【作者】 雷涵；

【导师】 邓乐；

【作者基本信息】 湖南师范大学 ， 微生物学， 2003， 硕士

【摘要】 固定化酶具有稳定性好，使用次数多等独特优点，已被用于生物反应器，生物催化，和生物传感器等众多领域。用于制备固定化酶的方法很多，其中共价结合法因具有结合牢固的优点，而被广泛采用，但是由于选择应用的载体类型不同，固定化酶的酶学性质也会随之不同。本研究首次提出了以纳米级磁性Fe₃O₄粒子为核心，苯乙烯（St）—丙烯酸（AA）共聚物为高分子壳层，合成了表面带羧基的磁性高分子复合微球，然后将这种微球用二氯亚砜进行活化处理，在其表面形成了反应性酰氯基团，该基团可以与游离酶的氨基形成肽键，从而将游离酶固定化。通过这类载体制备的固定化酶的酶活回收率在76％以上，而由一般载体制备的固定化酶的酶活回收率在45-65％之间，这表明活化后的磁性高分子复合微球是一类可以应用于酶固定化领域的高效载体。本研究的独特之处可概括如下： 1 优化了纳米级磁性Fe₃O₄粒子的制备工艺。以氨水（NH₃·H₂O）作沉淀剂，用共沉淀法制备出了反应型纳米级磁性Fe₃O₄粒子，经透射电镜观察测得其粒径分布在20-60nm。采用边沉淀边熟化的方式，优化出了最佳的制备流程。 2 采用分散聚合法，以上述反应型纳米磁性Fe₃O₄粒子为核心，苯乙烯（St）—丙烯酸（AA）共聚物为高分子壳层，合成了表面带羧基的磁性高分子复合微球。在合成过程中，充分考虑到水分补偿问题，防止了 中文摘要凝胶的产生。3建立了FreundliCh等温吸附模型。根据酶液浓度与载体吸附量的关系，结合时间与吸附量的关系，建立了Freundl ich等温吸附模型，并获得了该模型的动力学参数（lgK，n）。4在制备固定化酶的过程中不需加入交联剂（如:戊二醛等）。 此外，考察了影响固定化木瓜蛋白酶酶学性质的因素，并与游离酶进行了比较。结果表明:由于木瓜蛋白酶直接固定于微球表面而没有剩余空间，这给固定化酶提供了更高的稳定性，所以固定化木瓜蛋白酶在热稳定性与储藏稳定性方面优于游离酶。固定化酶的最适反应温度是80℃，最适pH是8.0，操作半衰期是28天，固定化酶的酶活回收率在76%以上，通过米氏方程测定了固定化酶和溶液酶的米氏常数，分别为21.64147和7.0836。更多还原

【Abstract】 Immobilized enzyme has been applied to numerous fields, including bioreactors, biocatalysts and biosensors because of its advantage such as good stability and reusability. There have many methods for preparation of immobilized enzyme, covalent combination has been widely adopted because of its excellence of combination fastness. But enzymatic properties of immobilized enzyme are different in connection with the different type of vector that has been selected. In this study, it has been put forward that taking reactive nanometer magnetic Fe304 particles as magnetic nucleus, and the copolymer of styrene(St)梐crylic acid(AA) as macromolecular shell, we could synthesize, magnetic polymer composite microspheres containing carboxyl groups on their surface, then microspheres are activated by thionylchloride, the surface of such magnetic composite microspheres thus produced had reactive acid chloride groups which then react with the free amino groups of the free soluble enzymes to give peptide bonds (桟O桸H?, finally enzyme is covalently immobilized. The reclaimed efficiency of immobilized enzyme that is prepared by thisvector is over 76%, but others are between 45% and 65%. It showsthat activated magnetic polymer composite microspheres is an efficient vector that can be applied immobilization field. The particular works are summarized as follows:1 The best preparation circuit was optimizedNH3?H20 was chosen to act as precipitating reagent, reactive nanometer magnetic Fe304 particles were prepared by co-precipitation method, the particles size distribution was between 20nm and 60nm through the detection of transmission electron microscope (TEM). The best preparation circuit was optimized through the combination process of precipitation and aging.2 With dispersion-polymerization, taking above reactive nanometer magnetic Fe304particles as nucleus, and the copolymer of styrene(St)梐crylic acid(AA) as macromolecular shell, we could synthesize, magnetic polymer composite microspheres containing carboxyl groups on their surface. In the process of synthesize, moisture compensation was fully considered and gel was prevented.3 Freundlich isothermal adsorption model was established. The model was based on the relationship between the concentration of freely soluble enzyme and adsorption quantity of the vector, combined with time and adsorption, some kineticparameters (IgK, n) related to the process were obtained by using the model.4 Immobilization process has no use for coupling reagent (e.g:glutaraldehyde).Apart from these, The factors involved with the enzymic properties of the immobilized papain were studied in comparison with freely soluble papain. The results showed that thermal and storage stabilities of the immobilized papain were higher than those of the freely soluble one, and that papain directly was immobilized onto the microspheres’ surface without any spare space provided a higher stability. The optimum temperature, the pH and storage stability were 80癈, 8. 0 and 28 days respectively. The reclaimed efficiency of immobilized enzyme that is prepared by this vector is over 76%, the Michaelis - Menten kinetic constants were evaluated for the immobilized and the freely soluble enzymes, they were 21.64147 and 7. 0836, respectively.更多还原