超顺磁性聚醋酸乙烯酯微球亲和分离纳豆激酶
文献类型：学位论文
| 作者 | 孔鹏 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-05-16 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 刘会洲 ; 谢渝春 |
| 关键词 | 纳豆激酶 磁性PVA微球 亲和分离 吸附动力学 |
| 其他题名 | Research on Superparamagnetic Polyvinyl Acetate Microspheres for Affinity Separation of Nattokinase |
| 学位专业 | 化学工程 |
| 中文摘要 | 血栓疾病严重危害人类的生命与健康，纳豆激酶作为一种潜在的新型口服溶栓药物备受关注。分离纯化技术是限制其大规模生产的瓶颈问题。本文通过改进悬浮聚合法制备了磁性聚醋酸乙烯酯微球，经水解、氨基化修饰，以戊二醛作间隔臂偶联配基对氨基苯甲脒，制得磁性PVA亲和微球，并用于纳豆激酶粗酶液的分离纯化，通过SEM, XRD, FT-IR, VSM对所合成的微球进行了表征。研究了纳豆激酶的酶活稳定性、吸附平衡、吸附和解吸动力学、酶的纯度和酶活回收。研究内容主要包括以下三个方面：（1）采用化学共沉淀法制备表面包覆油酸的磁性Fe3O4凝胶，采用悬浮聚合法合成了磁性聚醋酸乙烯酯（PVAc）微球，并考察了不同搅拌速率下，其形貌和粒径大小的不同。在800 r/min时，制备的磁性微球的平均粒径为3.3 μm，产物具有超顺磁性。（2）通过磁性聚醋酸乙烯酯微球醇解得到磁性聚乙烯醇微球，然后通过氯乙胺亲核取代使其表面氨基化，最后以戊二醛作为间隔臂接枝对氨基苯甲脒亲和配基，制得适合纳豆激酶分离的磁性亲和载体。其比饱和磁化强度为32.4 emu/g，具有超顺磁性，易于用外加磁场分离。采用在293 nm处测定接枝反应前后对氨基苯甲脒含量的变化来测定表面接枝亲和配基的量，计算得到磁性微球表面接枝配基含量为1.51 mmol/g。（3）使用固定化配基对氨基苯甲脒的亲和微球对纳豆激酶浸提液进行了纯化，每50 mg磁性亲和微球能分离纳豆激酶12.3 FU。微球表面偶联丰富的配基，吸附量明显增加。磁性微球具有较高的比饱和磁化强度，40 min即可达到吸附平衡。在1mol/L NaCl溶液中，15 min内完成解吸。 酶活回收率为73.4 %， 纯化因子为10.3。纳豆激酶纯度经电泳分析为一明显条带，分子量为28 kDa。 |
| 英文摘要 | Thrombotic diseases seriously endanger human life and health. Nattokinase as a potential new oral thrombolytic drug has received increasing concerns. Separation and purification technologies are bottlenecks to limit its large-scale production. In order to solve this problem, micro-size magnetic poly-vinyl acetate microspheres were prepared by a modification suspension polymerization. The magnetic beads were further modified with hydrolysis and aminization reaction, and magnetic affinity microspheres suitable for nattokinase purification were obtained through covalent immobilization p-aminobenzamidine to the amino-modified magnetic beads by the glutaraldehyde method. The properties of the magnetic beads were examined by SEM, XRD, FT-IR and VSM. Nattokinase activity stability with temperature and pH, adsorption equilibrium, adsorption and desorption kinetics, enzyme purity and activity recovery were also investigated.In this work，the main results are as follows: (1)Fe3O4 gel coated with oleic acid was prepared by chemical coprecipitation. Magnetic poly(vinyl acetate) (PVAc) microspheres were prepared by a modification suspension polymerization. Morphology and particle-size distribution were studied under different stirring rate. At 800 r/min, the average particle size of prepared magnetic microspheres is 3.3 μm with superparamagnetism. (2)Magnetic poly-vinyl alcohol microspheres were prepared by alcoholysis of PVAc microspheres on the surface, and then further modified with hydrolysis and aminization reaction.Finally covalent immobilization p-aminobenzamidine to the amino-modified magnetic beads with glutaraldehyde as a spacer. These magnetic affinity carriers are especially suitable for the separation of nattokinase. The saturation magnetization of the superparamagnetic affinity carriers is 32.4 emu/g, which is easy to separate with external magnetic field. The change of p-aminobenzamidine is determined by measuring ultraviolet absorption at 293 nm before and after grafting reaction. The amount of the ligand grafting to magnetic microspheres is calculated by 1.51 mmol/g. (3)The crude nattokinase extracted by deionized water was purified with microspheres immobilized p-aminobenzamidine. The purification capacity could reach 12.3 FU/50 mg. Adsorption capacity is significantly increased, as more ligands coupled. It took only 40 mins to reach adsorption equilibrium and less than 15 mins to achieve desorption equilibrium. The purification factor and the recovery of the enzyme activity were found to be 10.3 and 73.4 %, respectively. The purified nattokinase gave a single sharp band on SDS-PAGE. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1865]  |
| 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 | 孔鹏. 超顺磁性聚醋酸乙烯酯微球亲和分离纳豆激酶[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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