反胶团萃取牛血清白蛋白的研究
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摘要
本文对CTAB(十六烷基三甲基溴化铵)／异辛烷／正戊醇反胶团体系萃取牛血清白蛋白(BSA)的相关性能进行了研究，论文主要分三个方面进行。首先确定了反胶团萃取BSA的较优实验条件；然后研究了反胶团中BSA的反萃取情况；最后一部分考察了CTAB／异辛烷／正戊醇对BSA的特异选择性。
萃取实验主要集中在以下两点。(1)考察了表面活性剂浓度、原料相pH、离子浓度和种类、助溶剂比例、萃取温度、搅拌速率和油水比对牛血清白蛋白萃取率的影响，找到了适合BSA萃取的较优实验条件。实验表明，静电作用力是该萃取过程的主要推动力，随原料相离子浓度增加，萃取率降低，这主要是因为离子在反胶团内核和BSA之间产生了屏蔽效应。适宜比例的正戊醇有助于BSA萃取率的提高。在表面活性剂(CTAB)浓度为0.02-0.04mol／L、pH 9.0-11.0、离子浓度KCl／NaCl 0.05-0.2mol／L、正戊醇比例0.2-0.4、室温、搅拌速率200-300rpm、油水比0.67-1.5时，BSA的萃取率较高，最高可达98％；(2)为进一步的实验，进行了简单的反萃实验，证明BSA反萃后仍保持较好的活性。
BSA的反萃实验主要以萃取实验为依据，考察了反萃条件(反萃水相pH、离子浓度、反萃时油水比、反萃时间)对BSA反萃率的影响。通过实验得到，原料液中BSA浓度越低，其萃取率和反萃率越高。当反萃水相pH接近BSA等电点时，反萃率最高，这主要是由于该萃取过程受静电作用力控制。在条件选择适当的情况下，反萃率可达80％以上。并且通过实验可知，反萃水相离子浓度高时(＞0.4mol／l)，不仅无法提高反萃率，还会引起BSA的变性。
论文的最后通过对α-淀粉酶的萃取实验和BSA、溶菌酶的混合物分离实验，说明在一定实验条件下，CTAB／异辛烷／正戊醇对BSA具有一定的特异选择性，可以根据等电点不同进行目标蛋白的提取和分离。牛血清是一种复杂的混合物，除含有主要成分牛血清白蛋白外，还含有球蛋白、激素、氨基酸、葡萄糖等多种成分，自制的反胶团体系可以从这种牛混合溶液中提取牛血清白蛋白，并能重复利用。
Using BSA as a model protein, the extraction of the protein by reversed micelles system of CTAB/isooctane/1-pentane was studied in this paper. The research is focused on the following three aspects. Firstly, the optimal conditions for extraction of BSA are investigated, and then, back-extraction of BSA in reversed micelles is studied. Finally, BSA is extracted from bovine blood serum according to selectivity of CTAB/isooctane/1-pentane to BSA.
Extraction experiments include the two aspects. (1) Effect of some parameters, such as pH value, the type and concentration of ions in aqueous phase, the content of surfactant, proportion of co-solvent in reversed micelles, temperature, stirring velocity of extraction and ratio of oil to water, on extraction ratio of BSA was examined, and the optimal conditions for extraction of BSA ware obtained. Experimental results show that electrostatic interaction is the major driving force in the extraction process, the extraction ratio of BSA increased remarkably in the pH region higher than 5. For different kinds of ions, usually the extraction ratio of BSA decreased with increasing the ion concentration, such a decrease is due to electrostatic hindrance by the ions. Adding 1-pentane as co-solvent in the reversed micelles is beneficial to the extraction. The optimal conditions are as followings: CTAB 0.02-0.04mol/l, pH 9.0-11.0, ion concentration KCl/NaCl 0.05-0.2mol/l, proportion of co-solvent 0.2-0.4, room tempera
ture, stirring velocity 200-300rpm, ratio of oil to water 0.67-1.5. Under the optimum conditions, the extraction ratio of BSA can reach as high as 98%. (2) Simply back-extraction experiments were done to prove that BSA'S activity is well kept.
According to extraction conditions of BSA, the back-extraction is also investigated, the following parameters were examined, pH of back extraction aqueous phase, ion concentration, ratio of oil to water, back extraction time. According to the experiments, the lower concentration of BSA in the feed is, the higher extraction ratio and back extraction ratio would be obtained. The back extraction ratio can reach more than 80% as pH approaching to BSA'S pi. The main reason is that the process is controlled by electrostatic interaction. Increasing ion concentration is disadvantageous for BSA back extraction.
Extraction of a -amylase and extraction of BSA from a mixture protein solution, proving that CTAB/isooctane/1-pentane has certain selectivity to BSA, target protein can be extracted by difference of their pI. The reversed micelles system also was reused to extraction BSA from bovine blood serum.
萃取实验主要集中在以下两点。(1)考察了表面活性剂浓度、原料相pH、离子浓度和种类、助溶剂比例、萃取温度、搅拌速率和油水比对牛血清白蛋白萃取率的影响，找到了适合BSA萃取的较优实验条件。实验表明，静电作用力是该萃取过程的主要推动力，随原料相离子浓度增加，萃取率降低，这主要是因为离子在反胶团内核和BSA之间产生了屏蔽效应。适宜比例的正戊醇有助于BSA萃取率的提高。在表面活性剂(CTAB)浓度为0.02-0.04mol／L、pH 9.0-11.0、离子浓度KCl／NaCl 0.05-0.2mol／L、正戊醇比例0.2-0.4、室温、搅拌速率200-300rpm、油水比0.67-1.5时，BSA的萃取率较高，最高可达98％；(2)为进一步的实验，进行了简单的反萃实验，证明BSA反萃后仍保持较好的活性。
BSA的反萃实验主要以萃取实验为依据，考察了反萃条件(反萃水相pH、离子浓度、反萃时油水比、反萃时间)对BSA反萃率的影响。通过实验得到，原料液中BSA浓度越低，其萃取率和反萃率越高。当反萃水相pH接近BSA等电点时，反萃率最高，这主要是由于该萃取过程受静电作用力控制。在条件选择适当的情况下，反萃率可达80％以上。并且通过实验可知，反萃水相离子浓度高时(＞0.4mol／l)，不仅无法提高反萃率，还会引起BSA的变性。
论文的最后通过对α-淀粉酶的萃取实验和BSA、溶菌酶的混合物分离实验，说明在一定实验条件下，CTAB／异辛烷／正戊醇对BSA具有一定的特异选择性，可以根据等电点不同进行目标蛋白的提取和分离。牛血清是一种复杂的混合物，除含有主要成分牛血清白蛋白外，还含有球蛋白、激素、氨基酸、葡萄糖等多种成分，自制的反胶团体系可以从这种牛混合溶液中提取牛血清白蛋白，并能重复利用。
Using BSA as a model protein, the extraction of the protein by reversed micelles system of CTAB/isooctane/1-pentane was studied in this paper. The research is focused on the following three aspects. Firstly, the optimal conditions for extraction of BSA are investigated, and then, back-extraction of BSA in reversed micelles is studied. Finally, BSA is extracted from bovine blood serum according to selectivity of CTAB/isooctane/1-pentane to BSA.
Extraction experiments include the two aspects. (1) Effect of some parameters, such as pH value, the type and concentration of ions in aqueous phase, the content of surfactant, proportion of co-solvent in reversed micelles, temperature, stirring velocity of extraction and ratio of oil to water, on extraction ratio of BSA was examined, and the optimal conditions for extraction of BSA ware obtained. Experimental results show that electrostatic interaction is the major driving force in the extraction process, the extraction ratio of BSA increased remarkably in the pH region higher than 5. For different kinds of ions, usually the extraction ratio of BSA decreased with increasing the ion concentration, such a decrease is due to electrostatic hindrance by the ions. Adding 1-pentane as co-solvent in the reversed micelles is beneficial to the extraction. The optimal conditions are as followings: CTAB 0.02-0.04mol/l, pH 9.0-11.0, ion concentration KCl/NaCl 0.05-0.2mol/l, proportion of co-solvent 0.2-0.4, room tempera
ture, stirring velocity 200-300rpm, ratio of oil to water 0.67-1.5. Under the optimum conditions, the extraction ratio of BSA can reach as high as 98%. (2) Simply back-extraction experiments were done to prove that BSA'S activity is well kept.
According to extraction conditions of BSA, the back-extraction is also investigated, the following parameters were examined, pH of back extraction aqueous phase, ion concentration, ratio of oil to water, back extraction time. According to the experiments, the lower concentration of BSA in the feed is, the higher extraction ratio and back extraction ratio would be obtained. The back extraction ratio can reach more than 80% as pH approaching to BSA'S pi. The main reason is that the process is controlled by electrostatic interaction. Increasing ion concentration is disadvantageous for BSA back extraction.
Extraction of a -amylase and extraction of BSA from a mixture protein solution, proving that CTAB/isooctane/1-pentane has certain selectivity to BSA, target protein can be extracted by difference of their pI. The reversed micelles system also was reused to extraction BSA from bovine blood serum.
引文
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[2] 吴金川，何志敏．反胶团萃取蛋白质技术．化学工业与工程，1997，14(3)，50—54．
[3] B.A. Andrews, D.L. Pyle,, and J.A. Asenjo. The effect of pH and ionic strength on the partitioning of four proteins in reversed micelles system. Biotech and Bioeng. 1994(43), 1052—1058.
[4] Zhang T, Liu H, Chen J. Selective affinity separation of Yeast Alchol Dehydrogenase by reversed micelles with unbound triazine dye. Chinese J. Chem. Eng, 2001, 9(3), 314—318.
[5] Keri G A, Hustedt H. Exraction of emzymes by reversed micelles. Chem. Eng. Sci, 1992, 47—99.
[6] Kuboi R, Li D R, Yamada Y, Komasawa I. Effect of salt addition on hydrophobocity of reversed micelles partition system. Kagaku Kogaku Runbunshu, 1992, 18—66.
[7] 孙彦，史清洪，赵黎明．反胶团萃取蛋白质的平衡模型：静电和疏水作用的贡献．化工学报，2003，54(6)，796—801．
[8] Goklen, K. E, Hatton, T. A. Protein extration using reversed micelles. Biotechno[J]. Prog, 1985, 1, 69—74.
[9] Yasuhiro Nishii , et al. Transport behavior of protein in bulk liquid membrane using reversed micelles. Journal of Membrane Science. 2002, 195, 11—21.
[10] Giordana Marcozzi. Protein extraction by reversed micelles : a study of the factors affecting the forward and backword transfer of α-chymotrypsin and its activity. Biotechnol and Bioeng. 1991(38), 1239—1246.
[11] B.A. Andrews, D.L. Pyle,, and J.A. Asenjo. The effect of pH and ionic strength on the partitioning of four proteins in reversed micelles system. Biotech and Bioeng. 1994(43), 1052—1058.
[12] Koichiro Shiomori, et al. Extraction of proteins and water with Bis(2-ethylhexyl) Suifosuccinate/long Chain Alkyl Amines mixed micelles system. Sep Sci and Eng of Japan. 1999(32), 177—183.
[13] 严勇朝等，TRPO-AOT反胶团体系萃取牛血红蛋白的研究．生物工程学报，1997(13)，430—432．
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