功能性纳米纤维的制备及固定化酶研究
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摘要
近年来，静电纺纳米纤维由于具有高比表面积、高孔隙率等诸多特点备受关注，并逐渐应用于药物缓释、过滤、组织工程等诸多领域。本论文以静电纺纳米纤维为基材，通过形态结构及化学组成的有效调控，制备功能性纳米纤维，并将其作为固定化酶的载体，以期在增加酶的固定量的同时，进一步改善酶的催化性能。本论文还初步探索基于功能性纳米纤维构建高效酶膜反应器（EMR），以实现生物催化和膜分离过程的有效耦合。
通过对静电纺聚丙烯腈（PAN）纳米纤维的胺肟化改性，制备偕胺肟聚丙烯腈（AOPAN）纳米纤维，并以AOPAN纳米纤维为基体进行金属离子配合。利用扫描电镜、红外光谱对纳米纤维的微观形貌、化学组成进行表征。结果表明，在优化的改性条件下，偕胺肟基团被成功引入PAN纳米纤维表面，并保持良好的纳米纤维结构。根据动态接触角测量结果，改性后，纳米纤维的润湿性也显著上升。原子吸收光谱被用于测定金属离子的配位性能，通过对比分析，AOPAN纳米纤维对Cd2+、Cu2+和Fe~(3+)三种金属离子配合量大幅高于AOPAN普通纤维。利用多种等温模型分析实验数据，其中Langmuir模型具有更好的拟合性。在动力学分析的基础上得到的平均吸附能表明，吸附过程为化学吸附，其机理为AOPAN纳米纤维表面的偕胺肟基通过配位键与金属离子络合。
以Fe(Ⅲ)-AOPAN金属配合纳米纤维为载体，利用配位键固定过氧化氢酶，经倒置荧光显微镜及扫描电镜观察，过氧化氢酶被成功地固定于纳米纤维膜的表面。经过对酶催化反应动力学分析，分别得到自由酶和固定化酶的Vmax值和Km值，动力学参数体现了载体与固定化酶之间良好的生物亲和性。固定后的过氧化氢酶其酸碱稳定性、热稳定性、存贮稳定性均得到提高，并体现出良好的重复使用性能。
利用静电纺丝制备PVA/PA6复合纳米纤维。通过与Cu~(2+)离子配合反应得到Cu(Ⅱ)-PVA/PA6金属配合纳米纤维,并以Cu(Ⅱ)-PVA/PA6金属配合纳米纤维为载体，固定过氧化氢酶。实验结果表明：PVA/PA6具有良好的金属离子配合能力，并在金属离子溶液中保持稳定的纳米纤维形态；固定后的过氧化氢酶体现出更好的酸碱稳定性、热稳定性，其存贮及重复使用性能均有明显提高，其半衰期也由自由状态的8天提高到24天。本论文还通过环氧化反应制备环氧化PVA/PA6复合纳米纤维，并以此为载体利用环氧键共价固定过氧化氢酶。对比分析可知，以Cu(Ⅱ)-PVA/PA6金属配合纳米纤维为载体配位法固定化酶具有更高的固定量和更好的催化性能。
分别以Fe(Ⅲ)-AOPAN金属配合纳米纤维膜和Cu(Ⅱ)-PVA/PA6金属配合纳米纤维膜为基体构建固定化酶膜反应器。研究了操作压力、膜通量及过氧化氢转化率之间的关系，并对其重复使用性能进行分析，结果显示该酶反应器具有稳定高效的催化性能。
Nanofibers have gained a great deal of attention in recent years due to such properties as high surfacearea per unit mass, remarkable high porosity. The expanding applications of nanofibers include drugdelivery, filtration, tissue engineering and so on. In order to improve the load amount of enzyme andpromote the catalytic character of immobilized enzyme, functional nanofibers were obtained by regulatingthe structures and chemical composition of electrospun nanofibers, which were used as carrier for enzymeimmobilization in this work. In addition, novel enzyme membrane reactor (EMR) was formed on the baseof above work. It was expected that the EMR can integrate the process of biocatalysis and membraneseparation.
Amidoxime polyacryonitrile (AOPAN) nanofibrous membranes were generated by the reactionbetween electrospun polyacryonitrile (PAN) nanofibrous membranes and hydroxylamine hydrochloride,which were used as the matrix for metal ions chelation. FTIR spectra of the PAN nanofibers and AOPANnanofibers were recorded for analysis of the surface chemical structures, the surface morphologies of thenanofibers were observed by scanning electronic microscope (SEM). FTIR spectrum indicated theconversion of the nitrile to the amidoxime group, and the SEM images revealed that the diameter ofAOPAN nanofibers did not change substantially, and the fibrous structures was not distorted obviously onthe optimal condition. According to the results of dynamic contact angle measurement, the wettability ofAOPAN nanofibers have remarkably improved. Chelated amounts of metal ions were calculated byatomic absorption spectrometry (AAS). Results indicated that the chelated capacities of AOPAN nanofibersto Cd~(2+)、Cu~(2+)and Fe3+ions were higher than that of AOPAN conventional fibers. Many isothermal modelswere used to analyze the experimental data, the chelated isothermal processes of AOPAN nanofibers werefound to be in conformity with Langmuir model. Adsorption processes of AOPAN nanifibers to metal ionswere confirmed as chemical adsorption in accordance with average adsorption energies. The mechanismsof adsorption processes were described as metal ions were chelated with amidoxime group on the surfaceof AOPAN nanofibrous membrane by coordinate bond.
Fe(Ⅲ)-AOPAN nanofibrous membranes were used as carrier for catalases immobilization withcoordination bond. Surface morphologies of the nanofibrous membranes and immobilized catalases wereobserved by fluorescence microscopy and SEM. Enzyme molecules were successfully immobilized on thesurface of Fe(Ⅲ)-AOPAN nanofibrous membranes with coordination bond. Kinetic parameters Vmaxand Kmwere analyzed for both immobilized and free catalases., the results indicated that the immobilized catalaseshad a high affinity with the support. The immobilized catalases showed better resistance to pH andtemperature change than that of free catalases. The storage stability was significantly improved and thereusability was very high after catalases were immobilized onto the surface of Fe(III)-AOPAN nanofibrousmembranes.
PVA/PA6composite nanofibers were formed by electrospinning. Cu(Ⅱ)-PVA/PA6metal chelated nanofibers were prepared by reaction between PVA/PA6composite nanofibers and Cu2+solution, whichwere used as the support for catalases immobilization. The result of experiments showed that PVA/PA6composite nanofibers have excellent chelation capacity for Cu2+ions. At the same time, the structures ofnanofibers had not been distorted obviously during the reaction with Cu2+solution. The immobilizedcatalases showed better resistance to pH and temperature inactivation than that of free form, and the storagestabilities，reusability of immobilized catalases were significantly improved. The half-lives of free andimmobilized catalases were8d and24d repectively. Besides, the epoxy PVA/PA6nanofibers weregenerated by epoxidation reaction for catalases immobilization. Comparing the experimental data, theimmobilized catalases on matrix of Cu(Ⅱ)-PVA/PA6nanofibers presented higher activities thanimmobilized catalases on the surface of epoxy PVA/PA6nanofibers.
On the base of Fe(Ⅲ)-AOPAN metal chelated nanofibrous membrane and Cu(Ⅱ)-PVA/PA6metalchelated nanofibrous membrane, the EMRs were assembled. The relationships among operating pressure,membrane flux, and conversion ratio of H2O2were investigated, and results showed that the EMRs weresteady, efficient and repeatable.
通过对静电纺聚丙烯腈（PAN）纳米纤维的胺肟化改性，制备偕胺肟聚丙烯腈（AOPAN）纳米纤维，并以AOPAN纳米纤维为基体进行金属离子配合。利用扫描电镜、红外光谱对纳米纤维的微观形貌、化学组成进行表征。结果表明，在优化的改性条件下，偕胺肟基团被成功引入PAN纳米纤维表面，并保持良好的纳米纤维结构。根据动态接触角测量结果，改性后，纳米纤维的润湿性也显著上升。原子吸收光谱被用于测定金属离子的配位性能，通过对比分析，AOPAN纳米纤维对Cd2+、Cu2+和Fe~(3+)三种金属离子配合量大幅高于AOPAN普通纤维。利用多种等温模型分析实验数据，其中Langmuir模型具有更好的拟合性。在动力学分析的基础上得到的平均吸附能表明，吸附过程为化学吸附，其机理为AOPAN纳米纤维表面的偕胺肟基通过配位键与金属离子络合。
以Fe(Ⅲ)-AOPAN金属配合纳米纤维为载体，利用配位键固定过氧化氢酶，经倒置荧光显微镜及扫描电镜观察，过氧化氢酶被成功地固定于纳米纤维膜的表面。经过对酶催化反应动力学分析，分别得到自由酶和固定化酶的Vmax值和Km值，动力学参数体现了载体与固定化酶之间良好的生物亲和性。固定后的过氧化氢酶其酸碱稳定性、热稳定性、存贮稳定性均得到提高，并体现出良好的重复使用性能。
利用静电纺丝制备PVA/PA6复合纳米纤维。通过与Cu~(2+)离子配合反应得到Cu(Ⅱ)-PVA/PA6金属配合纳米纤维,并以Cu(Ⅱ)-PVA/PA6金属配合纳米纤维为载体，固定过氧化氢酶。实验结果表明：PVA/PA6具有良好的金属离子配合能力，并在金属离子溶液中保持稳定的纳米纤维形态；固定后的过氧化氢酶体现出更好的酸碱稳定性、热稳定性，其存贮及重复使用性能均有明显提高，其半衰期也由自由状态的8天提高到24天。本论文还通过环氧化反应制备环氧化PVA/PA6复合纳米纤维，并以此为载体利用环氧键共价固定过氧化氢酶。对比分析可知，以Cu(Ⅱ)-PVA/PA6金属配合纳米纤维为载体配位法固定化酶具有更高的固定量和更好的催化性能。
分别以Fe(Ⅲ)-AOPAN金属配合纳米纤维膜和Cu(Ⅱ)-PVA/PA6金属配合纳米纤维膜为基体构建固定化酶膜反应器。研究了操作压力、膜通量及过氧化氢转化率之间的关系，并对其重复使用性能进行分析，结果显示该酶反应器具有稳定高效的催化性能。
Nanofibers have gained a great deal of attention in recent years due to such properties as high surfacearea per unit mass, remarkable high porosity. The expanding applications of nanofibers include drugdelivery, filtration, tissue engineering and so on. In order to improve the load amount of enzyme andpromote the catalytic character of immobilized enzyme, functional nanofibers were obtained by regulatingthe structures and chemical composition of electrospun nanofibers, which were used as carrier for enzymeimmobilization in this work. In addition, novel enzyme membrane reactor (EMR) was formed on the baseof above work. It was expected that the EMR can integrate the process of biocatalysis and membraneseparation.
Amidoxime polyacryonitrile (AOPAN) nanofibrous membranes were generated by the reactionbetween electrospun polyacryonitrile (PAN) nanofibrous membranes and hydroxylamine hydrochloride,which were used as the matrix for metal ions chelation. FTIR spectra of the PAN nanofibers and AOPANnanofibers were recorded for analysis of the surface chemical structures, the surface morphologies of thenanofibers were observed by scanning electronic microscope (SEM). FTIR spectrum indicated theconversion of the nitrile to the amidoxime group, and the SEM images revealed that the diameter ofAOPAN nanofibers did not change substantially, and the fibrous structures was not distorted obviously onthe optimal condition. According to the results of dynamic contact angle measurement, the wettability ofAOPAN nanofibers have remarkably improved. Chelated amounts of metal ions were calculated byatomic absorption spectrometry (AAS). Results indicated that the chelated capacities of AOPAN nanofibersto Cd~(2+)、Cu~(2+)and Fe3+ions were higher than that of AOPAN conventional fibers. Many isothermal modelswere used to analyze the experimental data, the chelated isothermal processes of AOPAN nanofibers werefound to be in conformity with Langmuir model. Adsorption processes of AOPAN nanifibers to metal ionswere confirmed as chemical adsorption in accordance with average adsorption energies. The mechanismsof adsorption processes were described as metal ions were chelated with amidoxime group on the surfaceof AOPAN nanofibrous membrane by coordinate bond.
Fe(Ⅲ)-AOPAN nanofibrous membranes were used as carrier for catalases immobilization withcoordination bond. Surface morphologies of the nanofibrous membranes and immobilized catalases wereobserved by fluorescence microscopy and SEM. Enzyme molecules were successfully immobilized on thesurface of Fe(Ⅲ)-AOPAN nanofibrous membranes with coordination bond. Kinetic parameters Vmaxand Kmwere analyzed for both immobilized and free catalases., the results indicated that the immobilized catalaseshad a high affinity with the support. The immobilized catalases showed better resistance to pH andtemperature change than that of free catalases. The storage stability was significantly improved and thereusability was very high after catalases were immobilized onto the surface of Fe(III)-AOPAN nanofibrousmembranes.
PVA/PA6composite nanofibers were formed by electrospinning. Cu(Ⅱ)-PVA/PA6metal chelated nanofibers were prepared by reaction between PVA/PA6composite nanofibers and Cu2+solution, whichwere used as the support for catalases immobilization. The result of experiments showed that PVA/PA6composite nanofibers have excellent chelation capacity for Cu2+ions. At the same time, the structures ofnanofibers had not been distorted obviously during the reaction with Cu2+solution. The immobilizedcatalases showed better resistance to pH and temperature inactivation than that of free form, and the storagestabilities，reusability of immobilized catalases were significantly improved. The half-lives of free andimmobilized catalases were8d and24d repectively. Besides, the epoxy PVA/PA6nanofibers weregenerated by epoxidation reaction for catalases immobilization. Comparing the experimental data, theimmobilized catalases on matrix of Cu(Ⅱ)-PVA/PA6nanofibers presented higher activities thanimmobilized catalases on the surface of epoxy PVA/PA6nanofibers.
On the base of Fe(Ⅲ)-AOPAN metal chelated nanofibrous membrane and Cu(Ⅱ)-PVA/PA6metalchelated nanofibrous membrane, the EMRs were assembled. The relationships among operating pressure,membrane flux, and conversion ratio of H2O2were investigated, and results showed that the EMRs weresteady, efficient and repeatable.
引文
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