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.

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。

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.

Sorbitan trioleate was modified with Cibacron Blue F-3GA (CB) to create an affinity surfactant and to form affinity-based reverse micelles in nhexane. The partitioning equilibria and the extraction kinetics of lysozyme and bovine serum albumin (BSA) were then examined. The solubilization capacity of the reverse micellar system for lysozyme increased linearly with increasing the CB concentration from 0.1 to 0.5 mmol L−1. In contrast, the capacity for BSA at 0.5 mmol L-1 of coupled CB was only about one-fifth that for lysozyme. It indicates a strong steric hindrance effect of the micelles for the high molecular mass protein. The overall volumetric mass transfer coefficient of lysozyme in the forward extraction increased from 0.43×10−3 to 1.25×10−3 s−1 with increasing CB concentration from 0.1 to 0.5 mmol L−1. Due to the high molecular mass of BSA, its volumetric mass transfer coefficient in the forward extraction was only one-sixth that of lysozyme. The ratio of the coefficient in the back extraction to that in the forward extraction was less than 0.03, much lower than those in other micellar systems. It indicates that the interfacial resistance in this system was severer than in others.

Sorbitan trioleate (Span 85) modified with Cibacron Blue F-3GA (CB) was used as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. It was found that the addition of hexanol to the reversed micellar system resulted in a significant increase in water content and hydrodynamic radius of the affinity-based reversed micelles. Moreover, the reversed micelles with hexanol revealed broader aggregation number distribution and larger average aggregation number than the reversed micelles without hexanol addition. This is considered to be due to the decreases in the micellar curvature and rigidity of the micellar interfacial layer and the increase in the micellar interfacial fluidity. Consequently, the solubilization capacity of lysozyme increased about 70% in the reversed micellar solution with 3 vol% hexanol. On the other hand, the capacity of BSA was only 30% increased under the same conditions due to its larger molecular size than lysozyme. Kinetic analysis revealed that the increase in the micellar interfacial fluidity in the presence of hexanol resulted in faster release of lysozyme from the micelles, thus leading to an increase of the overall volumetric mass transfer coefficient in the back extraction.