人工细胞用于酶固定.doc

摘 要
目前，酶在生物体外的应用已经涉及到化工、医学、食品等各种行业。然而，由于游离酶不稳定，容易失活，限制了酶的应用，酶的固定化技术是解决这一问题的途径之一。随着固定化酶的发展，多酶的固定化已经成为一个新的挑战。本文结合生物仿生硅化，层层组装技术，模拟细胞核和细胞的关系，以具生物相容性的碳酸钙为模板，通过层层组装精蛋白和硅酸钠，制备出一种新型的的“壳中壳”载体，利用这种载体能够实现分区包埋两种不同的酶—葡糖糖氧化酶和辣根过氧化酶，能够避免两种酶之间的干扰，也可以减少中间代谢物的传质路径，提高反应速率。载体制备过程温和，能够保持较高的酶活。最后考察了将两种酶包埋在制备载体中的催化效果，发现这种包埋方法比混合包埋两种酶的反应速度要快约15-30%。在重复使用6次后，仍能保持约65%的活力。
关键词：多酶固定化；层层自组装；仿生硅化；人工细胞；葡萄糖氧化酶；辣根过氧化酶
ABSTRACT
Enzyme has been widely applied in industrial fields such as chemical engineering, food , biomedical fields. However, free enzyme is always vulnerable and easy to denature and difficult to reuse, which restrict the practical application of enzyme. Enzyme immobilization is one of the effective methods to solve this problem.
As the developing of the enzyme immobilization technique, multienzyme immobilization to carry cascade reactions emerges.
This work demonstrates a novel way to encapsulate two different enzymes in one capsule to carry a cascade reaction. These shell-in-shell structures were fabricated by alternative deposition of positively charged protamine layers and negatively charged silica layers on the surface of CaCO3 microparticles, followed by a coprecipitation with CaCO3, then by dissolution of the CaCO3 microparticles using EDTA. The microcapsule was used to encapsulate two different enzyme-glucose oxidase and horseradish peroxidase in separated and confined zones in the microcapsule like compartments in natural cells. Compared to the enzymes mixed in one capsule, the advantage of the structure is to avoid the interference between enzymes and to reduce the mass transfer distance of intermediate reagents, resulting in higher reaction velocity. The fabrication process was carried out on a rather mild condition. Besides, high enzyme loading was achieved to about 60%. Reaction velocity for enzymes in shell-in-shell microcapsule was about 15~30% higher compared to mix the enzymes in one microcapsule. After repeatedly