以壳聚糖为基质的天然两性膜的制备及其在蛋白质分离中的应用
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摘要
膜色谱技术是液相色谱和膜分离相结合的一种新技术,融合了二者之长,具有快速、高效、高选择性、易于放大等特点,能满足生物大分子高效分离与纯化的需要。多孔膜色谱最主要的优点是能够在相对较短的操作时间内,在低压降下获得高流速。由于膜色谱能够解决传统色谱柱的部分缺点,近年来在生物大分子的分离与纯化中已日益受到人们的重视。
在膜色谱技术中具有多种不同的分离机理,离子交换便是其中的一种。离子交换膜色谱主要是利用膜介质表面的离子交换基团与目标分子之间的离子交换作用进行分离的。两性膜是一种既带有碱性基团又带有酸性基团的特殊的荷电膜,其表观电荷具有pH可调节性,即表面电荷随着外部缓冲溶液pH的变化而变化,利用这一特性,可以用来分离不同等电点的蛋白质。
随着公众健康和环境意识的增强以及对于排放物越来越严格的环境标准,人们纷纷把目光投向了可持续发展的天然高分子材料,希望其可以代替合成高分子材料。纤维素、淀粉、甲壳素和木质素等聚多糖是最为广泛应用的天然高分子之一。商品化的聚多糖材料多为中性和酸性,只有甲壳素及其衍生物壳聚糖是碱性多糖。壳聚糖具备聚阳离子化合物性质和良好的生物相容性,分子链上存在羟基和氨基,通过化学改性可引入不同基团,也可以被用来固定具有生物活性的物质。
本文以壳聚糖为膜基质材料,通过与自制的O-羧甲基壳聚糖和羧甲基纤维素共混,制备出壳聚糖/羧甲基壳聚糖(CS/CMCS)和壳聚糖/羧甲基纤维素(CS/CMC)两种天然高分子两性荷电膜。同时,我们将这些天然高分子两性膜对不同等电点的蛋白质进行静态吸附和动态吸附的研究,并且根据等电点不同成功分离了蛋白质混合溶液。
通过壳聚糖溶液和羧甲基纤维素溶液共混,以硅胶作为制孔剂,戊二醛和环氧氯丙烷作为交联剂,制备了CS/CMC共混膜。吸附动力学研究表明对于溶菌酶而言,经过5个小时吸附达到平衡;而卵清白蛋白则需要6个小时达到吸附平衡。由于CS/CMC共混膜和蛋白质都是两性物质,表面带电荷情况均会随着环境pH值而变化,从而导致在不同pH值吸附条件下蛋白质在膜上吸附量的变化。当pH值为9.2时,溶菌酶达到最大吸附值;pH值为4.8时,卵清白蛋白达到最大吸附值。根据不同等电点蛋白质在相同pH值溶液中的吸附差异,我们从溶菌酶和卵清白蛋白的混合溶液中分别对这两种蛋白质进行了分离,并取得良好的效果。通过多次吸附.脱附实验后,我们发现该膜可以重复利用,并在适合的条件下能将绝大部分吸附物质脱附下来。
制备O-羧甲基壳聚糖的目的是为了尽量保留壳聚糖上的氨基不被反应,从而可以与壳聚糖上的氨基进行交联,提高CS/CMCS共混膜中CMCS的含量。将CMCS与壳聚糖溶液共混,硅胶为制孔剂,戊二醛和环氧氯丙烷为交联剂,制得CS/CMCS共混膜。CS/CMCS共混膜在适当的条件下能对溶菌酶、卵清白蛋白产生有效的吸附作用,经过动力学方程拟合,Lagergren二级方程可以较好地描述整个吸附过程。对于等电点不同的溶菌酶和卵清白蛋白,它们在CS/CMCS共混膜上吸附最大值分别出现在pH值为5.2和8.0。Langmuir模型和Freundlich模型都能够用来描述溶菌酶和卵清白蛋白的吸附等温线。CS/CMCS共混膜有良好的可重复利用性。依据不同等电点蛋白质在相同pH值溶液中在共混膜上吸附的差异来实现溶菌酶/卵清白蛋白双组分混合溶液的分离和蛋清液中溶菌酶的分离。
本文还研究了壳聚糖/羧甲基壳聚糖共混膜在动态吸附条件下对溶菌酶和卵清白蛋白吸附和分离的应用。在蛋白质原始液浓度均为0.5 mg/mL,流速2 mL/min(95.5 L/h·m~2)的条件下溶菌酶的动态吸附能力为9.19 mg/mL,卵清白蛋白的动态吸附能力为1.24 mg/mL。CS/CMCS共混膜在溶菌酶动态吸附-脱附循环中能被多次使用,且脱附率均在90%左右。由于在卵清白蛋白最佳吸附pH值范围内CS/CMCS膜都存在着较为严重的溶胀现象,这对共混膜在卵清白蛋白动态吸附中的应用造成一定影响,相对而言得到的结果不如溶菌酶的理想,但对研究CS/CMCS共混膜动态吸附卵清白蛋白的过程仍具有相当的价值。根据脱附条件的不同,共混膜对卵清白蛋白动态吸附-脱附过程中的脱附率约在45%～75%之间。在适当的条件下共混膜能从溶菌酶和卵清白蛋白的混合溶液中把两个组分完全分开,分别得到各自单一组分的溶液。
Nowadays more attention has been paid in membrane chromatography systems that function as short, wide chromatography columns, as some of the major problems associated with packed bed chromatography can be solved by using macroporous membranes as chromatography media. The main advantage of macroporous membrane chromatography is the high flow rate through the membranes at low pressure drops and relatively short operation time.
Different types of interactions have been utilized in membrane chromatography for bioseparation. One of them is ion-exchange, in which the separation can be achieved based on the electrostatic interaction between the surface charges of biomacromolecules and the charged groups on membranes. Amphoteric ion-exchange membranes contain both weak acidic (negative charge) groups and weak basic (positive charge) groups that are randomly distributed within the membrane matrix, and the sign of the net charge of the membrane can be controlled by environmental pH, resulting in some characteristic properties that can not be shown in a single-charged material.
In view of growing public health and environmental awareness accompanied by an increasing number of ever stricter environmental regulations on discharged wastes, attention has been focused on the use of natural polymers from renewable resources as alternative to synthetic polymers. One kind of the most widespread natural polymers is polysaccharides, such as cellulose, starch, chitin and lignin. Among the commercially available polysaccharides that are prevailingly neutral or acidic, but chitin and its primary derivative chitosan (CS) are special in that they are basic. CS has both reactive amino and hydroxyl groups with common characteristics of polycation nature and good biocompatibility. These two functional groups offer several possibilities for derivatization and immobilization of biologically active species.
In the present work, an amphoteric membrane matrix was prepared by a simple solution blending method of natural polyelectrolyte CS and carboxymethyl cellulose (CMC) or carboxymethyl chitosan (CMCS). These natural amphoteric membranes were studied in static adsorption and dynamic adsorption of proteins with different pIs. Both lysozyme and ovalbumin could be effectively separated from their binary mixture according to the difference between their pI.
The CS/CMC blend membrane was prepared through mixing the CS and sodium CMC solution. Silica particles were used to generate the pores in the membranes. Glutaraldehyde and epichlorohydrin were used as crosslinking agents. The time for lysozyme to reach adsorption equilibrium was about 5 h and 6 h for ovalbumin. Both the protein and the CS/CMC blend membrane are amphoteric, so the charges on their surface vary according to the environmental pH, and as a result the adsorption capacity of the protein on membrane is different at different pH. The maximum adsorption of lysozyme and ovalbumin was found at pH 9.2 and 4.8, respectively. According to the different adsorption capacity between lysozyme and ovalbumin in the solution with same pH value, lysozyme and ovalbumin could be effectively separated from their binary mixture. Four cycles of adsorption-desorption were performed. The result shows the high desorption ratio and good reusability of the membrane.
O-carboxymethyl chitosan was prepared in order to keep more amino groups of chitosan so the amino groups of chitosan and O-carboxymethyl chitosan could be cross-linked to increase the content of CMCS in CS/CMCS blend membrane. The preparation of CS/CMCS blend membrane was similar to that of CS/CMC blend membrane. CS/CMCS blend membranes could adsorb lysozyme and ovalbumin effectively under certain circumstance and Lagergren second-order kinetics model described the data better. The adsorption capacity varied with the change of environmental pH, and the maximum adsorption of ovalbumin and lysozyme was found at pH 5.2 and 8.0, respectively. Both Langmuir model and Freundlich model were suitable for describing the biosorption equilibrium of both proteins in the concentration ranges we studied. The stability and repeatability of the CS/CMCS blend membrane were found quite well after several adsorption-desorption cycles, which indicated the good reusability of the membrane. These membranes also could be effectively used to separate lysozyme-ovalbumin binary mixture only by changing the pH of the feed and the desorption solution. In addition, lysozyme could be separated from fresh chicken egg white by CS/CMCS blend membrane.
The dynamic adsorption behavior of lysozyme and ovalbumin on CS/CMCS blend membrane was also investigated. The dynamic adsorption of individual protein on blend membranes was carried out by loading 0.5 mg/mL protein solution at a flow rate of 2 mL/min. The dynamic adsorption capacity for lysozyme was 9.19 mg/mL and for ovalbumin was 1.24 mg/mL. CS/CMCS blend membrane could be used repeatedly in the dynamic adsorption-desorption process of lysozyme and the desorption ratios were all about 90%. The dynamic adsorption behavior of ovalbumin on CS/CMCS blend membrane was affected by the swelling of membrane when it was used in the favorable pH range for ovalbumin adsorption. Thus the results for ovalbumin were not so satisfied compared with the results for lysozyme, but these data were still instructive for the further study. The desorption ratios for ovalbumin varied from 45% to 75% according to the desorption condition. Under proper condition the CS/CMCS blend membrane could separate each protein from lysozyme-ovalbumin binary mixture.
在膜色谱技术中具有多种不同的分离机理,离子交换便是其中的一种。离子交换膜色谱主要是利用膜介质表面的离子交换基团与目标分子之间的离子交换作用进行分离的。两性膜是一种既带有碱性基团又带有酸性基团的特殊的荷电膜,其表观电荷具有pH可调节性,即表面电荷随着外部缓冲溶液pH的变化而变化,利用这一特性,可以用来分离不同等电点的蛋白质。
随着公众健康和环境意识的增强以及对于排放物越来越严格的环境标准,人们纷纷把目光投向了可持续发展的天然高分子材料,希望其可以代替合成高分子材料。纤维素、淀粉、甲壳素和木质素等聚多糖是最为广泛应用的天然高分子之一。商品化的聚多糖材料多为中性和酸性,只有甲壳素及其衍生物壳聚糖是碱性多糖。壳聚糖具备聚阳离子化合物性质和良好的生物相容性,分子链上存在羟基和氨基,通过化学改性可引入不同基团,也可以被用来固定具有生物活性的物质。
本文以壳聚糖为膜基质材料,通过与自制的O-羧甲基壳聚糖和羧甲基纤维素共混,制备出壳聚糖/羧甲基壳聚糖(CS/CMCS)和壳聚糖/羧甲基纤维素(CS/CMC)两种天然高分子两性荷电膜。同时,我们将这些天然高分子两性膜对不同等电点的蛋白质进行静态吸附和动态吸附的研究,并且根据等电点不同成功分离了蛋白质混合溶液。
通过壳聚糖溶液和羧甲基纤维素溶液共混,以硅胶作为制孔剂,戊二醛和环氧氯丙烷作为交联剂,制备了CS/CMC共混膜。吸附动力学研究表明对于溶菌酶而言,经过5个小时吸附达到平衡;而卵清白蛋白则需要6个小时达到吸附平衡。由于CS/CMC共混膜和蛋白质都是两性物质,表面带电荷情况均会随着环境pH值而变化,从而导致在不同pH值吸附条件下蛋白质在膜上吸附量的变化。当pH值为9.2时,溶菌酶达到最大吸附值;pH值为4.8时,卵清白蛋白达到最大吸附值。根据不同等电点蛋白质在相同pH值溶液中的吸附差异,我们从溶菌酶和卵清白蛋白的混合溶液中分别对这两种蛋白质进行了分离,并取得良好的效果。通过多次吸附.脱附实验后,我们发现该膜可以重复利用,并在适合的条件下能将绝大部分吸附物质脱附下来。
制备O-羧甲基壳聚糖的目的是为了尽量保留壳聚糖上的氨基不被反应,从而可以与壳聚糖上的氨基进行交联,提高CS/CMCS共混膜中CMCS的含量。将CMCS与壳聚糖溶液共混,硅胶为制孔剂,戊二醛和环氧氯丙烷为交联剂,制得CS/CMCS共混膜。CS/CMCS共混膜在适当的条件下能对溶菌酶、卵清白蛋白产生有效的吸附作用,经过动力学方程拟合,Lagergren二级方程可以较好地描述整个吸附过程。对于等电点不同的溶菌酶和卵清白蛋白,它们在CS/CMCS共混膜上吸附最大值分别出现在pH值为5.2和8.0。Langmuir模型和Freundlich模型都能够用来描述溶菌酶和卵清白蛋白的吸附等温线。CS/CMCS共混膜有良好的可重复利用性。依据不同等电点蛋白质在相同pH值溶液中在共混膜上吸附的差异来实现溶菌酶/卵清白蛋白双组分混合溶液的分离和蛋清液中溶菌酶的分离。
本文还研究了壳聚糖/羧甲基壳聚糖共混膜在动态吸附条件下对溶菌酶和卵清白蛋白吸附和分离的应用。在蛋白质原始液浓度均为0.5 mg/mL,流速2 mL/min(95.5 L/h·m~2)的条件下溶菌酶的动态吸附能力为9.19 mg/mL,卵清白蛋白的动态吸附能力为1.24 mg/mL。CS/CMCS共混膜在溶菌酶动态吸附-脱附循环中能被多次使用,且脱附率均在90%左右。由于在卵清白蛋白最佳吸附pH值范围内CS/CMCS膜都存在着较为严重的溶胀现象,这对共混膜在卵清白蛋白动态吸附中的应用造成一定影响,相对而言得到的结果不如溶菌酶的理想,但对研究CS/CMCS共混膜动态吸附卵清白蛋白的过程仍具有相当的价值。根据脱附条件的不同,共混膜对卵清白蛋白动态吸附-脱附过程中的脱附率约在45%～75%之间。在适当的条件下共混膜能从溶菌酶和卵清白蛋白的混合溶液中把两个组分完全分开,分别得到各自单一组分的溶液。
Nowadays more attention has been paid in membrane chromatography systems that function as short, wide chromatography columns, as some of the major problems associated with packed bed chromatography can be solved by using macroporous membranes as chromatography media. The main advantage of macroporous membrane chromatography is the high flow rate through the membranes at low pressure drops and relatively short operation time.
Different types of interactions have been utilized in membrane chromatography for bioseparation. One of them is ion-exchange, in which the separation can be achieved based on the electrostatic interaction between the surface charges of biomacromolecules and the charged groups on membranes. Amphoteric ion-exchange membranes contain both weak acidic (negative charge) groups and weak basic (positive charge) groups that are randomly distributed within the membrane matrix, and the sign of the net charge of the membrane can be controlled by environmental pH, resulting in some characteristic properties that can not be shown in a single-charged material.
In view of growing public health and environmental awareness accompanied by an increasing number of ever stricter environmental regulations on discharged wastes, attention has been focused on the use of natural polymers from renewable resources as alternative to synthetic polymers. One kind of the most widespread natural polymers is polysaccharides, such as cellulose, starch, chitin and lignin. Among the commercially available polysaccharides that are prevailingly neutral or acidic, but chitin and its primary derivative chitosan (CS) are special in that they are basic. CS has both reactive amino and hydroxyl groups with common characteristics of polycation nature and good biocompatibility. These two functional groups offer several possibilities for derivatization and immobilization of biologically active species.
In the present work, an amphoteric membrane matrix was prepared by a simple solution blending method of natural polyelectrolyte CS and carboxymethyl cellulose (CMC) or carboxymethyl chitosan (CMCS). These natural amphoteric membranes were studied in static adsorption and dynamic adsorption of proteins with different pIs. Both lysozyme and ovalbumin could be effectively separated from their binary mixture according to the difference between their pI.
The CS/CMC blend membrane was prepared through mixing the CS and sodium CMC solution. Silica particles were used to generate the pores in the membranes. Glutaraldehyde and epichlorohydrin were used as crosslinking agents. The time for lysozyme to reach adsorption equilibrium was about 5 h and 6 h for ovalbumin. Both the protein and the CS/CMC blend membrane are amphoteric, so the charges on their surface vary according to the environmental pH, and as a result the adsorption capacity of the protein on membrane is different at different pH. The maximum adsorption of lysozyme and ovalbumin was found at pH 9.2 and 4.8, respectively. According to the different adsorption capacity between lysozyme and ovalbumin in the solution with same pH value, lysozyme and ovalbumin could be effectively separated from their binary mixture. Four cycles of adsorption-desorption were performed. The result shows the high desorption ratio and good reusability of the membrane.
O-carboxymethyl chitosan was prepared in order to keep more amino groups of chitosan so the amino groups of chitosan and O-carboxymethyl chitosan could be cross-linked to increase the content of CMCS in CS/CMCS blend membrane. The preparation of CS/CMCS blend membrane was similar to that of CS/CMC blend membrane. CS/CMCS blend membranes could adsorb lysozyme and ovalbumin effectively under certain circumstance and Lagergren second-order kinetics model described the data better. The adsorption capacity varied with the change of environmental pH, and the maximum adsorption of ovalbumin and lysozyme was found at pH 5.2 and 8.0, respectively. Both Langmuir model and Freundlich model were suitable for describing the biosorption equilibrium of both proteins in the concentration ranges we studied. The stability and repeatability of the CS/CMCS blend membrane were found quite well after several adsorption-desorption cycles, which indicated the good reusability of the membrane. These membranes also could be effectively used to separate lysozyme-ovalbumin binary mixture only by changing the pH of the feed and the desorption solution. In addition, lysozyme could be separated from fresh chicken egg white by CS/CMCS blend membrane.
The dynamic adsorption behavior of lysozyme and ovalbumin on CS/CMCS blend membrane was also investigated. The dynamic adsorption of individual protein on blend membranes was carried out by loading 0.5 mg/mL protein solution at a flow rate of 2 mL/min. The dynamic adsorption capacity for lysozyme was 9.19 mg/mL and for ovalbumin was 1.24 mg/mL. CS/CMCS blend membrane could be used repeatedly in the dynamic adsorption-desorption process of lysozyme and the desorption ratios were all about 90%. The dynamic adsorption behavior of ovalbumin on CS/CMCS blend membrane was affected by the swelling of membrane when it was used in the favorable pH range for ovalbumin adsorption. Thus the results for ovalbumin were not so satisfied compared with the results for lysozyme, but these data were still instructive for the further study. The desorption ratios for ovalbumin varied from 45% to 75% according to the desorption condition. Under proper condition the CS/CMCS blend membrane could separate each protein from lysozyme-ovalbumin binary mixture.
引文
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