丁基琼脂糖疏水介质的制备及其表征
文献类型：学位论文
| 作者 | 孟庆强 |
| 学位类别 | 硕士 |
| 答辩日期 | 2004 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 马光辉 |
| 关键词 | 疏水介质 丁基琼脂糖 吸附 分离 动力学 |
| 其他题名 | Synthesis and Characterization of Butyl-agarose Media for Hydrophobic Interaction Chromatography(HIC) |
| 学位专业 | 生物化工 |
| 中文摘要 | 疏水层析是广泛应用于生物大分子分离纯化的技术，丁基琼脂糖（butyl-agarose） 介质是常用的疏水分离介质。本文制备了丁基琼脂糖介质，考察了其性能，并对“疏水 吸附动力学与平衡过程的理论进行了初步探讨。首先以国产快流速琼脂糖凝胶为基质，用活化偶联法和直接偶联法制备了丁基琼 脂糖介质。活化偶联法是指先活化琼脂糖凝胶，再将活化的琼脂糖凝胶与丁醇反应得 到丁基琼脂糖介质。在琼脂糖凝胶的活化中，选用环氧氯丙烷作活化剂，考察了反应 时间、环氧氯丙烷加入量、氢氧化钠浓度及加入量、反应温度等因素对产品环氧基密 度的影响。然后用环氧氯丙烷活化的琼脂糖凝胶与丁醇反应制备丁基琼脂糖介质，考 察了氢氧化钠和三氟化硼乙醚两种催化剂对偶联反应的催化效果。直接偶联法是指将 琼脂糖凝胶白球直接和丁基缩水甘油醚反应制备丁基琼脂糖介质。考察了直接偶联法 中催化剂三氟化硼加入量、反应时间和缩水甘油醚加入量的影响。最终得到了两种产 品：环氧活化的琼脂糖凝胶和丁基琼脂糖介质。制备实验结果表明：环氧氯丙烷对琼 脂糖的活化效果较好，环氧基密度可达0.68 mmol/g-dry-gel；三氟化硼乙醚适合作活化 偶联法的催化剂，而氢氧化钠不适合；直接偶联法是制备丁基琼脂糖介质的理想方法。考察了研制丁基琼脂糖介质的机械稳定性、吸附和分离性能进行。自制介质的压 强一流速曲线基本为直线，介质在较高压强下形变很小，机械稳定性好，耐压性强， 适用于大规模纯化生物大分子。自制疏水介质对牛血清白蛋白（BSA）和溶菌酶（lysozyme）饱合吸附量较高。硫酸按浓度为1.6mol／L-2.0 mol／L时，疏水介质对BSA 的吸附符合Langmtlir等温吸附方程，表明在此条件下，BSA在疏水介质上的吸附为 单分子层吸附。硫酸按浓度为1．4mol／L，溶菌酶浓度低于0.035μmol/ml时，疏水介 质对溶菌酶的吸附符合Freundlich等温吸附方程；硫酸按浓度为1.6mof／L，疏水介质 对溶菌酶的吸附既不符合Freundlich等温吸附方程也不符合Langmuir等温吸附方程。 对三种疏水性不同的蛋白质：BSA、卵清蛋白。（valbumin）和核糖核酸酶（RNase） 的混合体系进行了分离实验，得到了满意的分离效果。结果表明自制丁基琼脂糖介质 的性能与进口同类产品接近，具有广阔的使用前景。本文对疏水介质与蛋白质发生疏水相互作用的动力学和平衡过程作了基本假设，洲以BSA为模型蛋白质进行了验证。实验表明，在硫酸按浓度为1.6mol/L-2.0mol／L 时，BSA在疏水介质上的吸附动力学和平衡过程与假设基本相符，等温吸附线符合 Langmuir等温吸附方程。本研究以国产的快流速琼脂糖凝胶为原料，合成了用于生物大分子分离纯化的丁 基疏水介质，性能与进口同类介质接近，为疏水介质的国产化打下一定基础。 |
| 英文摘要 | Hydrophobia interaction chromatography(HIC) is widely used in protein separation and purification, and butyl-agarose is one of the most important HIC media. In this dissertation, the preparation and characterization of butyl-agarose media were investigated and the basic hypothesis of adsorption kinetics and equilibrium process of proteins in HIC was further discussed. Activated coupling and direct coupling were investigated. Activated coupling was carried out by activating the -OH of agarose beads by epichlorohydrin(ECH) at the first step. The factors affecting density of epoxy group on ECH-activated agarose such as reaction time, amount of ECH, concentration and amount of NaOH, reaction temperature were investigated. Then, the ECH-activated agarose was allowed to react with butyl alcohol to prepare butyl-agarose at the second step, the efficiency of two catalyst (BF3?Et2O and NaOH) were studied. In this method epoxy group was successfully induced on agarose media, but butyl group can not be induced by using NaOH as catalyst and only a small amount of butyl group was attached by using BF3?Et2O as catalyst. Direct couling was performed by butyl glycidyl ether which was reacted directly with agarose to prepare butyl-agarose. The factors affecting density of butyl group on butyl-agarose such as reaction time, amount of catalyst and butyl glycidyl ether were investigated. In this method, 40umol/ml butyl-group was successfully attached on agarose media. The mechanical stability, adsorption capability and separation performance of butyl-agarose which was homemade by direct coupling were also characterized. The flow rate-pressure curve of homemade butyl-agrose was approximately linear (0~0.1MPa)5 it implied that it had excellent mechanical stability and resistant to high pressure. Homemade butyl-agarose have high adsorption capability for BSA and lysozyme. When the concentration of (NHSOwas in the range of l.6mol/L ~2.0mol/L, the adsorption of BSA on butyl-agarose fit with Langmuir isotherm well. When the concentration of 2SO4 was 1.4mol/L and the concentration of lysozyme below 0.035|a,mol/ml, the adsorption oflysozyme on butyl-agarose fit with Freundlich isotherm well. However, when the concentration of (NHVhSO., was 1.6mol/L, the adsorption of lysozyme on butyl-agarose fit with neither Freundlich isotherm nor Langmuir isotherm. Furthermore, BSA, RNase A and ovalbumin were successfully separated by homemade butyl-agarose. The study demonstrated that homemade butyl-agarose was promising media and was expected to be widely used in future. The basic hypothesis of adsorption kinetics and equilibrium process of proteins in HIC was established. The experiment data showed that adsorption kinetics and equilibrium process of BSA in HIC were consistent with the basic hypothesis, the adsorption isotherm fitted with Langmuir adsorption equation well. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 81 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1425]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 孟庆强. 丁基琼脂糖疏水介质的制备及其表征[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004. |
入库方式： OAI收割
来源：过程工程研究所
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