纳微结构多酶催化体系的构建及研究
文献类型：学位论文
| 作者 | 张颖 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-03-04 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 王平 |
| 关键词 | 多酶体系 辅因子再生 固定化 纳米材料 纳微多尺度 |
| 其他题名 | Multi-Enzyme System Assisted by Nano and Macro Materials |
| 学位专业 | 生物化工 |
| 中文摘要 | 辅因子依赖的氧化还原酶在生物合成中具有很大的应用潜力，但辅因子的高成本及大量消耗阻碍了此类酶的工业化进程。将两种氧化还原酶偶联，构建包含辅因子再生的多酶催化体系，是降低生产成本的有效手段。论文的研究目标是构建一个经济、高效的多酶催化体系，实现具有商业应用价值的酶法生物转化；并利用纳微多尺度方法将多酶体系固定化，以利于其工业化应用。多尺度固定化首先将自由酶分子固定在纳米颗粒上，再将纳米载酶颗粒装入中空微囊中，形成以微囊为单位的多尺度生物催化单元。多尺度固定化的特点是：微囊中的纳米载酶颗粒保持原有的自由分散状态，纳米颗粒固定化酶的催化优势完全保留，胶囊的孔道直径从10-50 nm可控，既保证大分子底物的自由扩散，又能防止纳米载酶颗粒的泄露；此外，胶囊壳层为纳米颗粒表面的酶分子提供了一个保护屏障，有效提高了固定化酶在应用中的稳定性，而且微米载体回收便利，操作容易，有效弥补了纳米载酶颗粒在实际应用中回收困难的缺陷。论文首先制备了多尺度固定化的a-胰凝乳蛋白酶（a-chymotrypsin，a-CT），评价了多尺度固定化技术在实际应用中的可行性和先进性。a-CT是评价酶固定化技术中常用的模型酶，研究发现，多尺度固定化的a-CT活性回收率能达到50%左右，纳米载酶颗粒的催化特征在微囊内腔中得到了很好的保持；多尺度固定化的 a-CT在连续重复使用100次后，仍能够保持96%以上的初始活性，具有非常优异的生产稳定性。模型酶的工作充分体现了多尺度方法在酶固定化中的优势。以烟酰胺腺嘌呤二核苷酸（NAD+/NADH）为辅因子的甘油脱氢酶是甘油生物转化中的一种关键酶，构建高效、经济的甘油脱氢酶催化途径是甘油生物转化过程中的重要课题。在多尺度固定化模型酶的基础上，论文构建了多尺度固定化的甘油脱氢酶/谷氨酸脱氢酶（Glycerol Dehydrogenase/Glutamate Dehydrogenase，GDH/GluDH）多酶偶联体系，将甘油转化为高附加值的1, 3-二羟基丙酮（Dihydroxyacetone，DHA），同时实现辅因子再生。多尺度固定化过程中，固定化材料的介入没有影响辅因子循环效率，多尺度固定化的多酶偶联反应连续进行302天，仍可以保持60%的初始生产能力，在生产中的稳定性比纳米载酶体系提高了16倍。多尺度载酶与多酶体系的成功结合，实现了甘油的高效转化，为多酶体系固定化提供了全新的思路，同时有力证明了多酶体系巨大的应用潜力。为了进一步提高多酶催化体系的经济性，构建了甘油脱氢酶/木糖还原酶（Glycerol Dehydrogenase/Xylose Reductase，GDH/XR）多酶体系，此体系在辅因子再生的同时，可以同时实现甘油和木糖的生物转化，联产两种高附加值产物，为氧化还原酶提供了更经济的辅因子再生方法。为了提高多酶体系在实际生产中的稳定性及单元操作的可行性，用多尺度固定化方法将GDH/XR多酶体系固定化。研究工作表明，多尺度固定化对提高酶催化在批次反应中的催化效率有积极作用，多尺度固定化的GDH/XR多酶体系在连续生产中的半衰期为17天，其生产稳定性是自由多酶体系的8.5倍。总之，本论文构建了经济、有效的多酶催化体系，实现了甘油和木糖的酶法生物转化；多酶催化剂在多尺度固定化后保持了很好的催化能力，而且性质稳定、可重复使用、具有工业应用潜力。 |
| 英文摘要 | Cofactor-dependent oxidoreductases have great potentials in biosynthesis. However, the stoichiometrical consumption of cofactor has limited their commercial applications. Cofactors have to be regenerated to reduce the overall production cost. A natural way is the use of a secondary redox enzymatic reaction to construct a multi-enzyme system. In this paper, forward effective and economical multi-enzyme systems are constructed for value-added biotransformations, and a hierarchical immobilization method with nano- and micro- materials is investigated to facilitate the industrial application of enzymes. Through the hierarchical immobilization, enzymes were firstly immobilized onto nanoparticles and were then encapsulated into cell-like microcapsules. After immobilization, the catalytic properties of nanoparticle-supported enzymes will be guaranteed, and the porous shells of micro-capsules with pore sizes of 10 to 50 nm protect the enzymes from inactivation and leakage. The feasibility and an effectiveness of such hierarchical immobilization were tested with a-chymotrypsin (a-CT) which is a common model enzyme for evaluation of a new enzymatic immobilization method. The dispersity and catalytic properties of nanoparticle-supported a-CT were well preserved inside the microcapsules after the hierarchical immobilization with about 50% of original specific activity retained. Such hierarchically immobilized a-CT greatly facilitated the recycling operation, with more than 96% initial activity preserved after 100 times reuse. This observation indicated a high effectiveness of such a hierarchical immobilization method. NAD+-dependent Glycerol Dehydrogenase (GDH) can catalyze the conversion of glycerol to value-added product 1, 3-dihydroxyacetone (DHA). An economical and effective method to improve GDH catalysis is desirable for glycerol conversion besides chemical and microbiological approach. Based on the work of model enzyme, a hierarchically immobilized glycerol dehydrogenase/glutamate dehydrogenase (GDH/GluDH) multi-enzyme system with in situ cofactor regeneration was developed to convert glycerol to value-added 1, 3-dihydroxyacetone (DHA). The hierarchical multi-enzyme system showed a high cofactor regeneration efficiency similar to that of native system. The coupled reactions of the hierarchically immobilized multi-enzyme system could last about 302 days with 60% residual activity which is about 17 times that of nanoparticle supported system. The perfect combination of hierarchically immobilization and multi-enzyme system provides an efficient method in multi-enzyme biosynthesis. Further more, glycerol dehydrogenase/xylose reductase (GDH/XR) multi-enzyme system with in situ cofactor regeneration was designed and examined to simultaneously convert glycerol and xylose to value-added products, which propose for a more economical method for cofactor regeneration. The conversion and utilization of biorenewable feedstock for production of value-added chemicals are benefitial for the sustainability of the burgeoning bioeconomy. The half-life time of continuous production for hierarchically immobilized multi-enzyme system is about 8.5 times higher than that for native multi-enzyme system. To us, this work revealed an efficient and economical multi-enzyme system for simultaneous biotransformation of glycerol and xylose. This catalyst system showed good catalytic ability and excellent stability after hierarchical immobilization. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 页码 | 155 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1748]  |
| 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 | 张颖. 纳微结构多酶催化体系的构建及研究[D]. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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