N hydrochloric acid. The eluate was neutralized with 1N sodium hydroxide and decolorized with active charcoal, and then evaporated to a small volume. The D.P. substance was precipitated as a white powder by addition of an ethanol and ether (2:1)mixture. The substance was further purified by column chromatography on SephadexG-25.
Through static exchange experiments, static exchange parameters of ε -Poly-L-Lysine on D152 resin is discussed. Through dynamic exchange experiment, the technical process using Hcl as eluent and Hcl as regenerative agent is studied, and main technical parameters are determined.
本文采用D152弱酸性阳离子交换树脂(H型)对发酵法生产的ε-聚赖氨酸进行了分离提取的研究。参考外文文献设计了合理的工艺流程。离心分离发酵液(pH3．2)，调节滤液的pH值到8.5，过滤除去沉淀。滤液通过D152离子交换柱(H型)，用水和0.2N的醋酸洗涤，用0.1N的盐酸洗脱。用1N的NaON调节洗脱液的pH值到6.5，加入活性炭脱色后浓缩到小体积。加入乙醇和乙醚的混合物(体积比2.1)后，析出白色晶体。产品可进一步使用葡聚糖凝胶柱精制。
通过静态交换实验，研究了．D152树脂对ε-聚赖氨酸的静态交换的一些参数，通过动态交换实验，研究了以盐酸为洗脱剂，以盐酸为再生剂的工艺流程，确定了主要工艺参数。
ε -Poly-L-Lysine is comprised of lysine linked by amide bond through microbial fermentation. It has excellent antibacterial activity against Gram-positive and Gram-negative bacteria, yeast and some kinds of viruses. In additional ε -Poly-L-Lysine is soluble and attractive heat stable. At present ε -Poly-L-Lysine have been used as natural food preservative mainly, degradable biological matreria, coat of drug, carrier of garget drug and medical research.
The separation and extraction of ε -Poly-L-Lysine from fermentation liquid by using weak acid anion-exchange resin D152 was studied. The culture broth(pH3.2) was filtered and the filtrate was adjusted to Ph8.5. Then, the precipitate formed was removed by filtration. The filtrate was applied on a column of Amberlite IRC-50(H+-from) and washed with 0.2N acetic acid and water, successively. The substance was eluted with 0.1N hydrochloric acid. The eluate was neutralized with 1N sodium hydroxide and decolorized with active charcoal, and then evaporated to a small volume. The D.P. substance was precipitated as a white powder by addition of an ethanol and ether (2:1)mixture. The substance was further purified by column chromatography on SephadexG-25.
Through static exchange experiments, static exchange parameters of ε -Poly-L-Lysine on D152 resin is discussed. Through dynamic exchange experiment, the technical process using Hcl as eluent and Hcl as regenerative agent is studied, and main technical parameters are determined.
引文
[1]Shoji shima, Polylysine produced by Streptomyces, Agric Biol Chem [J], 1977, 41 (9): 1807-1809
[2]Prihardi kahar, toshiharuk iwata, Enhancement of ε -PL production by Streptomyces albulus Strain 410 using pH Control, Journal of bioscience and bioengineefing[J],2001,Vol.91, No.2:190 -194
[3]Shoji shima, Agric Biol Chem[J], 1981,45(Ⅱ): 2497-2502
[4]腾井正弘，公开特许公报，1988，JP63，112，981
[5]平木纯，公开特许公报，1993，JP5，219，925
[6]徐红华，王英东，赵新淮，多聚赖氨酸在食品抑菌方面的研究进展，粮油食品科技[J]，1999，Vol．7，33-34
[7]刘慧，徐红华，王明丽等，聚赖氨酸抑菌性能的研究，东北农业大学学报，Vol．31，No．4：294-298
[8]Shoji Shima, Antimicrobial action of ε -PL, Journal of antibiotics[J], 1984, Vol.37,1449-1455
[9]向智敏，天然食品防腐剂-聚赖氨酸，广州食品工业科技[J]，1993．1
[10]徐红华，刘慧，聚赖氨酸在牛奶保鲜中的应用研究，食品与发酵工业[J]，2000，Vol．26，No．2：33-35
[11]Ckenzie, Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides, Journal of peptide reserch[J],Oct 1999,Vol.54, No.4:311-318
[12]Hao zhen jun, In vitro reaction of penicilloyl and penicillanyl albumin and polylysineconjugates with igE-antibody, Journal of immunological methods[J],28 August,2000, Vol.242, No.1-2:43-51
[13]Nussionovitch-A, Gershon-Z, Nussinovith-M, Liquid-core hydrocolloid capsules, Food Hydrocolloids, 10(1): 21-26
[14]何立敏，薛毅珑，黎立，微囊化牛嗜铬细胞移植于脊髓蛛网下镇疼作用的研究，解放军医学杂志[J]，1999，24(4)：241
[15]H.B.Levy, G.Baer, A modified polyriboinodinic-polybocytidlic acid complex that induces interferon in primates, The Journal of infecticus diseases, 1975.10, Vol.132, No.4:434-439
[16]Fan-TSL, Zhang-GS, Chu-FS, An indirect enzyme-linked immunosobent
assay for T-2 toxin in biological fluids, Journal of food protection, 1984, 47(12)964-967
[17]赵亚刚，肿瘤的靶向基因治疗，广州军区广州总医院
[18]Sospedra.p, Physicochemical behavior of polylysine-[HAV-VP3 peptide]constructu-res at the air-water interface, Iangmuir[J],1999,15(15): 5111-5117
[19]徐铮奎，国外大力开发氨基酸新用途，中国化工报[J]，2001．3
[20]Diederich, Anke, Influence of polylydsine on the mputure of negative charged membranes, Langmuir[J],1998, 14(16): 4597-4605
[21]Roebner, u, Polylysine supports electrofusion, Bioelectrochemistry and Bioenergetics[J],1996,39(2): 181-184
[22]刘德伍，李国辉，以多聚赖氨酸为底物培养人表细胞的实验观察，华中医学杂志[J]，1998，Vol．22，No．1：30-31
[23]远藤刚，未来的聚赖氨酸及其在功能性材料方面的应用
[24]腾井正弘，日本对于天然食品防腐剂的开发与应用，翻译：吕绍杰
[25]Fujii-M, Hatakeyama-M, Hiraki-J, Method for treating food to control the growth of yeasts, Patent US5,009,907
[26]Shoii shima et.al. Agric Biol Chem[J], 1981, 45(Ⅱ): 2497-2502
[27]白燕，聚天冬氨酸的合成进展及用途，应用化工[J]，2000，Vol．29，No．3：1-2
[28]Low K C, Synthetic polyaspartic acid and its uses, Polym Mater Sci Eng[J], 1993, 69:253-254
[29]熊蓉春，董雪玲，魏刚，绿色生物高分子聚天冬氨酸的合成及其阻垢性能的研究，工业水处理[J]，Jan．2001，Vol．21 No．1：17-20
[30]扬革，细菌聚-γ谷氨酸的研究，江南大学博士学位论文
[31]南相日，多聚鸟氨酸介导外源基因的直接转化法，大豆科学[J]，Feb．2000， Vol．19 No．1：26-30
[32]周德庆，微生物学，高等教育出版社，1995：220—254
[33]李勇，郑武飞，生物化学与生物物理进展，3：54-56，1986
[34]安晓峰等，D301树脂分离衣康酸的新工艺，现代化工，1994， 11：30-32
[35]贾士儒等，离子交换树脂提取衣康酸的初步研究，天律轻工业学报，1997(7)：22-25
[36]苏拔贤等编，生物化学制备技术，科学出版社(1998)
[37]周加祥，刘铮，生物分离技术与过程研究进展，化工进展，2000，6：38-41
[38]周定，王建龙等，离子交换树脂提取柠檬酸的研究，哈尔滨工业大学学报，1994，26(5)：99-102
[39]吕仪军，离子交换树脂应用进展，四川化工与腐蚀控制，1999，12，16-35
[40]离子交换树脂系列商品手册，天津大学化工厂
[41]Fuijii-M, Hatakeyama-M, Hiraki-J, Method for treating food to control the growth of yeasts, Patent US5,009,907
[42]L．R．斯奈德，J．J．柯克兰著，扬明彪，瞿纯译，现代液相色谱法导论，化学工业部出版社(1980)
[43]上海化工学院等编，化学工程(第2册)，化学工业出版社，178页，(1980)
[44]别尔林著，有机化学实验操作技术，13页，化学工业出版社(1952)
[45]Laurent, T.C., Biochem.J.,9,253(1963)
[46]李良铸，李明晔等编著，最新生化药物制备技术，中国医药技术出版社 (2001)
[47]赵永芳编著，生物化学技术原理及其应用，武汉大学出版社(1994)
[48]蔡武城，李碧羽等，生物化学实验技术教程，上海复旦大学出版社(1983)
[2]Prihardi kahar, toshiharuk iwata, Enhancement of ε -PL production by Streptomyces albulus Strain 410 using pH Control, Journal of bioscience and bioengineefing[J],2001,Vol.91, No.2:190 -194
[3]Shoji shima, Agric Biol Chem[J], 1981,45(Ⅱ): 2497-2502
[4]腾井正弘，公开特许公报，1988，JP63，112，981
[5]平木纯，公开特许公报，1993，JP5，219，925
[6]徐红华，王英东，赵新淮，多聚赖氨酸在食品抑菌方面的研究进展，粮油食品科技[J]，1999，Vol．7，33-34
[7]刘慧，徐红华，王明丽等，聚赖氨酸抑菌性能的研究，东北农业大学学报，Vol．31，No．4：294-298
[8]Shoji Shima, Antimicrobial action of ε -PL, Journal of antibiotics[J], 1984, Vol.37,1449-1455
[9]向智敏，天然食品防腐剂-聚赖氨酸，广州食品工业科技[J]，1993．1
[10]徐红华，刘慧，聚赖氨酸在牛奶保鲜中的应用研究，食品与发酵工业[J]，2000，Vol．26，No．2：33-35
[11]Ckenzie, Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides, Journal of peptide reserch[J],Oct 1999,Vol.54, No.4:311-318
[12]Hao zhen jun, In vitro reaction of penicilloyl and penicillanyl albumin and polylysineconjugates with igE-antibody, Journal of immunological methods[J],28 August,2000, Vol.242, No.1-2:43-51
[13]Nussionovitch-A, Gershon-Z, Nussinovith-M, Liquid-core hydrocolloid capsules, Food Hydrocolloids, 10(1): 21-26
[14]何立敏，薛毅珑，黎立，微囊化牛嗜铬细胞移植于脊髓蛛网下镇疼作用的研究，解放军医学杂志[J]，1999，24(4)：241
[15]H.B.Levy, G.Baer, A modified polyriboinodinic-polybocytidlic acid complex that induces interferon in primates, The Journal of infecticus diseases, 1975.10, Vol.132, No.4:434-439
[16]Fan-TSL, Zhang-GS, Chu-FS, An indirect enzyme-linked immunosobent
assay for T-2 toxin in biological fluids, Journal of food protection, 1984, 47(12)964-967
[17]赵亚刚，肿瘤的靶向基因治疗，广州军区广州总医院
[18]Sospedra.p, Physicochemical behavior of polylysine-[HAV-VP3 peptide]constructu-res at the air-water interface, Iangmuir[J],1999,15(15): 5111-5117
[19]徐铮奎，国外大力开发氨基酸新用途，中国化工报[J]，2001．3
[20]Diederich, Anke, Influence of polylydsine on the mputure of negative charged membranes, Langmuir[J],1998, 14(16): 4597-4605
[21]Roebner, u, Polylysine supports electrofusion, Bioelectrochemistry and Bioenergetics[J],1996,39(2): 181-184
[22]刘德伍，李国辉，以多聚赖氨酸为底物培养人表细胞的实验观察，华中医学杂志[J]，1998，Vol．22，No．1：30-31
[23]远藤刚，未来的聚赖氨酸及其在功能性材料方面的应用
[24]腾井正弘，日本对于天然食品防腐剂的开发与应用，翻译：吕绍杰
[25]Fujii-M, Hatakeyama-M, Hiraki-J, Method for treating food to control the growth of yeasts, Patent US5,009,907
[26]Shoii shima et.al. Agric Biol Chem[J], 1981, 45(Ⅱ): 2497-2502
[27]白燕，聚天冬氨酸的合成进展及用途，应用化工[J]，2000，Vol．29，No．3：1-2
[28]Low K C, Synthetic polyaspartic acid and its uses, Polym Mater Sci Eng[J], 1993, 69:253-254
[29]熊蓉春，董雪玲，魏刚，绿色生物高分子聚天冬氨酸的合成及其阻垢性能的研究，工业水处理[J]，Jan．2001，Vol．21 No．1：17-20
[30]扬革，细菌聚-γ谷氨酸的研究，江南大学博士学位论文
[31]南相日，多聚鸟氨酸介导外源基因的直接转化法，大豆科学[J]，Feb．2000， Vol．19 No．1：26-30
[32]周德庆，微生物学，高等教育出版社，1995：220—254
[33]李勇，郑武飞，生物化学与生物物理进展，3：54-56，1986
[34]安晓峰等，D301树脂分离衣康酸的新工艺，现代化工，1994， 11：30-32
[35]贾士儒等，离子交换树脂提取衣康酸的初步研究，天律轻工业学报，1997(7)：22-25
[36]苏拔贤等编，生物化学制备技术，科学出版社(1998)
[37]周加祥，刘铮，生物分离技术与过程研究进展，化工进展，2000，6：38-41
[38]周定，王建龙等，离子交换树脂提取柠檬酸的研究，哈尔滨工业大学学报，1994，26(5)：99-102
[39]吕仪军，离子交换树脂应用进展，四川化工与腐蚀控制，1999，12，16-35
[40]离子交换树脂系列商品手册，天津大学化工厂
[41]Fuijii-M, Hatakeyama-M, Hiraki-J, Method for treating food to control the growth of yeasts, Patent US5,009,907
[42]L．R．斯奈德，J．J．柯克兰著，扬明彪，瞿纯译，现代液相色谱法导论，化学工业部出版社(1980)
[43]上海化工学院等编，化学工程(第2册)，化学工业出版社，178页，(1980)
[44]别尔林著，有机化学实验操作技术，13页，化学工业出版社(1952)
[45]Laurent, T.C., Biochem.J.,9,253(1963)
[46]李良铸，李明晔等编著，最新生化药物制备技术，中国医药技术出版社 (2001)
[47]赵永芳编著，生物化学技术原理及其应用，武汉大学出版社(1994)
[48]蔡武城，李碧羽等，生物化学实验技术教程，上海复旦大学出版社(1983)