多孔三元共聚物GMA-NVP-MBA的制备及其对豇豆酯酶的固定化研究-物理化学专业论文.docxVIP

I I 摘 要 摘 要 豇豆种子经浸泡，捣碎，冰箱 4 ℃下放置提取 12h 后，离心取上清液，对上清液进 行分段盐析，透析后得到豇豆酯酶粗酶液，测定了豇豆酯酶自由酶酶促反应的最适反应 温度、最适 pH 值，pH 稳定性，热稳定性，储存稳定性等相关性质。 以甲基丙烯酸缩水甘油酯（GMA）、N-乙烯基吡咯烷酮（NVP）为单体，N,Nˊ-亚 甲基双丙烯酰胺为交联剂，甲醇水溶液为致孔剂，采用悬浮聚合法制备了多孔聚合物微 球，并用于豇豆酯酶的固定化研究。通过实验摸索了 GMA、NVP、MBA 三者的用量关 系，酶的最适固定化时间、温度、pH 值以及酶液浓度等相关固定化条件及该固定化酶 酶促反应的最适温度、最适 pH 值，pH 稳定性，热稳定性，操作稳定性等相关性质。 结果发现：由于亲水性单体 NVP 的引入，载体小球亲水性得到提高，该性质更有利于 其与豇豆酯酶的结合，因而酶的固定化效果较好，且相对自由酶来说，固定化酶的酸碱 稳定性，热稳定性均有明显提高，操作稳定性表现良好。 采用固液双致孔方式制备了 GMA-NVP-MBA 高分子聚合物载体，利用扫描电子显 微镜对微球的表面结构进行了研究，并与单纯液相致孔所得微球进行比较，结果发现： 固液双相致孔制得的微球有更多的孔表面结构，有更大的比表面积，更有利于酶的固定 化。在最优化条件下，每克干燥载体上固定化酶酶活力值为 33.19U,活性回收率为 61.47%，而单纯液相致孔所得载体固定化酶活力为 18.97U/g，活性回收率为 33.10%。 动力学性质的测定表明该固定化酶 pH 稳定性，热稳定性，操作稳定性均较好。由此可 见，固液双致孔法制备的聚合物载体更有利于酶的固定化，具有非常广泛的应用前景。 关键词 甲基丙烯酸缩水甘油酯 N-乙烯基吡咯烷酮 N,Nˊ-亚甲基双丙烯酰胺 豇豆酯酶 固定化 Abst Abstract II II Abstract The cowpea esterase was extracted as follows: Cowpea was soaked in phosphate buffer solution and then was crushed and placed in fridge at 4 ℃ for 12 hours. The cowpea residue was separated from the cowpea grinding fluid by HYPERLINK /search?q=centrifugalizationamp;keyfrom=E2Ctranslation centrifugalization and then was discarded. The supernatant was retained. The effective protein of cowpea esterase was salted out from the supernatant and then was HYPERLINK /w/dialyze/ dialyzed. Finally relatively pure cowpea esterase solution was derived. Meanwhile, the optimum temperature and optimum pH of enzyme catalysis reaction were measured and the basic properties, such as the pH stability, thermal stability, storage stability, etc, were also studied for it. Porous poly(GMA-NVP-MBA) particles were synthesized with glycidyl methacrylate (GMA) and N-vinyl pyrrolidone (NVP) as monomer, N,N-methylene-bis-(acrylamide) (MBA) as cross linking agent, methanol aqueous solution as pore-forming agents via suspension polymerization. The optimum ratio of mole dosage of GMA, NVP and MBA was explored. The influence of the immobilization time、pH and enzyme concentration on activity recovery of imm