磁性微球的制备及其对BSA和酶的固定化研究
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摘要
磁性高分子微球是近年发展起来的一种新型功能高分子材料,它作为酶、细胞、药物的载体被广泛地应用到细胞学、生物工程和生物医学等领域。特别是磁性微球用于固定化酶技术,引起了研究者的极大关注。其中磁性高分子微球的结构及性能在很大程度上直接影响着固定化蛋白或酶的催化活性及操作稳定性。所以载体的制备和表面性质将成为固定化酶技术研究领域的重要内容。
本文在化学共沉淀法合成超细粉末Fe_3O_4的基础上,采用分散聚合法以苯乙烯(St)、丙烯酰胺(AM)和丙烯酸(AA)为共聚单体,以过硫酸胺为引发剂(AIBN),乙醇/水混合溶剂为分散介质,制备了微米级、单分散性较好的P(St/AM/AA)磁性高分子微球。对所制备的磁流体和磁性微球的外观形态、粒径和表面羧基含量进行了表征。红外光谱表明St、AM和AA在磁粒子表面发生了共聚合,扫描电镜的观察显示高分子微球的粒径为0.2μm-0.5μm。
同时将不同单体比例制备的微球作为固定BSA的载体,考察了不同表面性能的磁性微球对BSA固定化率的影响。结果表明,微球表面的亲疏水性不同,对BSA的固定化率也不同。此外还研究了微球表面羧基含量,活化酯时间,固定化时间,温度和pH值对BSA固定化率的影响。在此基础上进一步研究磁性微球对Candida rugosa脂肪酶(CRL)的固定化效果。结果表明,在固定了脂肪酶之后,磁性纳米微球仍具有优异的磁分离能力。当固定化时间为1 h时,酶活达到2.1 U/mg,酶活回收率达到95 %。为深入研究磁性微球固定化酶提供了有力的数据。
Magnetic polymer microsphere is a novel functional material that has been developed resently. As the carrier of enzyme, cell and drug, it can be widely applied in many fields, such as bio-engineering, cytology, biomedical science and so on. Especially, the application of magnetic microspheres is of great interest for researchers in enzyme immobilization technology. As an important part of immobilized enzymes, the structure and properties of magnetic polymer microspheres affect the activities and operation stabilities of the immobilized enzymes greatly. Therefore, the preparation of carrier materials as well as its surface characteristics will be one of the most significant researches for the development of immobilized enzymes in the future.
Here, the magnetic oxide (Fe_3O_4) was prepared by co-precipitation. Using ammonium persulfate as initiator, magnetic functional polymer microsphere P(St/AM/AA) was synthesized by the copolymerization of styrene, acrylamide and acrylic acid in aqueous ethanol medium. In this paper, external shape, size, superficial acrylic acid content of the microspheres and nano-particles of Fe_3O_4 were characterized. FTIR indicated that styrene, acrylamide and acrylic acid were successfully enfolded on the surface of the magnetic particles. SEM was employed to test the microspheres and the result showed that the average diameter was between 0.2-0.5μm. To investigate the effect of microsphere surface property on the immobilization of BSA, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. The results showed that microspheres with different hydrophobicity/hydrophilicity had different immobilized ratio of BSA. Meanwhile, the effects of ester activation, coupling times, temperature and pH value on the immobilized ratio of BSA were discussed.
Furthermore, these microspheres were used to immobilize lipase. It was found that the immobilized lipase possessed excellent ability of magnetic separation. When the molar percentage of St was 50 % and the immobilization time was 1 hour, the activity of immobilized enzyme was 2.1 U/mg. These results provided preliminary data for further study on the immobilization of lipase.
本文在化学共沉淀法合成超细粉末Fe_3O_4的基础上,采用分散聚合法以苯乙烯(St)、丙烯酰胺(AM)和丙烯酸(AA)为共聚单体,以过硫酸胺为引发剂(AIBN),乙醇/水混合溶剂为分散介质,制备了微米级、单分散性较好的P(St/AM/AA)磁性高分子微球。对所制备的磁流体和磁性微球的外观形态、粒径和表面羧基含量进行了表征。红外光谱表明St、AM和AA在磁粒子表面发生了共聚合,扫描电镜的观察显示高分子微球的粒径为0.2μm-0.5μm。
同时将不同单体比例制备的微球作为固定BSA的载体,考察了不同表面性能的磁性微球对BSA固定化率的影响。结果表明,微球表面的亲疏水性不同,对BSA的固定化率也不同。此外还研究了微球表面羧基含量,活化酯时间,固定化时间,温度和pH值对BSA固定化率的影响。在此基础上进一步研究磁性微球对Candida rugosa脂肪酶(CRL)的固定化效果。结果表明,在固定了脂肪酶之后,磁性纳米微球仍具有优异的磁分离能力。当固定化时间为1 h时,酶活达到2.1 U/mg,酶活回收率达到95 %。为深入研究磁性微球固定化酶提供了有力的数据。
Magnetic polymer microsphere is a novel functional material that has been developed resently. As the carrier of enzyme, cell and drug, it can be widely applied in many fields, such as bio-engineering, cytology, biomedical science and so on. Especially, the application of magnetic microspheres is of great interest for researchers in enzyme immobilization technology. As an important part of immobilized enzymes, the structure and properties of magnetic polymer microspheres affect the activities and operation stabilities of the immobilized enzymes greatly. Therefore, the preparation of carrier materials as well as its surface characteristics will be one of the most significant researches for the development of immobilized enzymes in the future.
Here, the magnetic oxide (Fe_3O_4) was prepared by co-precipitation. Using ammonium persulfate as initiator, magnetic functional polymer microsphere P(St/AM/AA) was synthesized by the copolymerization of styrene, acrylamide and acrylic acid in aqueous ethanol medium. In this paper, external shape, size, superficial acrylic acid content of the microspheres and nano-particles of Fe_3O_4 were characterized. FTIR indicated that styrene, acrylamide and acrylic acid were successfully enfolded on the surface of the magnetic particles. SEM was employed to test the microspheres and the result showed that the average diameter was between 0.2-0.5μm. To investigate the effect of microsphere surface property on the immobilization of BSA, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. The results showed that microspheres with different hydrophobicity/hydrophilicity had different immobilized ratio of BSA. Meanwhile, the effects of ester activation, coupling times, temperature and pH value on the immobilized ratio of BSA were discussed.
Furthermore, these microspheres were used to immobilize lipase. It was found that the immobilized lipase possessed excellent ability of magnetic separation. When the molar percentage of St was 50 % and the immobilization time was 1 hour, the activity of immobilized enzyme was 2.1 U/mg. These results provided preliminary data for further study on the immobilization of lipase.
引文
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