The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. The reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate were extensively characterized in water content, hydrodynamic radius, and aggregation number. The results show that the water content and hydrodynamic radius of the reversed micelles were significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate had a wider aggregation number distribution than the Span 85 reversed micelles. Using lysozyme as a model protein, protein solubilization by the reversed micelles was investigated. Lysozyme solubilization increased significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. High solubilization of lysozyme was obtained by the affinity-based reversed micelles of 62.7mmol/L Span 85 with coupled CB higher than 0.25mmol/L. Lysozyme recovery was carried out using a stripping solution of high ionic strength. The recovered lysozyme exhibited an activity equivalent to the native lysozyme and its secondary structure was also unchanged. The results indicate that the reversed micellar system would find potential application in protein separation.

研究了多种添加剂促进变性还原溶菌酶复性的作用。考察了添加剂浓度及变性剂盐酸胍浓度对复性收率的影响。结果表明，精氨酸、乙酰胺、丙酮、硫脲及甘油均能有效促进变性溶菌酶复性，并且存在最佳的添加剂浓度使变性溶菌酶的复性收率最大。在促进复性中，乙酰胺等结构类似物与盐酸胍具有相同作用，因此，在降低复性液中盐酸胍浓度的同时，适当提高乙酰胺浓度即可获得较高的复性收率。当盐酸胍浓度为0.2mol/L时，复性收率达到90%时的乙酰胺浓度为2mol/L，但降低盐酸胍浓度至0.06mol/L时，达到相同变性收率的乙酰胺浓度需要4mol/L。甘油与盐酸胍存在着协同作用，在一定浓度的盐酸胍存在下，添加适量的甘油能获得较高的复性收率。

It has been recognized that the artificial chaperone system, cetyltrimethylammonium bromide and β-cyclodextrin, is effective for enhancing protein renaturation. In this work, we studied the effect of the artificial chaperone system and guanidinium chloride (GdmCl) on the oxidative renaturation of lysozyme at 0.21-1.05mg/mL, and a kineticmodel based on the competition between protein folding and aggregation was employed to express the renaturation process. The refolding rate constant increased, while the aggregation rate constant decreased, with increasing concentration of the artificial chaperones. With increasing GdmCl concentration (0.28-2M), both rate constants decreased, but there existed a specific GdmCl concentration that maximized the ratio of the two rate constants and thus the renaturation yield. The results obviously indicated the cooperative effect of GdmCl and the artificial chaperones on enhancing protein renaturation.

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, thereexisted an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3ml of the lysozyme (1mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54mg per l gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12mg per 1gel perh), indicating the advantage of the present system for producing a high throughput in protein refolding operations.

研究了流加操作与稀释添加耦合辅助高浓度变性还原溶菌酶的复性，建立了相关的过程动力学模型。利用三态复性的过程模型对实验数据进行了拟合，获得了较好的拟合效果。利用模型分析了复性过程的动力学特性，结果表明，两者耦合可在较低的盐酸胍浓度（ mol·L-1）存在下使较高浓度的（5mg·ml-1）变性溶菌酶获得80%以上的复性收率；并且发现添加剂乙酰胺的辅助复性作用与盐酸胍相同，降低盐酸胍浓度，适当提高乙酰胺浓度即可使聚集体生成速率常数最小，酶的复性收率最大；但甘油与盐酸胍具有协同作用，只有在适当的盐酸胍浓度下添加甘油才可获得理想的复性效果。