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,在生物医学、分子生物学、分离工程、细胞学和生物工程等领域有着广泛的应用前景。用磁性高分子作为酶的固定化载体的研究对于磁性复合载体在固定化酶的应用中提供了理论和试验基础,同时也为降低脂肪酶利用成本,解决我国生物柴油酶法生产产业化问题进而提高其可行性和国际竞争力提供了较好的参考方案。和其他磁性材料相比,Fe304具备良好的氧化稳定性和较低的毒性,并且由于有不同的化学组成结构,从而可得到不同的磁性能,常用作磁性微球中的磁核。用磁性高分子作为酶的固定化载体的优点在于它在外加磁场的作用下被磁化而定向移动,可以从反应体系中被分离出来；当撤离外加磁场后,超顺磁性纳米颗粒又可以重新悬浮到反应体系中,具有良好的分散性能与可操控性。本论文主要研究了磁性复合载体的制备条件及其固定化脂肪酶的条件,并对固定化酶的酶学性质进行了探讨,主要研究结果如下：
磁性复合Fe3O4-SiO2载体制备。先利用改良的共沉淀法制备Fe3O4磁流体,再用溶胶-凝胶法制备磁性复合Fe304-SiO2载体。在扫描电镜观测的形态说明,该磁性复合载体呈球体状,单分散性良好,平均粒径约为300nm左右。磁性复合Fe3O4-SiO2载体经硅烷偶联剂在其表面氨基化后,在傅里叶变换红外光谱仪下检测氨基已加至载体表面。
磁性复合Fe304-SiO2载体固定化脂肪酶条件进行优化。由单因素实验确定各单素的最佳条件：酶与载体配比、戊二醛浓度、固定化温度、固定化时间分别为16：5g/g、8%、25℃、4 h。在此基础上,选取为酶与载体配比、戊二醛浓度和固定化温度进行Box-Behnken实验,对固定化脂肪酶的条件进一步优化,借助Design-Expert 7软件对实验数据进行统计分析,拟合出描述酶活与实验条件之间关系的数学模型,确定最适固定化条件为：酶与载体配比为3.32：1g/g、戊二醛浓度为8.20%、固定化温度为23.88℃时,固定化脂肪酶的酶活达到理论最大值3440.74 U/g。在以上确定的条件下进行重复的三组固定化试验,结果所得固定脂肪酶的平均酶活为3430±1.55U/g,与模型预测值相差为0.28%,说明所建立的模型与试验结果相符,此时蛋白固载量为79.21%,酶活回收率为72.46%,与响应面优化之前相比分别提高了7.93%和10.36%。
固定化酶酶学性质的探讨。通过比较游离酶与固定化酶的最适温度和热稳定性等性质,研究了固定化酶的重复使用率。实验结果表明,游离酶的Km为3.817mmol,Vm为0.041 nmol/min·L,固定化酶的Km为5.714mmol, Vm为0.087μmol/min·L。与游离脂肪酶相比,最适反应温度、热稳定性、最适反应pH值、pH稳定性等均无太大变化,但是热稳定性和pH稳定性均有所增加,说明固定化操作对游离脂肪酶的稳定性有一定的提高。固定化脂肪酶在重复使用8次后,酶活回收率仍为的36.4%(相对酶活,以4℃保存的固定化酶活力为100%)。
Magnetic polymer microspheres were new functional polymer materials developed in recent years, which have broad application prospect in the biomedical, molecular boilogy, separation engineering, cellular and bio-engineering and other fields. Magnetic polymer was used as enzyme immobilization carrier provides the theoretical and experimental basis in the immobilized enzyme application for reducing lipase cost, and the problems of biodiesel production were redused. Compared with other magnetic materials, Fe3O4 have good oxidation stability and low toxicity. And different magnetic properties were obtained because of its different chemical composition and structure. Magnetic microspheres were used for the magnetic core. As enzyme im-mobilization carriers, magnetic polymers can be magnetized in an external magnetic field and then directional movement, so it can be separated from the reaction system; when the external magnetic field was withdrawed, the superparamagnetic nanoparticles can be resuspended to the reaction system, with good dispersion properties and manipulation.
In this paper, the Fe3O4 magnetic fluid was prepared by modified coprecipitation, and then composite magnetic Fe3O4-SiO2 carriers were got by the sol-gel. The magnetic composite carriers observed by SEM showed that carriers were ballshaped, singlewell dispersed, the average size of about 300nm or so. Silane coupling agent, which was detected by FT-IR spectro-meter, was used for adding amino to magnetic Fe3O4-SiO2 surface.
Conditions of immobiling lipase by magnetic Fe3O4-SiO2 composite were studied, the optimal conditions got from single factor experiments were:the amount of enzyme was 8mL, concentration of glutaraldehyde was 8%,25℃,4h. The amount of enzyme, con-centration of glutaraldehyde and immobilized temperature were selected to do Box-Behnken experimental to further optimize the conditions, and experimental data was statistical analysis with the Design-Expert 7 software, the mathematical model was describing the relationship between activity and the expeimental conditions. The optimal conditions of immobilized showed by the model are:the amount of enzyme was 8.30mL, 8.20% glutaraldehyde concentration,23.88℃, then got the maximum activity 3440.74 U/g. Three sets of repeated immobilization test was studied under the conditions defined in the above, the results obtained the average fixed lipase activity was 3430±1.55U/g, and the difference between model prediction was 0.28%, indicating the mo-del consistent with the experimental results.
The enzymatic properties of immobilized enzyme was explored. Optimum temperature, thermal stabilities of immobilized enzyme were compared with the free enzyme. The immobilized enzyme were reused. The results showed that the Km for the free enzyme 3.817mmol, Vm was 0.041μmol/min·L, and which of immobilized enzyme were 5.714mmol and 0.087μmol/min·L, respectively. Compared with the free lipase, the optimum temperature, thermal stability, optimum pH value, pH stability of immobilized enzyme without much change, but the thermal stability and pH stability were increased, indicating operation of the immobilized increased the stability of free lipase. Immobilized lipase were reused 8 times, the activity recovery is still 36.4%.
引文
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