超大孔微球的固定化酶研究
文献类型：学位论文
| 作者 | 李燕 |
| 学位类别 | 硕士 |
| 答辩日期 | 2010-06-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 马光辉 |
| 关键词 | 超大孔微球 脂肪酶 固定化 孔径 亲疏水性 |
| 其他题名 | Research for enzyme immobilized on macroporous microspheres |
| 学位专业 | 生物化工 |
| 中文摘要 | 本论文针对常规多孔固定化酶载体孔径较小，而造成传质阻力等问题，设计和制备了新型超大孔固定化酶载体微球并将其用于酶固定化研究。系统地研究了载体材料的孔结构与亲疏水性能对固定化酶的稳定性、重复利用性、催化性能等的影响。本论文研究包括载体制备及表征和固定化酶两个方面。载体制备研究的主要内容如下: 使用实验室建立的新型超大孔微球的制备技术，合成了聚(苯乙烯-甲基丙烯酸缩水甘油酯)[P(ST-GMA)] 微球。考察了功能单体GMA比例、油溶性表面活性剂、稀释剂、交联剂等因素对聚合物微球孔结构的影响规律。通过优化实验条件，合成出具有特定孔径的不同功能单体GMA含量的超大孔P(ST-GMA)共聚微球。固定化酶研究的主要内容如下： 1. 选取三种不同孔径的聚苯乙烯微球（15 nm, 100 nm和300 nm）进行了固定化脂肪酶（Amano Lipase PS, from Burkholderia cepacia）的研究，首先优化固定化的条件，考察了时间、温度、酶浓度等因素的影响，获得了最优条件。在此基础之上，研究了微球孔径对酶活的影响，发现酶活性回收率随着微球孔径的增大而增加。超大孔微球不仅解决了传质阻力的问题，在酶活保持、酶的热稳定性和重复利用性等方面有着明显的优势。 2. 选取三种不同亲疏水性的载体，即PST、P(ST-GMA)、PGMA超大孔微球（孔径均为300 nm左右），研究其对脂肪酶（Candida antarctica Lipase B，诺维信提供）吸附行为的影响，并比较了物理吸附法和共价偶联法的区别，考察了固定化时间、初始酶浓度、反应温度、反应体系pH等的影响，发现超大孔的PGMA微球与其他两种微球相比无论是保持酶活，还是酶固载量上都有着明显的优势。这表明，相对亲水的材料更加有利于保持脂肪酶的活性，而且化学偶联的方法更加有利保持酶的稳定性。 |
| 英文摘要 | In order to solve the diffusion constraint problems existing in conventional porous immobilization carriers, we designed and synthesized a novel gigaporous carrier, and investigated its application in enzyme immobilization. We also studied how the pore sizes and the hydrophilicity/hydrophobicity of microspheres influenced the immobilization conditions and the properties of immobilized enzyme. The thesis included preparation and characterization of the carriers and enzyme immobilization. Gigaporous poly(styrene-glycidyl methacrylate) microspheres [P(ST-GMA)] were prepared using surfactant reverse micelles swelling method. The effects of GMA content, surfactant amount, diluent, and crosslinking agent amount on the pore distribution in microspheres were investigated. According to the optimization conditions, gigaporous P(ST-GMA) microspheres with special pore sizes and different contents of GMA were synthesized. The investigation of immobilized enzymes in this study is as follows. 1. A series of giga-/macro-/meso-porous polystyrene (PST) microspheres (15 nm, 100 nm and 300 nm) were employed as supports to immobilize lipase (Amano Lipase PS, from Burkholderia cepacia). Firstly, immobilization conditions were studied, such as immobilization time, reaction temperature, and the initial enzyme concentration, et al., and the optimal conditions were obtained. Based on these results, the effect of pore sizes on lipase activity was investigated in detail, and the activity yields were increased with increasing pore sizes. Notably, the diffusion constraint could be solved in gigaporous microspheres. The thermal stability, storage stability, and reusability were all improved significantly with the increase of pore sizes. Taken together, all these results in this study strongly supported that the gigaporous PST microspheres was an ideal candidate support for lipase immobilization. 2. Three different hydrophobic materials, that is, gigaporous PST, P(ST-GMA), and PGMA microspheres (300 nm pores), were selected to immobilize lipase (Candida antarctica Lipase B, Novozymes provided), and physical adsorption and covalent binding method were also compared. The immobilization behaviors were investigated, including immobilization time, the initial enzyme concentration, reaction temperature, and pH. Comparing with the other two immobilized lipases, the lipase immobilized on gigaporous PGMA microspheres exhibited much better in enzyme catalytic behavior. This indicated that the relatively more hydrophilic material was favorable to immobilize lipase, and the enzyme stability could be significantly improved by covalent binding method. |
| 公开日期 | 2013-09-17 |
| 页码 | 79 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1506]  |
| 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 | 李燕. 超大孔微球的固定化酶研究[D]. 北京. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：过程工程研究所
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