磁性亲和载体的制备及漆酶分离纯化研究
文献类型：学位论文
| 作者 | 刘兴华 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-06-04 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 刘会洲 |
| 关键词 | PEO-PPO-PEO 磁性纳米颗粒 吸附 亲和分离 漆酶分离纯化 |
| 其他题名 | Preparation of Magnetic Affinity Carriers and Application in Separation and Purification of Laccase |
| 学位专业 | 环境工程 |
| 中文摘要 | 磁性高分子载体因具有磁响应性和高分子聚合物特性，在细胞分离、蛋白质纯化、核酸分离等领域有广阔的应用前景，如何制备合适的磁性分离载体成为很活跃的研究领域，然而将其真正应用到生物产品的大规模分离纯化还面临着许多挑战，在如何制备高磁性、粒径小，如何降低其表面的非特异性吸附，如何选择合适的亲和配基等方面都值得研究探讨。本论文针对上述问题开展了磁性纳米载体的制备、表征及其在漆酶分离纯化中应用的研究工作。主要取得了以下几个方面的研究结果： 首先，选择了Pluronic嵌段共聚物P123和F88，并在其末端修饰阳离子高分子聚合物聚乙烯亚胺，连接到共沉淀法合成的羧酸根离子修饰的Fe3O4纳米颗粒表面，合成了粒径均一的磁纳米颗粒，磁核粒径平均为25 nm，粒径分布窄，磁响应性好，有较高的比饱和磁化强度(51.34 emu/g)和超顺磁性。此方法合成工艺简单，易于放大。 在上述基础上，将合成的两种磁性载体分别用于漆酶分离纯化。其中P123MNPs对漆酶的最大吸附量约为0.44 mg/mg，但随温度升高其最大吸附量变小；而F88MNPs的最大吸附量并没有随之下降，达到0.54 mg/mg，说明经F88修饰的磁性载体更能适应实际生产的操作温度，使磁性载体的吸附性能得到最大程度的发挥。在漆酶发酵液的分离纯化中，经F88MNPs分离纯化后酶活收率达62.9%。这些初步的实验结果为磁性载体吸附性能的研究及进一步的改进提供了依据。 最后，选择了对漆酶亲和力强的亲和配基丁香基偶联到上述合成的磁性载体表面，通过电位滴定法测定配基的偶联效率，丁香基的配基密度达到0.79 mg/g。亲和载体对漆酶的最大吸附量约为0.66 mg/mg。将固定有丁香基的嵌段共聚物磁性纳米颗粒用于变色栓菌漆酶发酵液的分离纯化，成功分离出了纯度很高的漆酶，并达到一定的酶活收率(70%)，电泳结果表明磁性亲和载体能从变色栓菌漆酶发酵液中一步分离纯化出漆酶且纯度超过了商品漆酶。 |
| 英文摘要 | The magnetic carrier which incorporates the merits of magnetism and property of polymers has wide application in some fields, such as cell isolation, protein purification and DNA separation. Synthesis of applicable magnetic carrier has become an active research area. However, challenges and problems still exist when magnetic carrier is applied in large-scale separation and purification of bio-product. Many aspects should be investugated, such as how to synthesize magnetic carrier with high magnetism and small size, how to get rid of non-specific adsortion and how to choose appropriate affinity ligand for the target protein. In order to solve these problems, this thesis includes preparation and characterization of magnetic nanoparticles and its application in purification and separation of laccase. Several results are achieved: Firstly, Pluronic block copolymer P123 and F88 were chosed to be modified by a cation polymer PEI. And then the compound was grafted to the Fe3O4 nanoparticles prepared by a co-precipitation method. The prepared Pluronic-based magnetic nanoparticles possessed relative narrow size distribution, size of 25 nm, high saturation magnetization (51.34 emu/g) and superparamagnetism. And this synthesis method had advantages of simple preparation and easy operation. Secondly, the magnetic carriers modified by the above Pluronic block copolymers were applied to purification and separation of laccase. The maximum adsorption capacity of laccase on P123MNPs was about 0.44 mg/mg, which decreased when temperature rised; while maximum adsorption capacity of laccase on F88MNPs was about 0.54 mg/mg, which didn’t decrease with the temperature rising. This result indicated that F88 modified magnetic nanoparticles acted better than those modified by P123 in preserving adsorption properties of the magnetic carriers. The enzyme recovery of laccase reached 62.9% after purification by F88-MNPs. These initial results provided an evidence for the research of adsorption property and further improvement of the magnetic carriers. Finally, syringic ligand, with great affinity towards laccase, was coupled at the surface of the prepared magnetic nanoparticles. The binding capacity of the ligands was tested by poteniometric titration, which was 0.79 mg/g. And the maximum adsorption capacity of laccase to the affinity magnetic carrier was about 0.66 mg/mg. when F88-MNPs-syringe was applied in the purification and separation of mycelium culture of Trametes versicolor, the total enzyme activity recovery of laccase reached 70.0%. The result demonstrated that syringic coupled affinity magnetic carrier separated high purity laccase by a single step from mycelium culture of Trametes versicolor. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 70 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1304]  |
| 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 | 刘兴华. 磁性亲和载体的制备及漆酶分离纯化研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009. |
入库方式： OAI收割
来源：过程工程研究所
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