磁性纳米材料的制备及固定化海洋脂肪酶adm47601的分析-preparation of magnetic nanomaterials and analysis of immobilized marine lipase adm 47601.docxVIP

上海海上海海洋大学硕士学位论文 PAGE PAGE VI饱和磁化强度和氨基含量分别为：19.5 emu/g 和 36.3 emu/g，20 %和 3 %。与Fe3O4@NH2 纳米微球相比，磁性 Fe3O4@SiO2-NH2 纳米微球粒径均一性良好，更 多的氨基含量，故选择磁性 Fe3O4@SiO2-NH2 纳米微球用于后期的脂肪酶固定化 研究。3.应用制备的 Fe3O4@SiO2-NH2 纳米微球作为固定化酶载体材料，进行了海 洋脂肪酶 ADM47601 固定化条件研究，实验结果表明，磁性 Fe3O4@SiO2-NH2 纳米微球固定化海洋脂肪酶 ADM47601 的最佳固定化条件为：酶蛋白浓度为 0.125 mg/ml，固定化温度为 30 ℃，固定化体系的 pH 值为 8.0，固定化时间为10 h，酶活力回收率可达到 87.7 %，单位面积载体的蛋白吸附率可达到 23.7 mg/g。4.以最佳固定化条件制备的固定化脂肪酶为样品，以对硝基苯基月桂酸酯作 为底物，对固定化脂肪酶的酶促反应条件进行了考察，结果表明，固定化脂肪酶 的最佳催化温度为 40 ℃，最佳催化 pH 为 8.0；固定化酶在 pH 8.0 缓冲液中，60 ℃ 保温 1 h 仍能保持 42%的活力，游离酶仅为 10%；固定化酶和游离酶在 pH 9.0 缓冲液中活力最稳定， 30 ℃保温 24 h，固定化酶剩余酶活力 94%，游离酶剩余 酶活力 56%；固定化酶在 pH 9.0 缓冲液中，25 ℃放置 30 天，仍然保持 81%的 活力，游离酶为 59%；固定化酶在 pH 9.0 缓冲液中，25 ℃下贮藏半衰期为 99 天，游离酶为 39 天；固定化酶连续操作八次仍然能够保持 44%的活性，其操作 半衰期为七次；通过米氏方程测定固定化脂肪酶和游离脂肪酶的米氏常数分别为0.79 mg/ml 和 0.23 mg/ml。关键词：磁性纳米粒子，表面改性，海洋脂肪酶 AMD47601，固定化酶Preparation Of Aminosilane Modified Magnetic Nanomicrospheres And Research On Immobilization Marine Lipase ADM47601ABSTRACTLipase (glycerol ester hydrolase, Lipase EC 3.1.1.3), an important biological catalyst, which can catalyze a variety of reactions and is widely used in the food, chemical and pharmaceutical industry. The catalytic technology of free lipase is very mature, but taking into account the issues of can not be reused, difficult to achieve a process of continuous. Magnetic nanoparticles were new functional materials, have gradually developed after 1970s, which have broad application prospect. As the properties of superparamagnetic, high saturation magnetization, anisotropy, high dispersion and biocompatibility properties, Magnetic nanoparticles were widely used in various fields of biotechnology, such as enzyme immobilization, drug targeting and drug transport, magnetic fluid, contrast media, immunoassay, cell separation, biosensors, separation and purification of proteins and nucleic acids, and cancer diagnosis. The technology of magnetic nanoparticles immobilized enzyme not only makes the enzyme be reused and achieve continuous p