多尺度载酶微反应器的构建与应用
文献类型：学位论文
| 作者 | 高飞 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-05-30 |
| 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 | 马光辉 |
| 关键词 | 纳米颗粒 固定化酶 复乳液 中空微囊 多尺度结构 |
| 其他题名 | Construction and Application of Multi-scale Enzymatic Microreactor |
| 学位专业 | 生物化工 |
| 中文摘要 | 近年来随着酶修饰与酶固定化研究的发展，纳米材料被广泛应用到酶技术领域中，并引起了越来越多的关注。其中，使用自由分散的纳米颗粒固定化酶，能够同时获得较高的载酶量和较小的传质阻力，同传统的载酶方法相比具有显著优势。此外，纳米颗粒在水相中的布朗运动行为，使催化体系处于均相催化与非均相催化之间，对固定化酶的活性保留也有积极的贡献。但是，由于载酶纳米颗粒尺度较小，不利于实际生产中回收再利用，一旦扩散到环境中，还会对环境构成潜在的危害。本论文提出 了一种多步固定的方法，先构建出自由分散的纳米载酶颗粒，再将载酶颗粒装入中空微囊，形成以微囊为单位的微反应器，来克服纳米载酶颗粒不利于回收的缺点，并保障纳米催化剂的安全性。 通过无皂乳液聚合结合种子聚合，制备了水相分散的聚（甲基丙烯酸甲酯-乙二醇二甲基丙烯酸甲酯-甲基丙烯酸）（P(MMA-EDMA-MAA)）纳米球，作为第一步固定化酶的载体。首先通过无皂乳液聚合制备纳米尺度的聚（甲基丙烯酸甲酯-乙二醇二甲基丙烯酸甲酯）（P(MMA-MAA)）种子球，再通过种子溶胀和种子聚合，实现纳米球内分子的交联和表面的功能化，制备成P(MMA-EDMA-MAA)纳米载体。通过选择不同的无皂乳液聚合配方，分别制备出170 nm和400 nm两种不同粒径的种子球，在此基础上分别制备出具有相似粒径分布的纳米载体。载体的表面羧基密度在0-0.70 mmol/g之间可控。制备的纳米载体不仅适合固定化酶，而且满足后续复乳包埋的要求，在微囊内腔中仍能够保持原有的分散性与运动性。 通过复乳化结合悬浮聚合，制备内腔中空、壳层多孔的聚（甲基丙烯酸甲酯-乙二醇二甲基丙烯酸甲酯-甲基丙三醇三甲基丙烯酸甲酯）（P(MMA-EDMA-TTMA)）微胶囊，作为微反应器的壳层。通过制备油包水包油（W/O/W）型复乳液滴获得胶囊的模板，再通过后续悬浮聚合实现乳滴固化，形成固体微囊。为了确保聚合过程的生物兼容性，悬浮聚合过程使用紫外照射引发来代替传统的热引发聚合。通过对油相配方的优化，制备出具有稳定内腔结构的聚合物微胶囊。通过致孔溶剂与壳层聚合物间的相分离，制备贯穿壳层的纳米孔道，其平均孔径在0-42 nm之间，既能满足小分子迅速传递的需要，又能避免装载的纳米颗粒向外扩散。 为了获得宽敞的胶囊内腔，以利于内腔中纳米颗粒的自由运动，论文以单腔复乳液滴为模板，来制备具有单一中空内腔的微囊。论文考察了影响复乳形态的主要因素，包括水相稳定剂、乳化剂和无机盐的配比，提出了一种在多腔复乳液滴的基础上，通过复乳形态演变制备单腔复乳液滴的方法。通过对制备工艺的优化，微囊产品中单腔比例能达到98%以上。通过调节内、外水相渗透压梯度，能进一步调节微囊的壳层厚度，可控范围为3.2-6.5 m。 使用-胰凝乳蛋白酶为模型，集成上述制备技术，验证了多步固定方法的可行性。首先通过共价交联将酶分子固定于纳米载体表面，再将载酶纳米颗粒装载在中空微囊中。微囊化过程中保持载酶颗粒的分散性不受破坏，使装载后的纳米颗粒在内腔中仍保持了原有的布朗运动的特点。通过中空微囊装载，能保持纳米载酶的活性优势，多步固定的活性回收率能达到50%左右。同纳米载酶相比，微反应器具有优良的重复使用性，重复使用100次仍能够保持96%以上的初始活性。 所提出的微反应器及其构建思路，能够进一步推广应用于其他纳米材料的包埋与放大，用于克服纳米催化剂回收利用的困难，并有望在微单元中集成更复杂的过程与功能。 |
| 英文摘要 | With the development of enzyme modification and immobilization in recent years, nanomaterials have been widely employed in the field of enzyme technology, and attracted more and more interests. Typically, enzyme-loaded nanoparticles in a dispersive state could accommodate the usual contradictory requirements: maximum loading capacity and minimum diffusion restriction, comparing to traditional immobilization. In addition, the mobility of the enzyme-loaded nanoparticles in solution also contributes to their catalysis performance, as is featured by Brownian motion, and is thus distinct from homogesous and heterogeneous catalysis. However, such enzyme-loaded nanoparticles are unfavorable in practical application, for their tiny sizes which usually lead to arduous task in recycling and potential risk to environment. This study proposed a hierarchical immobilization method, first load enzyme on nanoparticles and then encapsulate the aqueous-dispersed nanoparticles in a microcapsule, as a microreactor, to overcome recycling difficulty and to guarantee application security. Aqueous-dispersed poly(methyl methacrylate-ethylene dimethacrylate-methyl acrylic acid) (P(MMA-EDMA-MAA)) nanoparticles which served as primary carriers for enzyme, were fabricated by a soap-free emulsion polymerization and subsequent seeded polymerization. P(MMA-MAA) seed nanoparticles were first synthesized by a soap-free emulsion polymerization, and were further crosslinked and functionalized during the subsequent seeded polymerization, leading to P(MMA-EDMA-MAA) nanocarriers. Two groups of seed nanoparticles, 170 nm and 400 nm in diameter respectively, obtained with unique soap-free recipes, were employed as seeds for nanocarrier. Carboxyl groups exposing on the surface of nanocarriers could be designed at a density ranging from 0 to 0.70 mmol/g. Such polymer nanoparticles were not only suitable for enzyme immobilization, but also ideal core materials to be encapsulated by water in oil in water (W/O/W) double emulsion-templated microcapsules, in which they would maintain their original dispersity and mobility. Poly(methyl methacrylate-ethylene dimethacrylate-trimethylolpropane trimethacrylate) (P(MMA-EDMA-TTMA)) microcapsules featured with hollow cells and porous shell, were fabricated through a two-step emulsification and subsequent suspension polymerization, to serve as tanks of the microreactors. The W/O/W double emulsion globules, were applied as templates for microcapsule, and would be solidified during the subsequent polymerization. To guarantee a bio-friendly process, polymerization was initiated by UV irradiation rather than traditoanl thermal treatment. After an optimization on oil recipe, stable cell structure was relealized. Nanopores in the shell, which were fabricated by phase separation during polymerization, ranging from 0 to 42 nm in mean diameter, were proved capacious enough for rapid exchange of small molecules, but not allowing the enzyme-loaded nanoparticles to escape. To facilitate the mobility of encaged nanoparticles, single-cell microcapsules with large internal room were prepared from single-cell W/O/W emulsion globules. Parameters on morphology evolution of double emulsion globules, including the concentration of stabilizer, emulsifier and salt in the water phases, were particularly investigated, and a method to prepare single-cell globules from their multicell precursors was proposed. Single-cell microcapsules could occupy a majority proportion as high as 98% after optimization. It was also found that the shell thickness of could be adjusted from 3.2 to 6.5 m, by osmotic gradient between internal and external water phases. The feasibility of such hierarchical immobilization was tested by a model enzyme -chymotrypsin. Enzyme molecules were first immobilized on the surface of primary carriers and then encaged in the single-cell microcapsules. The dispersity of enzyme-loaded nanoparticles was guaranteed in the internal water phase during the encapsulation, and the Brownian motion was well preserved in the cells of enventual microcapsules. The size of the enzyme-loaded microcapsules, tens of micrometers after two-step immobilization, involved little difficulty in recycling. Compared to traditional enzyme encapsulation, the performance of nano-scale catalysts could be well preserved after encapsulation, with about 50% original specific acitivity recovered. Compared to enzyme-loaded nanoparticles, such microreactor could greatly facilitate the recycling operation, with more than 96% initial activity preserved after 100 reuses. It is believed that the method presented can be universalized to other functional materials in nano-scale to overcome the recycling difficulties, and are expected to integrate more complex processes in future development. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 152 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1254]  |
| 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 | 高飞. 多尺度载酶微反应器的构建与应用[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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