The Refolding Of Denatured Lysozyme Assisted By The Cooperative Effect Of Artificial Chaperones And Additives

Based on the improvement of measuring the standard curve of lysozyme activity, we optimized the conditions of the lysozyme refolding assisted by artificial chaperones. The further study was done in the high concentration lysozyme refolding assisted by the cooperative effect of artificial chaperones and additives.The data of the standard activity of lysozyme was measured by the improving method. In this way the standard activity of lysozyme of any temperature could be found by doing only a set of experiments,so that the veracity and the comparability could be enhanced.The conditions of the lysozyme refolding assisted by artificial chaperones were investigated. The results indicates that the ratio of GSSG/GSH to maximum refolding yield varies because of different concentration of lysozyme in refolding buffer. Without considering the effect of reduced DTT , the best refolding yield could be obtained when the concentration of lysozyme is 0.06,0.1,0.2 mg/mL and the GSSG/GSH remains 1:1. When the lysozyme concentration is 0.6 mg/mL, the effect of reduced DTT should be considered, the better refolding yield can be obtained while the transformed GSSG/GSH remains 1:1.The best refolding yield can be obtained as the lysozyme is 0.6 mg/mL, GdmCl is 0.4 mol/L and CTAB/β-CD is 1.2:9.6 mmol/L. Under the stillness and shake conditions, the effect was obviously different. In order to get higher refolding yield under the stillness conditions, it must be with longer time and higherβ-CD; under the shake conditions,higher refolding yield could be obtained with shorter time and lowerβ-CD, but it is lower than the refolding yield that getting from stillness with longer time and higherβ-CD.Arginine, glycerol, acetamide and thiourea could all be helpful in the refolding of denatured lysozyme assisted by the cooperation of artificial chaperones, among them, acetamide and thiourea were better. The cooperation of acetamide reacted faster, in this experiment, when the concentration of acetamide increased, the cooperation of acetamide increased too. The cooperation of thiourea reacted slower, and the best cooperative concentration of thiourea did exist.Orthogonal experiment indicates that the cooperative condition of obtaining the better refolding yield of lysozyme at 1 mg/mL: acetamide is 2.2 mol/L,GSSG/GSH is 5:2 mmol/L,CTAB/β-CD is 1.2:9.6 mmol/L, refolding yield can be 85% in 1h, and thiourea is 0.8 mol/L,GSSG/GSH is 5:2 mmol/L,CTAB/β-CD is 1.2:9.6 mmol/L, refolding yield can be 74% in 1h.