【作者】 刘杨；

【导师】 孙彦；

【作者基本信息】 天津大学 ， 生物化工， 2006， 博士

【摘要】 反胶团萃取技术在蛋白质等生物活性物质的分离领域中被广泛研究。但是传统的离子型反胶团在萃取过程中往往对蛋白质选择性低以及使蛋白质失活,因此本文研制出新型亲和非离子反胶团系统,并对此反胶团系统在蛋白质分离纯化过程中的萃取特性进行了系统的分析研究。通过两液相合成的方法将非离子型表面活性剂(Span 85)分子结合亲和型色素染料辛巴蓝(CB)分子,并以正己烷为溶剂制备出CB-Span 85亲和型反胶团系统。利用水分测定仪和激光光散射仪对亲和反胶团的性质进行研究。结果表明,由于亲和配基CB的引入,CB-Span 85反胶团的含水率(W0),流体力学半径(Rh),以及胶团聚集数(Nag)均比Span 85反胶团提高。亲和反胶团对蛋白质的萃取率也随CB浓度的增加而显著增加,说明亲和相互作用在萃取过程中起主导作用。被萃取的蛋白质通过1mol/L NaCl溶液进行反萃,回收得到的蛋白质显示出与天然态蛋白质完全相同的活性和二级结构,证明该亲和反胶团系统良好的生物相容性。助剂对反胶团萃取蛋白质的过程具有显著的影响,因此本文考察了正己醇作为助剂对CB-Span 85亲和反胶团性质及蛋白质萃取的影响。结果表明3 vol.%正己醇可增加反胶团的W0、Rh和Nag,从而使反胶团对蛋白质的萃取率提高16%,并且揭示出正己醇分子几乎参与亲和反胶团系统中每一个反胶团的形成,使反胶团界面层的曲率和硬度降低,界面层的流动性增加。对亲和反胶团系统中蛋白质分配平衡和萃取动力学进行了研究。结果表明蛋白质的分配平衡等温线可用Langmuir方程进行拟合,并且亲和配基CB和助剂正己醇浓度的增加可显著增加亲和反胶团系统的萃取容量。萃取动力学实验结果反映了液膜传质阻力、空间位阻相互作用、亲和相互作用和界面传质阻力这几种因素协同影响CB-Span 85亲和反胶团系统萃取蛋白质的动力学传质过程。最后将CB-Span 85亲和反胶团系统应用于实际蛋白体系的萃取分离。通过一次萃取可以使粗鸡卵清白蛋白中溶菌酶的纯化因子达到23。并且在反胶团相的多次循环利用中,溶菌酶的纯化因子并未随利用次数的增加而显著下降,说明CB-Span 85亲和反胶团系统在分离纯化蛋白质方面具有一定的应用前景。更多还原

【Abstract】 Reversed micelles for the protein extraction have been extensively studied. Ionic surfactants were mostly employed to form the extractive reversed micellar systems, and problems remained for the denaturation of proteins and the low selectivity to proteins. The dissertation develops a novel affinity-based reversed micelles system formulated by nonionic surfactant, which is applied in protein extraction and purification.The noninonic surfactant of Span 85 is modified with CB as an affinity surfactant by a two-phase reaction. A novel reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate are extensively characterized in the water content (W0), hydrodynamic radius (Rh) and aggregation number by water determination and laser scattering technology. The results show that the water content and hydrodynamic radius of the reversed micelles are significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate have wider aggregation number distribution than the Span 85 reversed micelles. Moreover, protein extraction yield by the affinity-based reversed micelles increase significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. Protein recovery is carried out using a stripping solution of 1mol/L NaCl. The recovered proteins exhibite an activity equivalent to the native proteins and their secondary structure are also unchanged, which implies that the reversed micellar system is biocompatible.The dissertation investigates the effect of hexanol on the affinity-based reversed micelles due to the significant impact of the assistant in reversed micellar system. The addition of n-hexanol to the reversed micellar system resultes in significant increase in water content and hydrodynamic radius of the affinity reversed micelles. Moreover, the reversed micelles with hexanol reveal broader aggregation number distribution and larger average aggregation number than the reversed micelles without hexanol addition. Lysozyme extraction yield increases 16% with n-hexanol concentration increasing from 0 to 3 vol.%. It is considered due to the decreases in the micellar curvature and rigidity of themicellar interfacial layer, and the increase in the micellar interfacial fluidity, which is indicated that hexanol molecules contribute to the formation of all reversed micelles.Partitioning equilibria and kinetic analyses in the affinity-based reversed micelles have been studied. The partitioning isotherms of proteins in CB-Span 85 reversed micellar system can be approximated by the Langmuir equation. The extraction capacity for protein increases significantly with CB concentration and n-hexanol concentration. Kinetic analyses in forward extraction and in back extraction have gained clearer insight into the contributions of liquid-film mass transfers, steric hindrance effect, affinity associations and the interfacial resistance for protein accommodation and release.Furthermore, lysozyme is separated from the natural chicken ovalbumin solution and the purification factor reaches 23 through the first extraction by the affinity-based reversed micelles. The affinity-based reversed micellar system is recycled three times for lysozyme purification and the purification factor for lysozyme doesn’t decrease greatly. The results indicate that the reversed micellar system would find potential application in protein separation.更多还原